Animal Life
Hedgehogs in
Hedgehogs in
Hedgehogs in
Hedgehogs in
Ireland
Ireland
Ireland
Ireland
The Hedgehog
Though hedgehogs
are common
throughout Ireland, they are not
Latin: Erinaceus europaeus ( Erinaceus means  spiky wall )
native to this country. It is thought
Irish: Gráinneog (meaning  horrible one )
they were introduced by humans,
possibly the Normans in the 13th
woodlands and gardens.
century.
In the wild, a hedgehog
The hedgehog in Ireland is the same
can live for about three
species as that found in the rest of
to five years, but some
Europe. There are about a dozen
can live up to 10 years.
other species of hedgehog and these
Hedgehogs are nocturnal
are found in South East Asia, China
and wander about at
and Africa.
night, travelling about 3
Hedgehogs are protected in Ireland. If
km in search of food. If
you want to keep one captive
you see one during the
(perhaps because it is ill)
day, it is possible that it
you need to apply to
could be ill.
the National Parks and
Hedgehogs hibernate in
Wildlife Service for a
winter when food
license. Hedgehogs
becomes scarce. They
cannot be sold.
wake up now and then
he hedgehog is easy (for its size), and a short and feed, often when the
Are hedgehogs and
Are hedgehogs and
Are hedgehogs and
Are hedgehogs and
Tto recognise. Its tail. Its small bright eyes weather is mild. They
T
T
T
head and back are cannot see very well but build nests out of leaves,
porcupines related?
porcupines related?
porcupines related?
porcupines related?
covered with sharp spines, it has a very good sense grass and other
As both hedgehogs and porcupines
each 2-3 cms long. These of smell and great vegetation, often under
have spines, you might think that they
spines are actually hearing. hedges, in compost heaps
are related, but they are not.
modified hair (much For food, hedgehogs eat and beneath piles of
Hedgehogs belong to a group of
harder than normal hair). caterpillars, earthworms, wood.
animals known as "insectivores", small
If frightened or attacked, slugs, beetles, snails and Few animals will eat a
mammals that feed mainly on insects
it will curl up into a ball, insects  and are very hedgehog because of its
and similar small creatures.
and uses the spines to noisy eaters! They also spines, but badgers will.
Porcupines are rodents and, being
protect its body. An make pig-like squeals The biggest killer of
herbivores, mostly eat plant food such
adult hedgehog has when distressed and hedgehogs are cars and in
as bark and leaves.
approximately 5,000- grunt when courting. the garden, slug pellets.
The hedgehog doesn't really have any
7,000 spines. Hedgehogs generally live Hedgehogs are a
close relatives. However, it is thought
A hedgehog is about 25 alone and only look for gardener's friend, eating
that there is some distant link to moles
cm long. It has a sharp company when they are slugs and caterpillars and
and shrews.
snout, relatively long legs mating. They live in not doing any damage.
Hedgehogs and their young
Hedgehogs and their young
Hedgehogs and their young
Hedgehogs and their young
The hedgehog will have four or five young at a time, one
litter between May and July and often another in August
or September. The young are born with soft spines,
which will soon harden.
A baby hedgehog is called a hoglet and is also known as a
pup, kit or piglet.
Porcupines (above) are not related to hedgehogs.
© 2010 Sherkin Island Marine Station & its licensors. All rights reserved.
www.naturesweb.ie Spring 2010
5
Courtesy Samuel Palmer CC-A-SA-2.5
Courtesy of www.naturespicsonline.org
Turk J Zool
34 (2010) 237-242
© TÜB0
TAK
Research Article
doi:10.3906/zoo-0810-17
The anatomy and histology of the atrioventricular conducting
system in the hedgehog (Hemiechinus auritus) heart
Abolghasem NABIPOUR*
Department of Anatomical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad,
P. O. Box: 91775-1793, IRAN
Received: 30.10.2008
Abstract: This study examined the atrioventricular conducting system in 4 adult male hedgehogs (Hemiechinus auritus).
The histological structure of these components was studied using routine histological methods. The AVN was located at
the lower and anterior part of the interatrial septum, near the root of the aorta. It was almost oval and consisted of twisted
cells. Internodal pathways in the hedgehog heart were not observed, but there were numerous purkinje-like fibers within
the myocardium of the atrium. The AVB was a continuation of the AVN, as a compact structure, extended obliquely
through the fibrous ring toward the apex of the interventricular septum, and was composed of many purkinje cells.
Key words: Hedgehog, heart, histology, atrioventricular node, atrioventricular bundle
Introduction Cardiovascular diseases are an important cause of
human mortality worldwide, especially in developing
No other exotic animal has caught the attention of
countries. In addition to high rates of mortality, the
the public quite like the hedgehog has. Their spines,
costs associated with treating these diseases are high.
friendly nature, and an ever-smiling expression have
The negative economic, social, industrial, and
endeared them to millions of confessed hedgehog
psychological effects of these diseases are significant. In
lovers around the globe.
order to understand cardiac function, research on the
In the evolutionary development of the vertebrate
histological structure of the cardiac conduction system,
heart, the specialized atrioventricular conduction
especially the atrioventricular system, is necessary. Two
system appears as a phylogenetically new structural
principal components of the atrioventricular
entity, which, to date, has been documented only in
conducting system are the atrioventricular node (AVN)
mammals and birds (Szabo et al., 1986). Moreover, in
and atrioventricular bundle (AVB). For example, some
considering its development, it is very important to
cardiac arrhythmias are due to pathological lesions and
compare the cardiac conducting system in different anatomical defects in the AVN and AVB, or in their
species. blood supply.
* E-mail: [e-mail]
237
The anatomy and histology of the atrioventricular conducting system in the hedgehog (Hemiechinus auritus) heart
The anatomy and histology of the AVN and AVB the heart was flushed with warm (40 °C) normal
have been studied in humans (Lev and Lerner, 1955; saline and for fixation was perfused with 10% neutral
Titus et al., 1963; James, 1970; Titus, 1973), dogs and buffered formalin solution. The lower part of the
monkeys (Nonidez, 1943; James, 1964), hoofed interatrial septum (from the level of the upper part
animals (Meyling and Terborg, 1957; Prasad and with the coronary sinus) along with the upper part of
Sinha, 1980), rabbits (James, 1967), birds (Szabo et al., the interventricular septum were removed. The
1986), lizards (Prakash, 1990), camels (Ghazi and samples were placed in the same fixative, and then
Tadjalli, 1993, 2002), cats (Ghazi et al., 1998; Tadjalli through a series of graded alcohols and xylene, and
et al., 1999), cattle (James, 1965), horses (Bishop and eventually into paraffin wax. Serial sections 6-µm
Cole, 1967), goats (Nabipour, 2002; Nabipour et al., thick were made longitudinally, starting from the
2002), guinea pigs (Nabipour, 2004a, 2004b), and right side of the samples. The sections were preserved
recently in ovine fetuses (Nabipour and Shahabodini, and then selected by the interval of 3, stained with
2007). However, precise data are not available on the Masson s trichrome green and PAS-Alcian blue
anatomy and histology of the AVN and AVB in (periodic acid Schiff-Alcian blue) (Luna, 1968). The
hedgehogs. The present study follows others on the stained sections were studied under a light
histological structure of the AVN and AVB in microscope.
different animal species. Histological knowledge of
the AVN and AVB provides the basis for
Results
understanding their physiological function, and
The hedgehog AVN was located at the lower and
delineation of their structure in the hedgehog will
anterior section on the right side of the interatrial
provide additional insights into the significance of
septum, near the root of the aorta, and was almost
their structure.
oval (Figure 2). Morphologically, the hedgehog AVB
was a continuation of the AVN. There was no
Materials and methods
detectable border between the node and the AVB. The
AVB extended obliquely through the fibrous ring to
The study included 4 adult male hedgehogs
the apex of the interventricular septum, as a compact
(Hemiechinus auritus) with an average weight of 357
structure (Figure 3).
g (Figure 1). They were euthanized with an overdose
Within the AVN there was a mass an interlacing
of sodium pentobarbital administrated
bundle of fibers that were smaller than ordinary
intraperitoneally. After removal of the pericardium,
Figure 1. The Hemiechinus auritus species of hedgehog studied Figure 2. Photomicrograph showing the location and shape of
in this research. the atrioventricular node (AVN); interatrial septum
(IAS); interventricular septum (IVS); fibrous ring (FR),
(green Masson s trichrome staining, ×160).
238
A. NABIPOUR
myocardial fibers. The myofibrils of the nodal cells The AVB was composed of many purkinje cells.
Myofibrils were located at the periphery of the cells
were a little smaller than ordinary myocardial fibers.
and a perinuclear clear zone was obvious, whereas the
As such, the difference in color between the node and
other cells of the AVB had darker cytoplasm (Figure
the surrounding myocardium was minimal (Figure
6). Intercalated discs between the cells of the AVB
2). There was a framework of collagen fibers between
were present. Internodal pathways were not observed
the AV nodal fibers. There were 2 types of cells in the
in the hedgehog heart, but there were numerous
hedgehog AVN: P (pacemaker-like) cells and other
purkinje-like fibers within the myocardium of the
cells with darker cytoplasm. The cytoplasm of P cells
atrium and auricle (Figure 7). Several arterioles, nerve
contained a perinuclear clear zone (Figure 4). There
fibers, and ganglions were present at the caudodorsal
were no detectable mucosubstances in the cells of the
section of the AVN and AVB to supply them.
atrial and ventricular myocardium, AVN, or AVB
Additionally, there was fibrous cartilage in the
(Figure 5). hedgehog atrioventricular fibrous ring (Figures 8, 9).
Figure 3. Photomicrograph showing the AVB that is passing Figure 4. Histological structure of the AVN in the heart
through the fibrous ring. Atrioventricular bundle hedgehogs. Pacemaker like cells (P); darker cells (D);
(AVB); interventricular septum (IVS); fibrous ring collagen fibers (arrows), (green Masson s trichrome
(FR), (green Masson s trichrome staining, ×160). staining, ×640).
Figure 5. The photomicrograph does not show detectable Figure 6. Showing the AVB in the heart of hedgehogs.
mucosubstances in the ventricular myocardium of Atrioventricular bundle (AVB); interventricular
hedgehog, (Periodic Acid Schiff-Alcian blue staining septum (IVS); fibrous ring (FR). Note the high number
×640). of the purkinje fibers (arrows), (green Masson s
trichrome staining, ×320).
239
The anatomy and histology of the atrioventricular conducting system in the hedgehog (Hemiechinus auritus) heart
Figure 7. Showing numerous purkinje-like fibers within the atrial Figure 8. Showing a parasympathetic ganglion near the AVN and
myocardium in the heart of hedgehogs. Purkinje-like AVB of hedgehogs. Capsule (C); perikaryon (P); nerve
fibers (arrows), (green Masson s trichrome staining, fibers (NF); amphicyte (arrow), (green Masson s
×640). trichrome staining, ×640).
vena cava in most mammals) is unusually large. This
effectively displaces the AVN and AVB anteriorly
toward the root of the aorta. However, in sheep
(Copenhaver and Truex, 1952), humans (Titus et al.,
1963), dogs (James, 1964), horses (Bishop and Cole,
1967), cattle (James, 1965), camels (Ghazi and
Tadjalli, 2002), cats (Tadjalli et al., 1999), and goats
(Nabipour, 2002) the AVN is located in the posterior
section of the interatrial septum, anterior to the
coronary sinus. The hedgehog AVN was oval, whereas
in ovine fetuses, as in adult sheep (Copenhaver and
Truex, 1952), the AVN is almost spherical. It is oval
Figure 9. Fibrous cartilage in the right atrioventricular fibrous
or fan-shaped in humans (Titus et al., 1963), is like a
ring of hedgehogs. Collagen fibers (CF); lacuna and
tiny spleen in dogs (James, 1964), has a flattened
chondrocyte (arrow), (green Masson s trichrome
oblong shape in horses (Bishop and Cole, 1967), is
staining, ×640).
ovoid in cattle (James, 1965), is an irregular elongated
oval in goats (Nabipour, 2002), is an irregular ellipse
in camels (Ghazi and Tadjalli, 2002), is an irregular
Discussion elongated oval in cats (Tadjalli et al., 1999), and is
almost spherical in guinea pigs (Nabipour, 2004a).
The anatomic location of the AVN in the
The AVN in avian hearts is not morphologically
hedgehog heart was similar to that in rabbits (James,
definable (Szabo et al., 1986).
1967), guinea pigs (Nabipour, 2004a), and ovine
fetuses (Nabipour and Shahabodini, 2007). Because The hedgehog AVB was displaced anteriorly, near
the ostium of the coronary sinus is so large in the the root of the aorta. This location is similar to that
rabbit (James, 1967) and guinea pig (Nabipour, in rabbits (James, 1967), guinea pigs (Nabipour,
2004a), the AVN is displaced anteriorly and occupies 2004b), and ovine fetuses (Nabipour and
the entire region, and the AVB is foreshortened. As Shahabodini, 2007). The shortness of the hedgehog
these animals normally have a left cranial vena cava, AVB is also similar to that in goats (Nabipour et al.,
the ostium of the coronary sinus (embryologically 2002), ovine fetuses (Nabipour and Shahabodini,
derived from the terminal portion of the left cranial 2007), and cattle and horses (Meyling and Terborg,
240
A. NABIPOUR
1957). Due to the absence of the membranous part of 1999), rabbits (James, 1967), goats (Nabipour, 2002),
the interventricular septum in these animals, the AVB and ovine fetuses (Nabipour and Shahabodini, 2007),
is short; however, in animals in which the ganglia are present in the posterior part of the AVN,
membranous part is present, e.g. cats (Ghazi et al., but not in the node. Additionally, there are no ganglia
1998), the AVB is long. at the periphery or within the node in the guinea pig.
In the present study internodal pathways in the
The AV node cells and their arrangement as a mass
hedgehog heart were not observed, but there were
of interlacing bundles interwoven with collagen fibers
numerous purkinje-like fibers within the myocardium
in the hedgehog is similar to that in humans (Titus et
of the atrium and auricle. The distribution of these
al., 1963), dogs (James, 1964), horses (Bishop and
fibers suggests that they may be involved in the
Cole, 1967), cattle (James, 1965), camels (Ghazi and
interatrial spread of excitation. In humans (James,
Tadjalli, 2002), goats (Nabipour, 2002), cats (Tadjalli et
1963), dogs (Glomset and Glomset, 1940), rabbits
al., 1999), rabbits (James, 1967), guinea pigs
(James, 1967), and guinea pigs (Nabipour, 2004a)
(Nabipour, 2004a), and ovine fetuses (Nabipour and
internodal pathways are connected to the margins of
Shahabodini, 2007). The difference in color between
the AVN.
the node and ordinary myocardial fibers we observed
in the hedgehog is less than has been reported in other
Histologically, there were 2 types of cells in the
animals, which is because there were more myofibrils
hedgehog AVB: purkinje cells and cells that did not
in the cytoplasm of the cells of the hedgehog AVN.
have the typical characteristics of purkinje cells. In
There is a small quantity of elastic fibers scattered
this respect it is similar to that in guinea pigs
within the AVN in humans (Titus et al., 1963), dogs
(Nabipour, 2004b) and ovine fetuses (Nabipour and
(James, 1964), and cats (Tadjalli et al., 1999).
Shahabodini, 2007). Typical purkinje cells
(Copenhaver and Truex, 1952), as seen in the AVB of
The number of P cells in the hedgehog AVN was
ungulates (James and Sherf, 1971), have a distinct
low, which is similar to other animals, while the AVN
perinuclear light zone and a much larger diameter
of the guinea pig (Nabipour, 2004a) and ovine fetus
than cardiac cells. Ungulate purkinje cells are almost
consisted of numerous P cells. The level of
spherical or polyhedral, and make contact with other
carbohydrates in the AVN cells of ovine fetuses is high
cells along virtually their entire periphery, whereas the
(Nabipour and Shahabodini, 2007); however, there is
cells in the AVB of canines and humans are elongated
no glycogen in the AVN cells in goats (Nabipour,
and oblong, and make contact to some extent along
2002), camels (Ghazi and Tadjalli, 2002), or guinea
their lateral margins, but more often at their terminal
pigs (Nabipour, 2004a). There is a small quantity of
ends (James and Sherf, 1971). Partitioning of the AVB
nerve fibers within the hedgehog AVN; in this respect
was not observed in the hedgehog heart, which is in
it is similar to that in humans (Titus et al., 1963), dogs
contrast to the results reported for humans and other
(James, 1964), cats (Tadjalli et al., 1999), guinea pigs
(Nabipour, 2004a), and ovine fetuses (Nabipour and animals.
Shahabodini, 2007). In contrast, in cattle (James,
1965), horses (Meyling and Treborg, 1957), and goats
Acknowledgements
(Nabipour, 2002) an abundance of nerve fibers are
present in the node. In the hedgehog heart, as in that The author wishes to express his appreciation to
of humans (Titus et al., 1963), dogs (James, 1964), the Ferdowsi University of Mashhad Research
horses (Bishop and Cole, 1967), cattle (James, 1965), Council for their financial support and to thank Mr.
camels (Ghazi and Tadjalli, 2002), cats (Tadjalli et al., Pouradibi for his technical assistance.
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Ghazi, S.R. and Tadjalli, M. 2002. Anatomy of the atrioventricular Nabipour, A., Khanzadi, S. and Banihassan, M. 2002. Anatomy and
node of camels (Camelus dromedarius). Iranian J. Vet. Res. 3: histology of the atrioventricular bundle in the heart of goats
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cardiac conduction system in ungulates, dog and man. Am. Nabipour, A. and Shahabodini, M.R. 2007. Histological study of the
Heart J. 20: 389-398. atrioventricular node and bundle in the heart of ovine fetus.
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242
Lorraine A. Corriveau,DVM
Purdue University Veterinary Teaching Hospital
Small Animal Community Practice Clinician
Hedgehog Medicine
Description
The African pigmy hedgehog (Atelerix albiventris) has become a very popular pet in the early 1990 s.
They were a fad that hit the  get rich quick crowd initially but now has a small but very loyal following. At
first these pets sold for $120-200, but as more people bred them the prices dropped and everyone who
wanted them had them. It is not difficult to find them given up for adoption now. Now color variations
exist, hedgehog clubs have been formed, and shows are held. Clinical information is becoming more
abundant and easy to find.
Hedgehogs are members of the insectivore family. They possess 36-44 teeth with the first
incisors being notably longer than the rest and are spaced apart. The lower incisiors  fit into this space
when the jaw is closed. {ðDental formula: 2(3/2,1/1,3/2,3/3)}ð. Their teeth are brachyodontic like
carnivores. The most obvious feature of the hedgehog is the dermal spines. These are modified hairs
that provide protection from predators. Beneath the spines is a thick layer of subcutaneous fat.
Hedgehogs will roll up when alarmed into an impermeable ball of spines, making them clinically
challenging. They have sharply pointed snouts and typically small eyes. All possess a collarbone
(clavicle). They are nocturnally active, and like to hide during daytime hours. The olfactory and auditory
senses of all insectivores are highly developed, making them good hunters and foragers. Hedgehogs
have a plantigrade gait, meaning that they walk on the entire soles of the feet rather than the toes alone.
The most common pet species in the United States is the African pigmy hedgehog. Adults of this species
generally weigh 300-600 grams. Longevity is about 5-7 years in captivity. Some color varieties are
available now and if the pattern follows true, many genetic defects and a generally weakening of the
species may follow from the inbreeding practices used to produce these. Hedgehogs are easy to sex,
because males have a distinct prepuce like in dogs but testicles are abdominal. Females have a vulva.
Hedgehogs are induced ovulators year round. Litters of 3-6 young are produced following a gestation of
34-37 days. The offspring have a protective coating over the spines during parturition, which is lost in the
first 24 hours to expose the spines. Babies wean in 30-40 days and become sexually mature at about 2-3
months of age.
Diet
In the wild, hedgehogs consume insects, small vertebrates, and carrion. In captivity, hedgehogs fare
well on formulated hedgehog or low fat dog or feline diets. Some recommend soaking the dry food prior
to feeding. Reduced calorie formulations should be used, as obesity is the most commonly encountered
nutritional problem. Fruits and vegetables should be added to the diet to dilute out the high calorie foods
and to offer extra fiber. Cottage cheese, eggs, and other such protein sources can be given to breeding
hedgehogs, but should be avoided or limited in sedentary animals. Insects are relished and can be given
as a treat, but are calcium deficient and due to improper Ca:P ratio should not be the sole diet.
Recommend feeding once daily in the evening. Also encourage evening exercise to prevent obesity.
Housing
Usually caged alone but can be housed in groups if given enough space. Excellent climbers so cages
need to be smooth walled and high enough to prevent escape. It is recommended to avoid wire floors.
Solid floors should have bedding such as newspaper, recycled newspaper, or wood shaving (pine or
aspen). A hide/sleeping area should be provided. One can be creative in the type of hide/sleep area
provided.
Examination
Hedgehogs can be very difficult to work with as patients. Their ability to roll up into a ball makes them
impossible to examine if they do not want to be examined. Lightweight leather gloves should be used to
prevent the spines from pricking the handler s hands. Sometimes, an aquarium with a shallow layer of
water can be used to allow for visual examination. When the hedgehog is placed in the water, it must
unroll so as to not breathe the water. This helps keep the hedgehog unrolled for examination. Some
hedgehogs uncurl with back stroking of the rump spines. Anesthesia is almost always required for a
complete examination. Isoflurane or Sevoflurane are the recommended gas anesthesia. A small mask,
fashioned from a syringe case can be slipped into the opening of the ball and over the snout.
Alternatively, the hedgehog can be placed in an anesthetic chamber for induction. Once anesthetized,
the mask can be positioned better or an endotracheal tube can be placed (very challenging). At this point
the hedgehog can be easily examined. The examination should be systematic. A visual of the eyes,
nose, ears, oral cavity, teeth, spines, anal and urogenital openings, palpation of lymph nodes and
abdominal cavity, and auscultation of the thorax and abdomen will detect most disorders. In many cases,
the problem will require further evaluation.
Clinical Pathology
Hematology and serum biochemistry are often the first stop in evaluating more vague illnesses in
hedgehogs. Blood collection can be a bit challenging but can be mastered with a bit of practice. The
jugular or cranial vena cave is generally used for venipuncture. The jugular is relatively short and runs
from the thoracic inlet to the ear. A large lymph node lies just lateral to the jugular. It usually cannot be
palpated, but can be blindly entered in the anesthetized hedgehog. With the hedgehog anesthetized and
in dorsal recumbency, the neck should be extended. While it cannot usually be visualized, the jugular
vein will generally course from the manubrium sterni to the ear. Large lymph nodes lie just lateral to the
vein and serve as a landmark. The jugulars should be entered blindly with slight suction on the syringe
until a flash is seen. Slow and steady pressure is used until the desired quantity of blood is obtained.
One percent of the body weight can be taken, but 1ml should be adequate for routing analysis. Pressure
should be applied briefly following venipuncture. Fecal examination, urinalysis, cytology are run and
interpreted in the same manner as the more familiar mammals. Clinical pathology data is limited but
empirically, most values are similar to canine and feline patients.
Radiographs
Radiographs are most easily taken in the anesthetized hedgehog. A ventrodorsal and lateral view can be
taken on a single small film. It may be beneficial on the lateral view to elevate the excess fat pad that
holds to dorsal spines. The can be accomplished by using a  chip clip . Knowledge of normal anatomy is
necessary for interpretation of radiographs. A radiographic atlas of exotics is available for normal
anatomy.
Therapy
The diagnosis is not the only challenge in hedgehog medicine. The same defense that prevents
examination can also make treatment difficult. Once a diagnosis is made, or at least initial diagnostic
procedures are finished, therapy should be started. Early treatment is crucial to success. The small size
of hedgehogs makes them very susceptible to starvation and dehydration. If they are not eating or
drinking, they should be force-fed and administered fluids. If shocky or critically dehydrated, fluids should
be given intraosseously. A needle can be placed in the femur, in the same fashion as an intramedullary
pin, and fluids or drugs can be administered in this fashion. The fluids are taken up into circulation so
rapidly that this technique is equivalent to intravenous infusions, which is very difficult in hedgehogs.
Less severely ill pets can be given subcutaneous or oral fluids. Maintenance fluid requirements are 60-
100ml/kg/day. Therefore a 400gram hedgehog will need 24-40ml per day for maintenance plus the
deficit. Force-feeding energy requirements can be calculated with the following formula:
Basal energy requirement (BER) = 68*(body-weight in kg.75)
Maintenance energy requirement (MER) = 1.25*(BER)
Actual energy requirements will vary from 1-2 times maintenance energy, depending on the medical
condition. A 400gram hedgehog will require 45-90 kcal of energy per day for maintenance. Using the
higher calorie formula, this means about 23-45ml per day.
Normal room temperature should be used for housing hospitalized mammals unless they are
hypothermic or hyperthermic. Hedgehogs will go into a torpid state when cold which could be dangerous
if they are sick. Care should be taken, however, that they are not overheated in an avian or reptile
intensive care unit. Minimizing stress is critical and somewhat more difficult when treating  prey type
2
species. These secretive creatures will feel more comfortable when adequate hiding spaces are
provided. They preferably should be kept in a quieter location, away from barking dogs.
Information regarding drug dosing and efficacy is largely anecdotal at this point. There are no
label-approved drugs for hedgehogs. All medications should be used with caution and with informed
consent of the owner. Generally drugs and dosages published for ferrets are appropriate for hedgehogs.
When exotic animals are treated, follow up evaluation takes on a critical role since we are still feeling our
way along. The safest choice of available drugs should always be made. Often, injectable medications
are preferred since they can be given when the hedgehog is in  spiny ball mode. Oral medications are
given in liquid suspensions or solution form. Very tame hedgehogs can be medicated easily by their
owners, but some will have to have the medications put on a favorite food item. Eye and ear medications
are very difficult to administer. Solution preparations are easier to apply but ointments will last longer and
require less frequent administration.
Dental care for hedgehogs is difficult. Few owners can brush the teeth and yet periodontal
disease, tooth root abscesses, and decay are all common problems. Recommendations include using
dry food formulated for dental care, using crisp vegetables, and if possible, the use of oral cleansing gels
(example: Maxiguard®).
Client education is critical to proper treatment. In addition to demonstrating the proper method of
treatment, owners should be informed of predisposing factors and how to correct them.
Anesthesia
Certain diagnostic and therapeutic procedures may require anesthesia for restraint and prevention of
pain. In most cases, isoflurane gas, administered by mask is the simplest, safest, and most rapid
method. Endotracheal intubation is difficult and requires that the hedgehog be deeply anesthetized, so
not recommended for novice clinicians. About 1.5-2 mm endotracheal tube is needed for an adult
hedgehog. Injectable anesthetics carry the inherent disadvantage of greater difficulty to control depth of
anesthesia. Hedgehogs loose much heat more rapidly, the tracheal lumen occludes more easily, the
patient is more difficult to monitor. A supplemental heat source is essential, and cotton tipped applicators
should be available to swab out the throat. A small endotracheal tube should be available for emergency
intubation. Clear adhesive drapes facilitate monitoring. Most importantly, a technician should be
dedicated to the constant monitoring of the patient. The tidal volume is generally too low to move the bag
on most systems so respiration cannot be monitored in this way. The low tidal volume also leads to a
large amount of dead space within the delivery system. Semi-open, non-rebreathing systems must be
used for hedgehogs. Special bags are available that adjust to the small tidal volume. Alternatively, a
balloon can be used as a bag on the non-rebreathing circuits. A respiratory monitor helps detect early
changes in respiratory rate or tidal volume. An ECG facilitates monitoring well.
Surgery
Preparation of the surgical site can be a challenge if surgery is performed on the dorsal part of the body.
The spines must be removed so a surgical field can be formed. They can be plucked or clipped at the
base. These spines are very difficult to pluck. Clipping is done with a pair of scissors just at the base,
leaving a little behind. The skin is then prepared as in other animals. Surgery of hedgehogs can be
enhanced by the use of several types of instrumentations not commonly used in traditional pets. Their
small size requires finer instruments, methods of controlling small amounts of hemorrhage, magnification
and directed source of light. Microsurgical or ophthalmic instruments are frequently used. Microsurgical
instruments should be counterbalanced and have rounded handles to allow them to be manipulated by
gently rolling them between the fingers. There should be no locking mechanism on needle holders as
releasing these causes considerable jarring. Delicate surgery should be performed while seated with the
wrists supported on the table to minimize motion. Hemorrhage can be controlled by the use of
electrocoagulation. Bipolar instrumentation is preferred. Vessels must be isolated and then coagulated.
If bipolar instruments are not available, the vessel can be isolated with forceps and the electrode is then
touched to the forceps. Care must be taken not to cause excessive time destruction. Ligatures will be
required on larger vessels (>2mm). Small sizes of suture (4-0 to 10-0) can be used for these when they
are in an accessible area. Vasculare clips (Hemoclips) are preferred when working in a restricted area
such as the body cavity or when speed is required. Occasionally hemorrhage will occur when none of the
above will be applicable. In these cases, the area can be packed off with absorbable foam sponges (Gel
Foam). Due to the small size of hedgehogs, magnification of the surgical field is advantageous. Optical
loupes can be used for many procedures and are reasonably priced. Operating microscopes will provide
greater magnification and also lighting and are very beneficial for many procedures. Working under
magnification is very different from standard surgery. Every movement is magnified and the field is very
restricted. It is sometimes difficult to even find instruments. Surgical procedures performed under a
3
microscope can be done with much greater precision, but they also take more time. Considerable
practice is required to master microsurgery. The most commonly performed surgeries are tumor removal
procedures and pyometra sugery.
Common Problems
Ectoparasites are the single most commonly presented problem of hedgehogs. Mites, especially
Chorioptes and Caparina sp. are very common in hedgehogs. These parasites are just barely visible to
the naked eye and the crusts can be seen to move in heavily infested hedgehogs. Loss of spines,
pruritus, and scaling of the skin are the primary clinical signs of this disease. A skin scraping should be
performed routinely on hedgehogs as these mites can take a long time to cause overt disease. When
diagnosed, hedgehogs should be treated with an ivermectin product. A dose of 0.5mg/kg given
subcutaneously once weekly for 3-6 weeks is generally effective in eliminating the parasite. Revolutionâð
(Selamectin) used topically at 9mg/pound once and then repeated in 3 weeks has shown good success
also in the treatment of these mites.
Endoparasites were quite common in imported hedgehogs but appear to be uncommon in the
domestic raised ones commonly encountered in private practice now. Fecal examinations should be
routinely performed to determine if any are present and then they should be treated appropriately.
Periodontal disease occurs frequently in hedgehogs. This also frequently progresses to tooth rot
abscesses. Affected hedgehogs will lose weight, show abnormal feeding behavior, may have swelling of
the jaw, maxilla, or periorbital region or may become anorectic. A thorough examination of the mouth,
including probing the periodontal pockets will detect most problems. Skull or dental radiographs may be
needed in some cases. Minor cases may respond to supragingival and subgingival scaling, antibiotics,
and nutritional support. More often, extensive extractions must be performed. Hedgehogs appear to do
well with few or no teeth. If no teeth are present, softer foods may be needed.
Hedgehogs are emerging as rivals of budgerigars, ferrets, and boxers in the arena of tumor
production. Neoplasias of many different types have been reported in hedgehogs, despite the relatively
sparse literature on them in general. Squamous cell carcinoma of the oral cavity and lymphosarcoma of
any body part are over represented in the literature. Whenever a lump is encountered, it should be
aspirated or removed for histopathology. Vague clinical signs of illness often result in a final diagnosis of
cancer. Thorough palpation of the lymph nodes, neck, abdomen, and auscultation of the thorax should
be done on each exam. Radiographs and ultrasound are helpful for early detection or gaining further
information about a tumor. Currently, the treatment of choice for tumors is surgical resection. If done
early, this can achieve a cure in many cases.
Obesity is a common occurrence in pet hedgehogs. The combination of unlimited supply of high
calorie food and the lack of exercise results in a calorie excess (fat deposition). Some are so fat that they
cannot or have difficulty rolling into a ball. Treatment of obesity includes changing the diet to a low calorie
cat/dog food, limiting the amount given to 2/3 of the current ration, giving fresh fruits and vegetables for
fiber, and increasing exercise by giving a wheel and allowing more play-time outside of the cage.
Trauma can also occur to the limbs of hedgehogs. One must be careful to not have string-like
material on the floor or in the cage. There have even been reports of even human hair causing
constriction bands around the limbs/digits leading to avascular necrosis.
Enteritis has been sporadically encountered in hedgehogs. Often clostridial organisms are seen
on cytological examination of the feces. Amoxicillin and metronidazole are both affective in elimination of
the clostridium and the diarrhea in most cases.
Preventive Medicine
Client education is the most important key to maintaining a healthy hedgehog. A combination of written,
spoken, and visual aids are usually necessary for adequate retention of information. Information about
housing, nutrition, sanitation, behavior, and health care should be given to each client.
Nutrition is key to keeping any animal healthy and with exotic pets, where formulated diets are not
available; the task is much more difficult. Common sense is important. A diet that would not be balanced
for more familiar species would not be for hedgehogs as well.
Parasite control should be routinely applied/performed. Fecal samples should be checked once
or twice annually and any parasites treated. Skin scrapings are very important for detecting external
parasites.
Early detection and treatment of diseases is critical. Small  prey species try to hide any signs of
illness or weakness as a mechanism to prevent predation. In captivity, this trait leads to presentation late
in the course of the disease. A hedgehog that looks sick is often very sick. A  wait and see what
happens approach is very dangerous in exotic animals. Rapid diagnosis and treatment is essential.
4
Suggested reading
1. Gregory MW, Stocker L: Hedgehogs. In Beynon PH, Cooper JE (eds); Manual of exotic pets.
Gloustershire, England, British Small Animal Veterinary Association, 1991, pp 63-68.
2. Hoefer HL: Hedgehogs. In Vet Clin North Am Sm Animal Pract 24 (1)pp 113-120, 1004.
3. Reeve, N: Hedgehogs. T&AD Poyser Ltd, London, 1994.
5
1
Caring for Your Hedgehog
By Mark Burgess, DVM
Hedgehogs are small, shy, insect-eating mam-
mals with quills on top and fur on the face and
belly. The quills resemble those of porcupines,
but are not barbed and do not imbed in the skin
when touched. When frightened, hedgehogs roll
into a ball and emit an odd rattling hiss. They
may bounce to try to poke you with their quills,
but they rarely attempt to bite. They can make
decent pets if socialized and they typically live
two to four years.
There are many species of hedgehogs, including the large European hedgehog and
many African species. The African pygmy hedgehog is the species commonly acquired
as pets. It is illegal to have hedgehogs as pets in certain areas of the U.S., so please
check your state, city, and county ordinances before getting a hedgehog.
If you do decide to get one, please adopt from a rescue group rather than buying from a
pet store or breeder. There are many wonderful hedgehogs out there just waiting to be
adopted. To find a hedgehog rescue, do a search for  hedgehog rescue on the Internet
or visit the Hedgehog Welfare Society at www.hedgehogwelfare.org.
Housing
Since African hedgehogs are native to warm regions, they do not tolerate cold tempera-
tures very well. They should be kept above 70 degrees Fahrenheit.
They may be housed in wood or wire cages with solid floors; a minimum cage size is 24
x 24 inches; cage height is not critical. Avoid wire floors, since their feet may become
caught and bones broken as the pet tries to free himself. Wire floors also tend to cause
sores on the feet. Provide a thick layer of good bedding, such as recycled paper or
hardwood shavings (not cedar or pine). Soft, clean towels can be used, but should be
removed if your pet chews them or if they become frayed; the hedgehog may become
tangled in any loose threads.
Hedgehogs are quiet animals, but they can be very active, especially at night. Ideally,
give them daily exercise outside of the cage for at least 30 minutes. Hedgehogs occa-
sionally can be aggressive with each other if housed together, so they may need to be
kept separate to prevent fighting, especially with males.
Feeding
The bulk of the hedgehog s diet should be low-fat hedgehog pellets (not a seed and fruit
mix). Pellets may be fed free-choice, unless obesity occurs. Use a formula with no more
than 7 percent fat  Pretty Pets is a good brand of food. You can also use a very low-fat
cat food, such as Hill s W/D (7 percent fat). The hedgehog s natural diet is mostly bee-
" 435-644-2001 " www.bestfriends.org
2
tles. In captivity, occasional insects such as crickets or mealworms can be fed, but these
are not nutritionally balanced and should be used sparingly. Invertebrates with better
nutritional content would include slugs, earthworms, and silkworms.
Fresh water, of course, should always be available. Ball-bearing bottles are cleaner and
not as easily spilled as bowls. Remember to clean your hedgehog s cage, food bowls
and water sources regularly.
Common Diseases
Obesity. The most common medical problem seen in captive hedgehogs is obesity. The
usual cause is feeding high-calorie foods, such as regular cat food, seeds or nuts, or
fatty insects such as mealworms and crickets. But hedgehogs can overeat even on a
low-fat hedgehog diet, so food intake often must be restricted to maintain proper body
weight. Healthy hedgehogs should appear twice as long as they are wide; they are not
round in shape, except when rolled up. Obesity increases the risk of tumor development
and may shorten your pet s lifespan. Weight loss should be accomplished slowly, since
obese pets who lose weight too rapidly may develop liver disease.
Weight loss. Because hedgehogs are aggressive eaters and rarely lose weight unless
put on a strict diet, spontaneous weight loss is a cause for concern. It usually indicates
significant illness, such as dental disease, cancer, heart disease, or uterine disease.
Seek immediate veterinary advice if you notice your pet losing weight for no apparent
reason.
Dental diseases. Hedgehogs develop gum disease and tooth infection with age. Signs
may include salivation, difficulty eating, and bad breath. Infected teeth may be loose and
painful. Treatment is cleaning and/or extraction of the affected teeth, and oral antibiot-
ics. Oral odor and salivation can also be a sign of oral cancer, which is common in older
hedgehogs.
Mites. These are common skin parasites, but they often produce no symptoms until the
mite population grows large. When numerous, the microscopic mites cause itching, flak-
ing, and quill loss. A hedgehog with a severe case of mites may develop scabs or sores
from intense scratching and biting at the skin. Mites respond to treatment with ivermectin
(oral or injectable) given weekly for 6-8 weeks. Lyme sulfur dip may help when applied
once or twice weekly for 6-8 weeks, but it is more labor-intensive and must be applied
thoroughly.
Cleaning the cage weekly when treating the mites may help reduce re-infestation, but
long-term environmental treatment is unnecessary, since the parasites die if they are off
their host for long. These parasites are species-specific but highly contagious between
hedgehogs, so use caution when introducing new pets to an existing group.
Respiratory infection. Signs include sneezing, wheezing, lethargy, nasal or eye dis-
charge, or difficulty breathing. Various bacteria may cause this type of infection. Treat-
ment is with antibiotics. You can minimize your hedgehog s risk of respiratory disease
by providing her with a warm, clean cage; avoiding the use of wood-chip beddings; and
feeding her a balanced diet.
" 435-644-2001 " www.bestfriends.org
3
Head tilt (wry neck, torticollis). This is usually due to internal ear infection, and is
sometimes secondary to a respiratory infection. The hedgehog usually tilts his head to
one side and loses his balance, often falling or circling when trying to walk. Bacteria are
the usual cause and treatment is with antibiotics.
Fight wounds. Hedgehogs can occasionally be aggressive with each other, and some-
times fight if housed together. This may result in bite wounds. Any visible wounds require
immediate medical treatment; the risk of infection is great, and early antibiotic therapy to
prevent infection is the safest option.
Cancers. Older hedgehogs are highly prone to many types of cancer. Common types
include oral tumors, and mammary tumors (breast cancer) in females. Any visible lump
should be checked immediately by a veterinarian. Oral odor, drooling, or difficulty eat-
ing are also cause for concern. Many tumors are curable if caught early and removed.
Weight control may reduce the risk of some tumors. Spaying female hedgehogs most
likely reduces the risk of mammary tumors, and eliminates the risk of uterine cancer.
Heart disease (cardiomyopathy). This degenerative disease of the heart is seen in
many pet species, including dogs, cats and ferrets, and also in humans. The causes are
unknown, but some forms in dogs and cats have been linked to nutritional deficiencies.
Signs of heart failure include bloating, lethargy, and difficulty breathing. Treatment may
control symptoms for months, but isn t likely to cure the disease. The risk of heart dis-
ease might be reduced by feeding a balanced diet and preventing obesity.
Progressive paralysis (degenerative myelopathy). This poorly understood disease
of unknown origin causes slow deterioration of the spinal cord. A gradual weakness and
paralysis begins in the rear legs and usually progresses to the front legs over time. Total
paralysis can result. There is no effective treatment in most cases, and the condition is
often fatal.
Hair or thread entanglements. Hedgehogs are prone to becoming entangled in long
pieces of thread (from bedding, such as frayed towels) or in strands of their people s
hair. The strands wrap around a leg or foot (or occasionally the penis in males) and act
like a tourniquet, cutting into the skin and cutting off blood flow. Infection and loss of the
foot may result. Minimize exposure to long hairs, strings or threads in the cage environ-
ment. If your pet is limping or has a swollen foot, seek immediate veterinary care.
Veterinary Care
No vaccines are given, but regular exams are recommended for early disease detection.
Have an exam done when you first get your hedgehog, then at one year old, then every
six months after that. With good care, your hedgehog can be a happy and lovable pet!
Dr. Mark Burgess is owner of Southwest Animal Hospital/The Exotic Animal Practice in
Beaverton, Oregon. Ninety-five percent of his practice is small exotic pets, including fer-
rets, rabbits, rodents, reptiles, hedgehogs, marsupials, and some wildlife. He lectures at
conferences and has published articles on exotic pet disease in medical journals.
" 435-644-2001 " www.bestfriends.org
Introduction to the African Pygmy Hedgehog
Heidi L. Hoefer, DVM, ABVP
Introduction
Hedgehogs are small mammals that belong to the order Insectivora. There are
several species that are found throughout the British Isles, Europe, and Asia and
Africa. Hedgehogs are not native to North America. The African pygmy hedgehog
(Atelerix albiventris) originated in the African
savanna, and is now widely bred in North
America for the pet industry. The European
hedgehog (Erinaceus europaeus) is a larger
species, native to England and Europe and
protected by law in many areas.
The African pygmy hedgehog is small,
nocturnal, and spiny-coated. They vary in color
from brown to almost black with a white ventrum. The adult ranges in weight from
300-600 grams (1 lb = 450 grams). Life expectancy averages 3-4 years in the wild
but up to 10 years in captivity.
Anatomy & Physiology
Hedgehogs are characterized by the short, grooved white and brown spines
that cover the upper part of the body. The face and belly are covered with soft, light-
colored fur. The hedgehog can assume a defensive posture by rolling up and
erecting the spines to resemble a tight ball of sharp spines.
Gender (sex) is easy to identify in hedgehogs. The male has a penis and
prepuce located midway along the abdomen. The testes are usually intra-abdominal
and are not easily seen. The female has a vulva located close to the rectal opening.
African hedgehogs breed year-round. The gestation period is 34-37 days. Litter size
ranges from 1-7 with an average of 3-4 pups. The young are born blind with soft
white spines. New brown spines appear in 2-3 days and the eyes open in 2-3
weeks. Weaning occurs at 4-6 weeks.
In their native habitat, a hedgehog will dig its burrow under logs, leaves, among
rocks, or tree roots and sleep most of the day. They are solitary and nocturnal,
emerging at dusk to forage for insects. When undisturbed, it moves with an
unsteady, waddling gait but can run quickly. The hedgehog has a keen sense of
hearing and smelling, making it an adept hunter and forager. The European species
will hibernate in the winter and the African species will aestivate in the hot, dry
season. Hibernation is not essential and is not recommended for captive hedgehogs.
Housing & Diet
Hedgehogs are usually caged alone but can be housed in groups if given
enough space. Hedgehogs are excellent climbers, so cages should be smooth-
walled and high enough to prevent escapes. Wire flooring should be avoided due to
the potential for toe and limb injury. Newspaper or wood shavings (pine or aspen)
can be used as bedding, but it must be changed frequently. A sleeping area can be
made from cardboard boxes, hollowed logs, wooden boxes, or plastic flowerpots and
filled with hay or leaves.
The native diet consists of a variety of insects, occasional small vertebrates,
and carrion (dead animals). Captive pets can be fed a diet of soaked low-fat dog or
cat chow, smaller amounts of mealworms or earthworms, and a small amount of
chopped fruit and vegetables. Because of the calcium-phosphorus imbalance, a diet
solely of insects must be avoided. Hedgehogs should be fed once daily in the
evening. Captive hedgehogs have the tendency to become obese; early evening
exercise should be encouraged.
Physical Examination & Restraint
Hedgehogs typically roll-up during clinic visits making a full physical
examination impossible without sedation. Light leather gloves are often used to
protect against the sharp spines. Some hedgehogs can be scruffed behind the ears
if caught before they ball up, however, the majority will need to be sedated.
The preferred method of sedation is isoflurane gas. The hedgehog is initially placed
in a small plastic box and then switched to a face mask to allow inhalation of the gas.
Isoflurane is generally safe for most hedgehogs.
Common Conditions And Diseases
There is very little information in the literature regarding the diseases of pet
African pygmy hedgehogs. The following list of conditions represents a review of the
literature as well as the author's own clinical experience.
Cancer of almost any body part, but especially the mouth and skin
Dental disease
Heart disease
Kidney failure
Leg and foot injuries
Obesity
Overgrown nails
Quill loss (mites are a common cause)
Respiratory problems
References
1. Allen ME: The nutrition of insectivorous mammals. In Proceedings of the Annual
Meeting of the American Association of Zoo Veterinarians, Oakland, CA 1992, pp
113-115
2. Done LB, Dietze M, Crnafield M, et al: Necropsy lesions by body systems in
African hedgehogs: Clues to clinical diagnosis. In Proceedings of the Annual
Meeting of the American Association of Zoo Veterinarians, Oakland, CA 1992, pp
110-112
3. Gregory MW, Stocker L: Hedgehogs. In Beynon PH, Cooper JE (eds): Manual of
Exotic Pets. Gloustershire, England, British Small Animal Veterinary Association,
1991, pp 63-68
4. Hoefer HL: Hedgehogs. In Vet Clin North Am Sm Anim Pract 24 (1) pp 113-120,
1994
5. Isenbugel E, Baumgartner R: Diseases of the Hedgehog. In Fowler, ME (ed): Zoo
and Wild Animal Medicine, Current Therapy 3. WB Saunders, Philadelphia, 1993,
pp 294-302
6. Smith AJ: Husbandry and medicine of African hedgehogs. J Small Exotic Anim
Med 2: pp 21-28, 1992.
VETERINARSKI ARHIV 72 (4), 213-220, 2002
Macro-anatomical investigations on the skeletons of hedgehog
(Erinaceus europaeus L.). II. ossa membri pelvini
Zait Ender Özkan*
Department of Anatomy, Faculty of Veterinary Medicine, Fżrat University, Elazżg, Turkey
ÖZKAN, Z. E.: Macro-anatomical investigations on the skeletons of hedgehog
(Erinaceus europaeus L.). II. ossa membri pelvini. Vet. arhiv 72, 213-220, 2002.
ABSTRACT
In this study, three adult male hedgehogs (Erinaceus europaeus Linnaeus) were used to
investigate the bones of hind limb. The spina iliaca ventralis caudalis was absent. The symphysis
pelvis was formed by symphysis pubis and it was an interpubic ligamentous tissue in hedgehogs.
The average Vialleton angle was measured as (8º). Average distance between the midacetabulum
and tuber coxae was 39.2 mm and average distance between the midacetabulum and ischial
tuberosity (tuber ischiadicum) was 13.8 mm. There were three trochanters on the femur. The
tibia and fibula were fused almost in the distal half. There were 7 tarsal bones and the pedis was
complete with five digits.
Key words: hedgehog, Erinaceus europaeus L., ossa membri pelvini
Introduction
Hedgehogs (Erinaceus europaeus L.) belong to the Erinaceidae family,
order Insectivora (VAUGHAN, 1972; DEMŻRSOY, 1996; 1997; 1998). The most
important features of hedgehogs are the bristles transformed to spine form
on the dorsal and lateral sides of the body, forming a quite round body
* Contact address:
Dr. Zait Ender Özkan, Department of Anatomy, Faculty of Veterinary Medicine, Fżrat University, 23159, Elazż
,
Turkey. Phone: [phone], Fax: [phone]
213
ISSN 0372-5480
Printed in Croatia
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
when threatened and in order to sleep in winter under a 4 °C environmental
temperature (DEMŻRSOY, 1997; 1998).The literature on the macro-anatomical
features of the skeletal system in hedgehogs is very meagre.
There have been some macro-anatomical investigations carried out on
the skeletal systems of wild animals such as lagomorphs (DUBRUL, 1950),
African rhinocerus (GOMER
IĆ and HUBER, 1982), hyena (TECŻRLŻO
LU,
1983), wolf and fox (GŻRGŻN et al., 1988), mink (DURSUN and TIPIRDAMAZ,
1989), raccoon dogs and badgers (HIDAKA et al.,1998), porcupine (YILMAZ et
al., 1998; 1999), otter (DÅ»NÇ et al., 1999), but the skeletal systems of hedgehogs
of the order Insectivora have not been investigated in detail.
The aim of this study is to investigate the ossa membri pelvini part of
the skeletal system in hedgehogs and to contribute to the fund of information.
Materials and methods
The bones examined were obtained from three adult male hedgehogs
caught by the villagers in Elazż
. Maceration of bones was carried out by
the method of BARTELS and MEYER (1991), and TAb
BAb
and TECŻRLŻO
LU
(1966).
For terminology, Nomina anatomica veterinaria (ANONYMOUS, 1994)
was used.
Vialleton angle (LESSERTISSEUR and SABAN, 1967) was measured as an
angle between the line lying from the midacetabulum to the midpoint of the
crista iliaca and the line lying from the midacetabulum to the centre of the
facies auricularis.
Results
Os coxae. Linae glutae were not prominent. Spina iliaca dorsalis
cranialis, spina iliaca dorsalis caudalis, spina iliaca ventralis cranialis were
prominent; spina iliaca ventralis caudalis was not present. The spina
ischiadica was not well developed. The great sciatic notch (incisura ischiadica
major) was deeper and wider than the lesser sciatic notch (incisura
ischiadica minor). The iliac tuberosity (tuberositas iliaca) on the sacropelvic
214 Vet. arhiv 72 (4), 213-220, 2002
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
surface (facies sacropelvina) was prominent, and facies auricularis was
formed as a small area.
Tuber ischiadicum was prominent and had a single process. There was
a small notch on the caudal side of the tabula ossis ischii. The symphysis
pelvis was formed by symphysis pubis and was in the form of a ligament
connecting the two caudal branch of the pubic bones (ramus caudalis ossis
pubis) (Fig. 1).
Fig. 1. Ventro-lateral aspect of os coxae of hedgehog (Erinaceus europaeus L.)
a) ala ossis ilii, b) crista iliaca, c) spina iliaca dorsalis cranialis, d) spina iliaca dorsalis
caudalis, e) spina iliaca ventralis cranialis, f) acetabulum, g) incisura acetabuli, h) for.
obturatum, ż) tabula osis ischii, i) a small notch on the caudal side of the tabula osis ischii,
j) incisura ischiadica major, k) incisura ischiadica minor, l) tuber ischiadicum, m) interpubic
ligamentous tissue.
The Vialleton angle was measured as (8). Average distance between
the midacetabulum and tuber coxae was 39.2 mm, and the average distance
between the midacetabulum and ischial tuberosity (tuber ischiadicum) was
13.8 mm. The average sagittal length and width of the foramen obturatum
were 11.4 mm and 6.6 mm, respectively.
Vet. arhiv 72 (4), 213-220, 2002 215
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
Femur. There were three trochanters on the femur: the greater
trochanter (trochanter major), the lesser trochanter (trochanter minor) and
the third trochanter (trochanter tertius). The trochanteric fossa (fossa
trochanterica) was wide and deep and the trochanteric ridge (crista
intertrochanterica) was present between the lesser and the greater
trochanters. Condylus lateralis, condylus medialis, epicondylus lateralis,
epicondylus medialis, linea and fossa intercondylaris were prominent (Fig.
2).
Fig. 2. The femur, ossa cruris and the patella in hedgehog (Erinaceus europaeus L.)
i- caudal aspect of the femur; ii- cranio-lateral aspect of the tibia and fibula
a) caput ossis femoris, b) trochanter major, c) trochanter minor, d) trochanter tertius, e)
crista intertrochanterica, f) fossa trochanterica, g) condylus lateralis, h) condylus medialis,
ż) epicondylus lateralis, j) epicondylus medialis, k) linea intercondylaris, l) fossa
intercondylaris, m) tibia, n) fibula, o) tuberositas tibiae, p) patella, r) facies cranialis, s)
basis patellae, t) apex patellae
Patella. The cranial surface of the patella was convex; apex patellae
was pointed.
Skeleton cruris. The tibia and fibula were fused almost in the distal
half and there was a wide spatium between the tibia and fibula in the
216
Vet. arhiv 72 (4), 213-220, 2002
Table 1. Specification of myomorphus mammals examined by renoculture and
microscopic agglutination acording to the trapping area with corresponding results
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
proximal half. Tuberositas tibiae was prominent. There was a prominence
near the lateral malleolus and the cochlea tibiae was sagittal (Fig. 2).
Ossa tarsi. There were 7 tarsal bones. The proximal row consisted of
the talus and calcaneus. Os tarsi centrale was in the distal of the talus.
Facies articularis navicularis of the talus was convex and there was a
small pit on the trochlea tali. The distal row bones from medial to lateral
were os tarsale I, os tarsale II, os tarsale III, and os tarsale IV. The
comparative sizes of the distal tarsal bones were: IV>I>III>II.
Ossa metatarsalia I-V. The pedis was complete with five digits and
there were five distinct metatarsal bones lying between the tarsal bones
and phalanges. The comparative lengths of the metatarsal bones were:
IV>III>II>V>I.
There were two plantar located sesamoid bones in pairs at each of the
metatarsophalangeal joints.
Ossa digitorum pedis. There were two phalanges in the first and fifth
digit and the other three digits comprised three phalanges. The distal
Fig. 3. Dorsal aspect of the tarsal and metatarsal bones of hedgehog (Erinaceus europaeus
L.). a) talus, b) calcaneus, c) os tarsi centrale, d) os tarsale I, e) os tarsale II, f) os tarsale III,
g) os tarsale IV, h) os metatarsale I, ż) os metatarsale II, i) os metatarsale III, j) os metatarsale
IV, k) os metatarsale V
217
Vet. arhiv 72 (4), 213-220, 2002
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
phalanges were arched and pointed to accommodate the curved nails. The
comparative lengths of nails were: II>III> IV>V>I.
Discussion
Any sexually dimorphic character can be used to distinguish males
from females, including differences in genitalia, body size, pelage,
ornamentation, behaviour. In practice, males are 20% larger than females
on average (KUNZ et al., 1996). In the present study, certain measurements
were taken in male hedgehogs.
In the Erinaceidae family, symphysis was formed by a cartilage or
interpubic ligament (LESSERTISSEUR and SABAN, 1967) and in the order
Insectivora the symphysis is sometimes non-existent, and always weak, as
in Erinaceus for example, where it is confined to the pubis (SAUNDERS and
MANTON, 1969). In the present study, an interpubic ligamentous form was
observed in hedgehogs.
The presence of a large obturator foramen bounded by the pubis and
ischium is characteristics of mammals (WEICHERT, 1970). In hedgehogs,
this foramen was also large and had an almost hemicycle form. Average
sagittal length and width of the foramen obturatum were 11.4 mm and 6.6
mm, respectively.
ROMER (1970) reported that the fourth trochanter in the femur is absent
in mammals, and SAUNDERS and MANTON (1969) mentioned that the femur
of the Insectivora has a third trochanter which is particularly well developed
in Erinaceus and Centetes. In this study, three trochanter in the femur in
hedgehogs were observed: the greater (trochanter major), the lesser
(trochanter minor) and the third (trochanter tertius).
The fovea capitis on the caput ossis femoris and fossa supracondylaris
are absent in porcupines (YILMAZ et al., 1999). The fovea capitis on the
caput ossis femoris is absent in the African rhinocerus ( Diceros bicornis
L.), too (GOMER
IĆ and HUBER, 1982). Similar findings were observed in
this study.
The fibula is a slender bone and is usually seperated from the tibia but
is, however, fused at the distal end in Erinaceus (SAUNDERS and MANTON,
1969; DEMŻRSOY, 1998). In porcupines the fibula is fused with the tibia at the
Vet. arhiv 72 (4), 213-220, 2002
218
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
europaeus L.). II. ossa membri pelvini
proximal portion (YILMAZ et al., 1999). In our study the slender fibula was
fused only at the distal half of the tibia in hedgehogs.
In some species of the Erinaceidae family the pedis is comprises four
digits (KURU, 1999). In our study, the pedis was complete with five digits.
References
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DEMŻRSOY, A. (1998): Yac
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GŻRGŻN, A., H. KARADA
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GOMER
IĆ, H., 
. HUBER (1982): Articulus coxae u Africkog nosoroga. XIX th. Congress
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ti., Ankara.
LESSERTISSEUR, J., R. SABAN (1967): Generalites sur le Squelette. Traite de Zoologie,
Anatomie, Systematique, Biologie. Publie Sous la Direction de Grasse , P. P. Masson
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219
Z. E. Özkan: Macro-anatomical investigations on the skeletons of hedgehog (Erinaceus
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Fac. Vet. Med. Univ. Ankara 12, 324-330.
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von Hunden der Hyäne. I: Truncus. J. Fac. Vet. Med. Univ. Ankara. 30, 149-166.
VAUGHAN, T. A. (1972): Mammalogy. W. B. Saunders Company, Philadelphia, London,
Toronto.
YILMAZ, S., Z. E. ÖZKAN, D. ÖZDEMÅ»R (1998): Macro-anatomical investigations on
the skeletons of porcupine (Hystrix cristata) I. ossa membri thoracici. Tr. J. Vet. Anim.
Sci. 22, 389-392.
YILMAZ, S., G. DÅ»NÇ, A. AYDIN (1999): Macro-anatomical investigations on the
skeletons of porcupine (Hystrix cristata) II. ossa membri pelvini. Tr. J. Vet. Anim. Sci.
23, 297-300.
WEICHERT, C. K. (1970): Anatomy of the Chordates. Mc Graw-Hill Book Company.
New York
Received: 5 July 2002
Accepted: 29 August 2002
ÖZKAN, Z. E.: Makroanatomska istra~
ivanja kostura je~
a (Erinaceus europaeus
L.). II. ossa membri pelvini. Vet. arhiv 72, 213-220, 2002.
SA%7Å„ETAK
Istra~
ena je anatomska gra
a kostiju stra~
nje noge u tri odrasla je~
a (Erinaceus europaeus
L.). Nedostajala je spina iliaca ventralis caudalis. Symphysis pelvis je bila oblikovana od symphysis
pubis, a ustanovljen je interpubi%0Å„ni ligament. Prosje%0Å„ni Vialleton kut iznosio je 8o. Prosje%0Å„na
udaljenost izme
u sredia
ta acetabuluma i bo%0Å„ne kvrge iznosila je 39,2 mm, a prosje%0Å„na udaljenost
izme
u sredia
ta acetabuluma i sjedne kvrge bila je 13,8 mm. Na bedrenoj kosti su ustanovljena
tri trochantera. Goljenica i lisnja%0Å„a su bile spojene u distalnoj polovici. Utvr
eno je 7 tarzalnih
kostiju, a stopalo je bilo potpuno s pet prstiju.
Klju%0Å„ne rije%0Å„i: je~
, Erinaceus europaeus L., kosti stra~
nje noge
220 Vet. arhiv 72 (4), 213-220, 2002
DEVELOPMENTAL DYNAMICS 237:1923 1934, 2008
PATTERNS & PHENOTYPES
Sonic Hedgehog Is Required for the Assembly
and Remodeling of Branchial Arch Blood
Vessels
Hana Kolesová,1 Henk Roelink,2* and Miloa
Grim1
Sonic hedgehog (Shh) is a morphogen involved in many developmental processes. Injection of cells (5E1)
that produce a Shh-blocking antibody causes an attenuation of the Shh response, and this causes vascular
malformations and impaired remodeling characterized by hemorrhages and protrusions of the anterior
cardinal vein and outflow tract, delayed fusion of the dorsal aortae, impaired branching of the internal
carotid artery, and delayed remodeling of the aortic arches. Distribution of smooth muscle cells in the vessel
wall is unchanged. In 5E1-injected embryos, we also observed impaired assembly of endothelial cells into
vascular tubes, particularly in the sixth branchial arch, around the anterior cardinal vein and around the
dorsal aorta. In 5E1-treated embryos, increased numbers of macrophage-like cells, apoptotic cells, and a
decreased level of proliferation were observed in head mesenchyme. Together, these observations show that
Shh signaling is required at multiple stages for proper vessel formation and remodeling. Developmental
Dynamics 237:1923 1934, 2008. © 2008 Wiley-Liss, Inc.
Key words: Shh; blood vessels; branchial arches; 5E1 hybridoma cells; quail embryo; endothelium
Accepted 8 May 2008
Smo inhibitor cyclopamine show de- early to assess the role for Hedgehog
INTRODUCTION
fects in vascular remodeling (Nagase signaling for pharyngeal vessel devel-
The inductive events controlling the
et al., 2006), indicating an ongoing re- opment (Zhang et al., 2001; Wijgerde
formation and remodeling of the first
quirement for Hh signaling. et al., 2002).
intra-embryonic vessels are not well
Among the first intraembryonic ves- Vessels of the branchial region un-
understood. Classical embryological
sels induced by endodermally derived dergo extensive remodeling in stages
experiments have demonstrated that
signals are the vessels of the branchial 15 to 23. At stage 15, caudal parts of
signals derived from endoderm can in-
(pharyngeal) region. Mouse embryos the paired aortae fuse to form a single
duce vessel formation in adjacent me-
lacking Shh have hypoplastic first descending aorta, while rostrally they
soderm (Pardanaud et al., 1989), and
branchial arches that prematurely become the distal parts of the left and
it appears that the Hedgehog (Hh) sig-
fuse in the midline (Yamagishi et al., right internal carotid arteries. The
naling mediates at least some of this
2006). The second and third branchial ventral aorta after the branching into
endoderm-derived activity since Smo
arches are hypoplastic, while the the aortic arches continues as the left
null embryos, which cannot respond to
Hedgehog (Hh), exhibit severe vascu- fourth and sixth arches do not appear and right external carotid arteries.
lar defects (Byrd et al., 2002). Em- to develop at all (Washington Smoak The first to sixth aortic arches de-
bryos treated at later stages with the et al., 2005). Smo null embryos die too velop in a cranio-caudal gradient (Hi-
1
Institute of Anatomy, First Faculty of Medicine, Charles University, Prague, Prague, Czech Republic
2
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California
Grant sponsor: Charles University; Grant number: GAUK 54/203209; Grant sponsor: NIH; Grant number: 1R01HD042307; Grant sponsor:
The Ministry of Education of The Czech Republic; Grant number: MSM 0021620806.
*Correspondence to: Henk Roelink, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley,
CA 94720-3204. E-mail: [e-mail]
DOI 10.1002/dvdy.21608
Published online 21 June 2008 in Wiley InterScience (www.interscience.wiley.com).
© 2008 Wiley-Liss, Inc.
1924 KOLESOVÁ ET AL.
ruma and Hirakow, 1995) from stages is subsequently covered with a layer of sels associated with the branchial
12 to 23. They form in branchial arch smooth muscle cells. The dorsal aorta arches, and found that decreased lev-
mesenchyme from cords of angioblasts is covered by sclerotome-derived cells els of Shh signaling result in angio-
around the foregut, which subse- (Wiegreffe et al., 2007), the aortic genic malformations. Ongoing devel-
quently become luminized and serve arches by cells derived from the neu- opment of existing vessels is disrupted
as a communication between the ven- ral crest (Le Lievre and Le Douarin, by attenuated Shh signaling. These
tral and dorsal aorta. In fish and am- 1975). Shh can induce angiogenic fac- vessels lose their ability to remodel,
phibian larvae, most branchial arches tors such as VEGFs and Angiopoietins fuse, and form branches. The vessel
develop into the gill arches (Kolesova in mesenchyme (Pola et al., 2001) and walls, in particular of the anterior car-
et al., 2007); in amniotes, aortic arches thus affect the smooth muscle distri- dinal veins, appear to be malformed;
undergo significant remodeling. bution and vessel stabilization (van hemorrhages are common in this area,
The first, second, and fifth aortic Tuyl et al., 2007). The anterior cardi- and these vessels cannot contain ink.
arches undergo regression that starts nal veins have no smooth muscle The area around the anterior cardinal
at stage 21. The third, fourth, and layer. veins has increased levels of apoptotic
sixth aortic arches are gradually rear- The aortic arches are located within cells and macrophage-like cells. More-
ranged. The third aortic arch is incor- the corresponding branchial arches, over, new vessel formation is im-
porated into the common and internal which are formed from neural crest paired, and endoderm cells can often
carotid arteries. In birds, the right cells and cells of paraxial mesoderm been seen lining incomplete vessels, or
fourth arch contributes to the arch of and are lined with an ectoderm on the as aggregates. Altogether, our results
aorta. The remainder of the left one outside, and endoderm on the surfaces demonstrate a varied and continual
incorporates in the subclavian artery. surrounding the developing pharynx. requirement for Shh signaling in the
The sixth aortic arch becomes part of Endothelial cells and striated muscle development of the vessels in and
the pulmonary artery. The main veins cells are derived from mesoderm, around the branchial arches.
of the cranial region are the paired while other tissues in the branchial
anterior cardinal veins, which drain arches are of neural crest origin
RESULTS
blood from the head and neck to the (Evans and Noden, 2006). According
common cardinal vein. The anterior to some observations, mesenchymal
Vessel Formation in
cardinal veins develop at stage 12. cells of branchial arches are stimu-
Branchial Arches
The endothelial cells lining the ves- lated to proliferate and prevented
sels in the branchial region originate from apoptosis by Shh (Ahlgren and The development of vessels in the
from paraxial mesoderm (Noden and Bronner-Fraser, 1999; Jeong et al., branchial region was extensively de-
Trainor, 2005; Evans and Noden, 2004). scribed in chicken embryos (Hiruma
2006). Initially, presumptive vessels We tested the requirement for Shh and Hirakow, 1995), and is very sim-
consist of an endothelial lining, which for the correct development of the ves- ilar in the quail embryo, although the
Fig. 1. A D: Normal development of head and neck vessels in quail embryos. Vessels stained with QH1 Ab. E F: Expression of Shh, visualized with
5E1 Ab. A: A stage-15 embryo with a formed first aortic arch (I), which connects the ventral (VA) with the dorsal aorta (DA). The anterior cardinal vein
is also indicated (CV). B: Stage 18, with the second (II), third (III), and the fourth (not visible on this section) aortic arches indicated. The anterior part
of the dorsal aorta (DA) continues as the internal carotid artery (IC). C: At stage 21, the fourth (IV) aortic arch is indicated. D: Stage 23, with the fifth
(V) and sixth (VI) aortic arches. E,F: Shh expression visualized with anti-Shh antibody in normal embryos. Stage 15 (E), stage 18 (F). Shh expression
is visible in the notochord, floor plate (arrows), and foregut (E, arrowheads) and parts of the branchial arch endoderm (F, arrowheads). Transversal
sections. D F are counterstained with hematoxylin. Scale bar 400 m in all panels.
Fig. 2. Inhibition of the Shh response by 5E1 (anti-Shh) Ab produced by hybridoma cells. A,B: Sections of stage-18 embryos injected at stage 11 with
hybridoma cells. Sections incubated with secondary antibody. A: An embryo injected with control 12CA5 hybridoma cells. No staining is visible in the
embryo. Secondary antibody reacts with hybridoma cells (arrowheads). B: An embryo injected with 5E1 hybridoma cells producing anti-Shh antibody.
Shh-expressing structures, floor plate, notochord, and endoderm of the branchial arches are indicated (arrows). Hybridoma cells are also stained
(arrowheads). Counterstained with hematoxylin. Scale bar 400 m in both panels. C,D: Expression of Ptch1, which is induced in response to Shh
signaling. C: An embryo injected with 12CA5 hybridoma cells shows normal distribution of Ptch1 in the ventral part of the neuroepithelium, in the
endoderm of branchial arches, and in adjacent mesenchyme (arrows). D: An embryo injected with 5E1 hybridoma cells shows a decreased level of
Ptch1 expression. Residual expression is observed only in the ventral part of the nervous system and in a few areas of branchial arch endoderm
(arrows). No expression is detected in the mesenchyme. Scale bar 200 m in both panels.
Fig. 3. Ink injection into blood vessels at stage 18. A: In a 12CA5-injected control embryo, the first and the second aortic arches have regressed and
formed capillary plexuses (white arrows I, II). The main artery conducting blood from the heart to the dorsal aorta is the third aortic arch in this stage
(white arrow III). The fourth aortic arch starts to develop (white arrow IV). Normal capillary plexuses drained into anterior cardinal vein are visible in the
head. B: Embryo injected with 5E1 hybridoma cells has a smaller head compared to the control. Development of the aortic arches is delayed. The first
and the second aortic arches are still present and form functional communication between the heart and the dorsal aorta (white arrows I, II). The third
and the fourth aortic arches are normally developed (white arrows III, IV). A significant amount of ink has leaked from the tributaries to the anterior
cardinal vein (white arrowheads). Scale bar in A and B 500 m. C, D: Paraffin sections of the anterior cardinal vein region, stained for acid
phosphatase. N, neuroepithelium. C: Red blood cells (arrowheads) are visible only in vessels; macrophage-like cells are indicated (arrow). D: Red blood
cells localized extravascularly (arrowheads), macrophage-like cells are more numerous than in control embryos (arrow). Scale bar in C and D 200
m.
SHH AND BRANCHIAL VESSEL DEVELOPMENT 1925
timing of their development is some- stage 15, the first aortic arch is ond arch starts to develop. The third
what different. Generally, in quail em- present as a vessel connecting the aortic arch develops around stage 18
bryos the aortic arches remodel faster; ventral and dorsal aorta, and the an- (Fig. 1B) and the fourth one develops
they form approximately one stage terior cardinal vein is already devel- at stage 20. At stage 21, the fifth and
later, and they regress a few stages oped (Fig. 1A). Immediately after the sixth aortic arches start to develop
earlier than in chick embryos. At first aortic arch has formed, the sec- and are fully formed at stage 23 (Fig.
1C,D). The first and second aortic
arches start to regress at stage 18.
The fifth aortic arch, a minor bypass of
the sixth aortic arch, starts to regress
at stage 24. While the first, second,
and fifth aortic arches are transient
structures, the third, fourth, and sixth
arches persist.
At stage 15, only the aortic arches
Fig. 1. Fig. 2.
Fig. 3.
1926 KOLESOVÁ ET AL.
supply the branchial arches (Fig. 1A), sion is invariably upregulated in re- animals, which were able to contain
while at stage 18, the dorsal aorta, the sponse to Shh signaling (Marigo and the ink within the vessels (Fig. 3A,B).
aortic arches, as well as the anterior Tabin, 1996). In control embryos, In addition to the ink-permeability,
cardinal vein give off smaller Ptch1 is expressed in areas adjacent to the lumen of either anterior cardinal
branches and capillaries (Fig. 1B). At Shh sources, such as branchial arch vein in 5E1-injected embryos is sinu-
stages 21 and 23, the development of mesenchyme, around the foregut, in soidal with endothelium-lined protru-
small vessels and capillaries contin- the ventral part of the neural tube, sions (Fig. 4A D). This demonstrates
ues in the whole region, resulting in a and around domains of Shh produc- that Shh plays an important role in
dense capillary network in branchial tion in the brain (Fig. 2C). In embryos the establishment of a functional wall
arch mesenchyme and in the regions injected with 5E1 cells, Ptch1 expres- in the anterior cardinal veins and its
surrounding the brain and eyes (Fig. sion in the neural tube and endoderm tributaries.
1C,D). is decreased and no Ptch1 expression Several arteries show abnormal de-
Besides luminized vessels and cap- is found in the abutting mesenchyme velopment as a consequence of 5E1
illaries, we also observed an increas- (Fig. 2D), demonstrating a significant injection as well. The internal carotid
ing number of isolated angiogenic attenuation of the Shh response. Re- arteries are characterized by the pres-
cells at stages 18  23 (Fig. 1B D). sidual expression of Ptch1 could be the ence of a transverse septum over a
These cells are mainly present around result of incomplete inhibition, but length of up to 40 m(in 2 of 6 em-
the anterior cardinal vein and in also be caused by other Hh ligands, bryos). This septum consists of two
branchial arches, while in the area in which are not recognized by 5E1 layers of endothelium, with mesen-
the vicinity of the dorsal aorta, only a (Goodrich et al., 1997; Carpenter et chyme in between, dividing the inter-
few of these cells are detected. These al., 1998).
nal carotid artery into two separate
isolated angiogenic cells are evenly Embryos injected with 5E1 and
vessels, which merge again further
distributed in the mesenchyme. with control 12CA5 hybridoma cells
rostrally (Fig. 4E G). Similarly, an
develop slightly slower than unin- abnormal septum is present in the
jected embryos, and staging was per- dorsal aorta (in 4 out of 6 embryos).
Inhibition of Shh Signaling
formed based on anatomical land- This aortic septum is usually about 60
With Anti-Shh Antibodies
marks. The 5E1 antibody-injected
m long, and is covered with endothe-
Produced by 5E1 Hybridoma
embryos largely exhibit a normal
lium on both sides (Fig. 4H J). Al-
Cells
gross morphology, although at least
though we assume that this septation
In general, we detected Shh in a pat- half of them have a smaller head com- is a result of delayed fusion, it re-
pared to control (Fig. 3B). Similar ce- mains possible that it has formed af-
tern and timing consistent with that
phalic phenotypes have been reported
observed in the chick (Roelink et al., ter the initial fusion of the left and
(Ahlgren and Bronner-Fraser, 1999).
1995). The notochord is a prominent right dorsal aortae. The same domain
Embryos injected with control 12CA5
site of early expression, and starting of the dorsal aorta in control embryos
hybridoma cells have a macroscopic
at stage 15, Shh is expressed in is already fused and has a single lu-
and microscopic anatomy identical to
endoderm lining the branchial arches men. Vessel abnormalities are also
found in outflow tracts of the heart.
and the foregut (Fig. 1E,F). To condi- untreated embryos.
tionally attenuate Shh signaling, we Besides vessels with irregular lumina
injected 5E1 (anti-Shh) hybridoma and unusual invaginations (Fig. 4N
Vessel Malformations in
cells under the vitelline membrane of P), we have also observed curving
Anti-Shh Antibody-Treated
stage-10  12 embryos, which were an- strands of endothelial cells in succes-
Embryos
alyzed at stages 18  23. 5E1 hybrid- sive sections, possibly malformed ves-
oma cell-derived anti-Shh antibodies Even at the anatomical level, the ef- sels with an incomplete vessel wall
distribute widely in injected embryos. fect of inhibiting Shh on vessel devel- (Fig. 4K M) (in 4 of 6 embryos). Em-
Visualizing the 5E1 antibodies by sim- opment is remarkable. Generally, in bryos injected with the control hybrid-
ply using an anti-IgG secondary anti- embryos injected with 5E1 hybridoma oma cells had no obvious vessel mal-
body on sectioned embryos, staining is cells development of the aortic arches formations.
detected at the sites of Shh expres- is delayed. While in stage-18 control To further assess malformations in
sion, such as the notochord, floor embryos, the first and the second aor- vessel endothelium, we determined
plate, and endoderm of the branchial tic arches start to regress into capil- the expression VEGFR2, which is ex-
arches and foregut. The secondary an- lary plexi, these aortic arches are still pressed in endothelial cells (Jaffredo
tibodies also react with 5E1 hybrid- present in 5E1-injected embryos, indi- et al., 1998) albeit not ubiquitously. At
oma cells, which always remain at the cating a delay in remodeling (Fig. stage 18, VEGFR2 is expressed in all
embryonic surface (Fig. 2A,B), just as 3A,B). endothelial cells of small developing
the control 12CA5 hybridoma cells A consequence of Shh inhibition is vessels as capillaries, such as brain
(Fig. 2C,D). the failure of the anterior cardinal capillaries, while only about half the
To verify if the Shh response is effi- vein and its branches to form func- endothelial cells of bigger arteries ex-
ciently blocked after 5E1 injection, we tional vessel walls. Hemorrhages were press VEGFR2. Similarly, the dorsal
analyzed the expression of the gene observed frequently (Fig. 3A D), and aorta is lined by VEGFR-positive
coding for its receptor Ptch1 by mRNA these vessels were permeable to ink, cells, but only in its ventrolateral as-
in situ hybridization. Ptch1 expres- unlike the control hybridoma-injected pect, where the aortic arches are con-
SHH AND BRANCHIAL VESSEL DEVELOPMENT 1927
nected. Besides endothelial cells,
VEGFR2 is highly expressed in the
outflow tract myocardium, while out-
flow tract endothelium contains only
few VEGFR2-positive cells. These
myocardial cells are probably derived
from endothelium (Wilting et al.,
1997). VEGFR2 was also expressed in
the notochord, as it was previously re-
ported (Nimmagadda et al., 2004). We
did not detect any difference in
VEGFR2 expression in 5E1-injected
embryos compared to control, indicat-
ing that VEGF is not a critical medi-
ator of the effects of Shh (not shown).
Following the formation of an en-
dothelial layer, the smooth muscle
cells start to surround the forming
vessels. At stage 18, smooth muscle
cells completely cover the endothe-
lial lining of the dorsal aorta and the
internal carotid arteries (Fig. 4A).
The aortic arches have smooth mus-
cle cells only on their lateral side,
while an incomplete layer of smooth
muscle cells is associated with the
outflow tract. Smooth muscle actin is
also present in the myotome (Fig.
Fig. 4. QH1 staining of vessel endothelium
showing lumen malformations in anti-Shh hy-
bridoma cell injected embryos. A D: Lumen
malformation in the anterior cardinal vein. A:
Control embryo. B: The anterior cardinal vein
exhibits irregularities and contains protrusions
of the endothelial layer extending into the ves-
sel lumen (arrow) at stage 18. C, D: Protrusions
in the anterior cardinal vein (arrow) are also
found in embryos injected with anti-Shh anti-
body and harvested at stage 23. E G: Internal
carotid artery malformations at stage 18. E:
Control embryo. F: The internal carotid artery is
divided by a horizontal septum (arrow) into two
separate vessels. G: Detail of septum covered
in endothelium from both sides (arrow). H J:
Dorsal aorta malformations, stage 18. H: Con-
trol embryo. I: Failure of complete fusion be-
tween the left and right dorsal aortae. The per-
sistent septum is indicated (arrow). J: Detail of
septum (arrow). K M: Malformations of outflow
tract endothelium at stage 18. K: Control em-
bryo. L: The endothelium of the outflow tract
fails to form a luminized vessel (arrow), and
instead forms an isolated endothelial wall with
red blood cells on one side. M: Detail of patent
vessel endothelium (arrow) and adjacent mal-
formed vessel (arrow). N P: Malformations of
the outflow tract at stage 18. N: Control em-
bryo. O: The outflow tract has developed a
protrusion of tissue surrounded by the endothe-
lial layer, which invaginates into the lumen (ar-
row). P: Detailed view of the endothelial protru-
sion (arrow). Transversal sections. B D, K M
are counterstained with hematoxylin. Scale
Fig. 4. bar 400 m in all panels.
1928 KOLESOVÁ ET AL.
5C). At stage 23, a continuous
smooth muscle layer surrounds aor-
tic arches and the outflow tracts
(Fig. 5E). Also, all branches of the
internal carotid arteries have a con-
tinuous layer of smooth muscle cells,
while the anterior cardinal veins and
their tributaries are devoid of
smooth muscle cells. The formation
of the smooth muscle cell lining of
the vessels is unaffected by injection
of 5E1 hybridoma cells (Fig. 5B,F),
despite the presence of obvious ves-
sel malformations. Smooth muscle
cells also line the abnormal aortic
septum (Fig. 5D). This is consistent
with our observation that the forma-
tion of the smooth muscle lining of
the dorsal aortae precedes the sub-
sequent fusion of these vessels. Alto-
gether, this indicates that the loss of
Shh signaling has little effect on the
process in which smooth muscle cells
form around new vasculature.
Angiogenic and Macrophage-
Like Cells in Anti-Shh
Antibody-Treated Embryos
The main effect of conditional Shh
inhibition on blood vessel develop-
ment in embryos injected at later
stages (injected in stage 13 15 and
harvested at stage 21 23) is the
presence of an increased number of
free, round endothelial cells, positive
for QH1. These cells are not inte-
grated into functional vessel lumina,
but aggregate into multicellular
clusters. Such aggregates usually
are found around the anterior cardi-
nal veins, around the dorsal aorta,
and in the branchial arches around
the aortic arches. Such cell aggre-
gates are not present in control
12CA5 hybridoma-injected embryos,
where angiogenic cells are fewer and
isolated. The increased number of
aggregates is most significant in the
sixth branchial arch (Fig. 6G L),
around the anterior cardinal vein,
Fig. 5. Distribution of vascular smooth muscle cells visualized with smooth muscle actin Ab. A,B:
and just ventral to the dorsal aorta
Smooth muscle cells in a stage-18 embryo are present around the dorsal aorta, internal carotid
(Fig. 7A L), while the anterior
artery, and the lateral part of the aortic arches (arrows). 5E1-injected embryos show no difference
branchial arches are the least af- in the distribution of smooth muscle cells (B) compared to control injected embryos (A). Scale bar
400 m. C,D: Stage-18 embryos. Smooth muscle cells also cover the endothelial malformation
fected. In the first and second
caused by 5E1 injection. C: Normal dorsal aorta. D: Persistent septum in the dorsal aorta (arrow).
branchial arch, we have not observed
Arrowheads show expression of the smooth muscle actin in the myotome. Scale bar 400 m.
any significant difference compared
E,F: Smooth muscle cells in a stage-23 embryo are present in the dorsal aorta, internal carotid
to control in the number of endothe- artery, aortic arches, and the outflow tracts (arrows). 5E1-injected embryos show no difference in
the distribution of the smooth muscle cells (F) compared to control-injected embryos (E). Scale
lial cell aggregates (data not shown).
bar 200 m.
This might either indicate that at
the moment of injection the Shh re-
SHH AND BRANCHIAL VESSEL DEVELOPMENT 1929
Fig. 6. QH1-positive cell abnormalities in branchial arches at stage 21 (A D, F) and stage 23 (E, G U); Characterization of QH1-positive cells:
macrophage-like cells stained for acid phosphatase (M O); apoptotic cells positive for cleaved caspase-3 (P R); proliferating cells positive for phospho
histone H3 (S U). A F: QH1-positive cells in the mesenchyme and ectoderm of third and fourth branchial arches at stage 21 (stage 23-E). A,B: Control
embryos. C,D: Larger aggregates of QH1-positive cells (arrows) in anti-Shh-injected embryos at stage 21 or (E) stage 23. F: Graph shows that
anti-Shh-treated embryos have a similar number of QH1-positive cells as the controls. However, QH1-positive cells are present in large aggregates
compared to single cells in control embryos. Y-axis: average number of QH1-positive cell aggregates in the third and fourth branchial arches. G L:
Increased number of QH1-positive cells in the sixth branchial arch of stage-23 embryos injected with anti-Shh hybridoma cells. G,H: Control embryos.
I: QH1-positive cells (arrows) in the distal region of the sixth branchial arch and (J) detail. K: Increased number of QH1-positive cells (arrows) in the
proximal region of the branchial arch. L: Graph shows the increased number of QH1-positive cells in anti-Shh-injected embryos in the sixth branchial
arch. Y-axis: average number of QH1-positive cell aggregates in the sixth branchial arch. M O: Very few macrophage-like cells are present in the
branchial arches. Anti-Shh injection has no significant effect on the number of macrophage-like cells (O). P R: No significant difference in the number
of apoptotic cells (arrow) in response to anti-Shh injection (R). S U: No significant difference in the number of proliferating cells in the branchial arches
in anti-Shh injected embryos (arrows). Transversal sections. C E, K, P, Q counterstained with hematoxylin. Scale bar 200 m in all panels. Graphs:
Standard deviation is indicated. *Significant difference (P 0.05); ns, no significant difference.
1930 KOLESOVÁ ET AL.
Fig. 7. Abnormalities in the number of QH1-positive cells around the main vessel trunks at stage 23: the anterior cardinal vein and the dorsal aorta
(A L); Characterization of QH1-positive cells: macrophage-like cells stained for acid phosphatase (M O); apoptotic cells positive for cleaved caspase-3
(P R); proliferating cells positive for phospho histone H3 (S U). A F: Anterior cardinal vein malformations. A, B: Control embryos. C: Increased number
of QH1-positive cells around the anterior cardinal vein (arrows) in anti-Shh hybridoma cell injected embryos. D: Increased number of QH1-positive
cells around a branch of the anterior cardinal vein (arrows). E: Free QH1-positive cells are present around the anterior cardinal vein already at stage
18 (arrows). F: Graph showing a higher number of QH1-positive cells in the anterior cardinal vein region in embryos injected with anti-Shh antibody.
Y-axis: average number of QH1-positive cells per section. Standard deviation is indicated. G L: Increased number of QH1-positive cells around the
dorsal aorta at stage 23. G,H: Control embryos. I: Aggregate of QH1-positive cells on the ventral side of the dorsal aorta (arrow) and (J) detail of the
aggregate (arrow). K: QH1-positive cells are present (arrow), but hematopoietic regions in dorsal aorta appear normal (arrowhead). L: Graph shows
the increased number of QH1-positive cells in anti-Shh-injected embryos in the dorsal aorta region. Y-axis: average number of QH1-positive cells per
field. Standard deviation is indicated. M O: Increased number of macrophage-like cells stained with acid phosphatase (arrows) in the region of the
anterior cardinal vein in anti-Shh-injected embryos. O: Graph indicating the increased number of macrophage-like cells. P R: Increased number of
cleaved Caspase-3 (apoptotic) cells (arrows) around the anterior cardinal vein in anti-Shh-injected embryos. R: Graph showing the increased number
of apoptotic cells. S U: Decreased number of dividing cells (arrows) in the anterior cardinal vein region in anti-Shh-injected embryos. U: Graph showing
the difference in proliferating cells. Transverse sections. C, E, P, Q, counterstained with hematoxylin; M, N, counterstained with Fast green. Scale bar
200 m in all panels. Graphs: Y-axis: average number of positive cells per field. Standard deviation is indicated; *significant difference (P 0.05).
SHH AND BRANCHIAL VESSEL DEVELOPMENT 1931
multaneously study the effects of Shh
quirement for initial vessel forma- Apoptosis and Proliferation
tion has passed, or that the absence inhibition in the further developed an-
in Anti-Shh Ab-Treated
terior branchial arches (I III) as well
of de-novo vascularization associ-
Embryos
as the newly formed posterior arches
ated with the regression of the first
In many instances during develop- (IV VI). In the posterior arches, we
and second aortic arches results in
ment, loss of Shh leads to decreased
observed a failure of vessel luminiza-
an absence of these aggregates of
proliferation and increased apoptosis,
tion, represented by formation of nu-
QH1-positive cells.
possibly explaining some of the vessel
merous aggregates of endothelial
In the third and fourth branchial
malformations we observed as a con- cells. This shows Shh requirement in
arches, Shh inhibition results in an
sequence of 5E1 injection.
the early stages of vessel develop-
increased number of larger endothe-
A significant increase in the number
ment. A similar effect that blocked
lial aggregates compared to 12CA5-
of apoptotic, caspase-3-positive cells
Shh signaling in the early stages of
injected control embryos. These aggre-
(Fig. 7P R) is observed around the an-
vasculogenesis was observed before
gates are mainly localized in the
terior cardinal veins in 5E1-injected
mice and chick embryos (Vokes and
mesenchyme, sometimes in close prox-
embryos, suggesting a role for Shh in
Krieg, 2002; Vokes et al., 2004).
imity to branchial arch ectoderm and
cell survival in this region and, conse-
Already formed vessels are affected
endoderm. Such aggregates are not
quently, an increased number of mac-
by Shh inhibition in their remodeling.
present in control embryos, where we
rophage-like cells is detected. In con-
This effect is characterized by delayed
observed only solitary QH1-positive
trast, in the branchial arches and
fusion, impaired branching, and un-
cells, in comparable numbers to the
around the dorsal aorta, the frequency
usual invaginations of the vessel
solitary cells in anti-Shh-treated em-
of cleaved caspase-3-positive apoptotic
walls. Such malformations are ob-
bryos (Fig. 6A F).
cells do not differ from control em-
served in the dorsal and ventral aorta,
The sixth branchial arch is the most
bryos (Fig. 6P R). Since the domains
the internal carotid arteries, and an-
affected as measured by the number of
of apoptosis coincide with the areas
terior cardinal veins, which are ac-
QH1-positive cells not part of an obvi- where vessel integrity is compro-
tively remodeled at that time. These
ous vessel wall. These cells are mised, it remains a possibility that the
disturbances of vessel remodeling are
present along the whole extent of the hemorrhages increase apoptosis in the
not known to be affected by Shh sig-
arches and are concentrated in mes- surrounding tissues (Figs. 6,7S U).
naling, although it has been observed
Coincident with the increased apo-
enchyme as aggregates and solitary
that cyclopamine treatment affected
ptosis, we observed decreased prolifer-
cells. In control embryos, only solitary
fusion of the dorsal aortae (Nagase et
ation near the anterior cardinal vein
QH1-positive cells are observed. The al., 2006). Alterations of vessel remod-
(Figs. 5, 6S U), but not in the other
total number of nonintegrated QH1- eling are found in arteries that have
regions studied.
already formed smooth muscle layer.
positive cells is significantly higher in
However, the wall of malformed ante-
5E1 hybridoma-injected embryos than
rior cardinal veins does not contain
in controls (Fig. 6G L).
DISCUSSION
smooth muscle cells. This suggests
The biggest increase of noninte-
The widespread expression of Shh in that the presence or absence of a
grated endothelial cells as a conse-
endoderm, floor plate, and notochord, smooth muscle layer does not play a
quence of 5E1 injection is detected
as well as the proximity of the dorsal critical role in the generation of vessel
ventral to the dorsal aorta (Fig. 7G
aortae, anterior cardinal veins, and malformations. These results show
L). It is possible that this abundance
aortic arches to these Shh-producing that Shh is essential to maintain the
of angiogenic cells is related to the
structures, prompted us to assess the stability and coherence of the endo-
area of hemangiogenesis within the
role of Shh signaling in pharyngeal thelial layer of the veins, as we de-
wall of the ventral aorta (Jaffredo et
vessel development. tected in the anterior cardinal vein. A
al., 1998). Increased numbers of endo-
Previous examinations of Shh null similar phenotype is observed in ze-
thelial cell aggregates were also found
embryos (Jeong et al., 2004; Yama- brafish with disrupted Shh signaling
in the vicinity of the anterior cardinal
gishi et al., 2006) have demonstrated (S.J. Childs personal communication).
veins. The QH1-positive cells in this
a Shh requirement for normal In our study, we expected to find
area (Fig. 7A F) are the only cells that
branchial arch formation. However, increased level of apoptosis and de-
are predominantly identified as mac-
global defects in these animals pre- creased proliferation as a consequence
rophage-like cells, rather than angio-
vent assessment of the involvement of of Shh signal attenuation, as it was
genic cells (Fig. 6M O). Macrophage-
Shh in later stages of branchial arch reported by previous observation at
like cells are the phagocytic cells of
development (Washington Smoak et earlier stages of avian development
the early embryo, are derived from he-
al., 2005). Using a method of inhibit- (Ahlgren and Bronner-Fraser, 1999)
mangioblasts (Cuadros et al., 1992),
ing Shh response at later developmen- and in mice (Jeong et al., 2004; Wash-
and are characterized by their expres-
tal stages, we demonstrate a continual ington Smoak et al., 2005). However,
sion of acid phosphatase (Fig. 7M O).
requirement for Shh for the correct our results do not generally show this
Direct co-localization of acid phospha-
formation of the pharyngeal vascula- effect. We observed increased level of
tase and QH1 staining is not possible
ture. Taking advantage of the ante- apoptosis and decreased proliferation
due to incompatible fixation and pro- rior-posterior developmental gradient only around the anterior cardinal
cessing requirements. of the branchial arches, we could si- veins at stage 23. At stage 18, there
1932 KOLESOVÁ ET AL.
was no significant difference in num- aries induced in the mesenchyme be- vidin peroxidase was used rather than
bers of proliferating and apoptotic cell tween the Shh sources and the ves- Extravidin peroxidase as it decreased
between control and Shh-inhibited sels. Nevertheless, the molecular background levels significantly. As a
embryos (data not shown). It seems, events following the initial Shh re- secondary antibody, Rabbit anti
thus, that Shh signaling influences sponse, which instructs endothelial mouse IgG1-Biotin was used.
cell proliferation and survival more cells to form blood vessels, remains to Apoptosis was detected with Anti-
profoundly at earlier developmental be solved. Cleaved Caspase-3 monoclonal anti-
stages. body (BD Pharmingen 559565) at
Interestingly, sources of Shh are not 1:500, in paraffin sections and cryo-
EXPERIMENTAL
necessarily in the direct vicinity of the sections. The secondary antibody was
PROCEDURES
vessels affected by the application of Goat anti-Rabbit Biotin (1:500) and
5E1 anti-Shh hybridoma cells. There tertiary antibody was Extravidin Px
Embryos
are several sites of Shh expression in (Sigma 2886) (1:100), and visualized
Fertilized quail (Coturnix coturnix ja-
the vicinity of the anterior cardinal using DAB.
ponica) eggs were obtained from the
veins, including the floorplate of the Proliferating cells were detected
Research Institute of Animal Produc-
hindbrain and midbrain, the domains with anti-Phospho Histone H3 poly-
tion, Prague, Czech Republic, and
of Shh expression in the forebrain, as clonal antibody (Upstate 06570) 1:200
from B&D Farm (Harrah, OK). For in
well as the notochord and the pre- in cryosections. As a secondary anti-
situ hybridization of Ptch1 mRNA, we
body we used Goat-anti Rabbit Rhoda-
chordal plate. Shh derived from the
used chick embryos. Eggs were incu-
mine (Cappel Pharmaceutical) (1:500).
notochord and floor plate influences
bated at 38°C and embryos ranging
Periendothelial smooth muscle cells
dorsal aortic development (Nagase et
from stage 6 to 23, as defined by Ham-
were detected using anti-smooth mus-
al., 2006), and it remains unsolved
burger and Hamilton (1951) (23 100
cle actin Ab (Sigma A2547) 1:500 and
how Shh reaches these distant sites.
hr of incubation), were studied. We
Goat anti Mouse TRITC as a second-
Intermediary factors such Angiopoi-
injected over 150 embryos with 5E1
ary Ab (1:150). Phagocytic function of
etin-1, VEGFs (Pola et al., 2001),
hybridoma cells. As a control, we used
macrophage-like cells was detected
Fox1, and BMP4 (Jeong et al., 2004;
approximately 100 embryos injected
with histochemical staining for Acid
Astorga and Carlsson, 2007) could me-
with 12cA5 hybridoma cells and as an
Phosphatase resistant to Tartaric
diate the effects of Shh. However, di-
additional control we used uninjected
Acid. Embryos were fixed in acetone
rect signaling of Shh to the endothe-
embryos.
overnight at 4°C, transferred into xy-
lial is likely to occur, consistent with
lene, and subsequently embedded in
the relatively large area over which
paraffin. Sections were deparaffinized
Ptch1 expression is decreased after
Immunohistochemistry and
with xylene and rehydrated through
Shh inhibition. Also, the Shh indepen-
Histochemistry
acetone and acetone/distiled water (1:
dence of VEGFR2 expression indi-
1). Sections were incubated overnight
cates that VEGF does not play a crit- Shh was detected in cryostat sections
at room temperature in solution pre-
ical intermediary role between Shh derived from embryos fixed in 4%
and its effect on the endothelial cells, Phosphate Buffered Paraformalde- pared with 10 mg of Naphtol-AS-BI
phosphate (Sigma, 70491-Fluka) in
altogether supporting a model in hyde using monoclonal antibody 5E1
0.5 ml N,N-dimethyl formamide. This
which Shh signals the vessel endothe- (Hybridoma Bank) diluted at 1:50. As
lium directly. Furthermore, in experi- a secondary antibody, Goat anti- solution was resuspended in 50 ml of
0.1M acetic acid buffer, pH 5.2 (Wal-
ments with mutant mice, in which Mouse Biotin (Sigma B7264) (1:500)
pore acetate), and 20 mg of Fast Red
neural crest derived mesenchyme in was used and tertiary antibody was
Violet (Sigma, F3381) was added, as
the branchial arches was rendered in- Extravidin Px (Sigma 2886) (1:100)
sensitive to Hh signaling, initial vas- and DAB (Sigma D5905) as a chromo- well as a minimum of 140 mg of Tar-
taric Acid (Sigma Aldrich, 14314DE).
cularization appeared normal (Sasai gen.
et al., 2001; Jeong et al., 2004), show- The endothelium of vessels was vi-
ing that a possible intermediary sig- sualized with monoclonal antibody
mRNA In Situ Hybridization
nal is not generated in crest-derived QH1 (Hybridoma Bank) in paraffin
A plasmid containing chick Ptch1
mesenchyme. However, the possible sections (1:1,000). As a secondary an-
(clone 200; a gift from M. Scott) (Xie et
role of non-crest mesenchymal cells tibody, we used Goat anti-Mouse Px
al., 1997) was linearized with SalI and
remains to be determined. IgG (Sigma A 4416) and the reaction
transcribed using T3 polymerase. Hy-
In conclusion, our results demon- product was detected with DAB.
bridization in situ on paraffin sections
strate a continual requirement for Alternatively, endothelium was visu-
was carried out as described (Nieto et
Shh signaling for vascular develop- alized with VEGFR2 monoclonal anti-
al., 1996; Nanka et al., 2006).
ment and remodeling. We have ob- body (kindly provided by Dr. Eich-
served minor differences at best in mann; Eichmann et al., 1997) diluted
apoptosis and proliferation, and no 1:1 in the cryostat sections. For en-
Vascular System Ink
difference in smooth muscle actin and hancing the signal Tyramide Signal
Injection
VEGFR2 expression, supporting the Amplification system (TSA, Dako) was
idea that Shh acts directly on vessel used according to the manufacturer s The vascular system was visualized in
endothelium and not via intermedi- recommendation except that Strepta- vivo by injecting black ink diluted 1:20
SHH AND BRANCHIAL VESSEL DEVELOPMENT 1933
in PBS via the vitelline vein using a starting caudally in the heart and con- Goodrich LV, Milenkovic L, Higgins KM,
Scott MP. 1997. Altered neural cell fates
glass capillary. The beating heart dis- tinuing cranially to the branchial
and medulloblastoma in mouse patched
tributed the ink completely through- arches.
mutants. Science 277:1109 1113.
out the vascular system. After injec- Cells were counted in every fourth
Hamburger V, Hamilton HL. 1951. A se-
tion, embryos were collected, fixed in section, at a minimum of 20 sections
ries of normal stages in the development
of the chick embryo. J. Morphol. 88:49
4% PFA, and analyzed. per region. Six embryos were analyzed
90.
for the number of QH1-positive cells,
Handley-Gearhart PM, Stephen AG,
four embryos for macrophage-like
Inhibition of Shh Function Trausch-Azar JS, Ciechanover A,
cells and for apoptotic cells and two
Schwartz AL. 1994. Human ubiquitin-
With Anti-Shh (5E1)
embryos for the number of proliferat- activating enzyme, E1. Indication of po-
Antibody
tential nuclear and cytoplasmic subpopu-
ing cells. Counts were averaged and
lations using epitope-tagged cDNA
Just before the injection, around 108 the standard deviation was deter-
constructs. J Biol Chem 269:33171 33178.
mined.
hybridoma cells were collected by cen-
Hiruma T, Hirakow R. 1995. Formation of
the pharyngeal arch arteries in the chick
trifugation and resuspended in about
embryo. Observations of corrosion casts
200 l of Liebovitz L-15 medium
by scanning electron microscopy. Anat
ACKNOWLEDGMENTS
(Sigma). This suspension was loaded
Embryol (Berl) 191:415 423.
We thank Ms. E. Kluzáková, M. Plesch-
into a small capillary and injected un-
Jaffredo T, Gautier R, Eichmann A, Diet-
nerová, A. Kvasilová, and Mr. M. Tsma
der the vitelline membrane near the erlen-Lievre F. 1998. Intraaortic hemo-
for excellent technical assistance. This
poietic cells are derived from endothelial
branchial region of quail and chick
cells during ontogeny. Development 125:
work was supported by GAUK 54/
embryos. Injecting the cells to the
4575 4583.
203209 (Grant Agency of Charles
paraxial mesoderm caused the same
Jeong J, Mao J, Tenzen T, Kottmann AH,
University) to Hana Kolesová, by
level of Shh inhibition as with injec-
McMahon AP. 2004. Hedgehog signaling
MSM 0021620806 (project from The
tion in the proximity of branchial in the neural crest cells regulates the
Ministry of Education of The Czech patterning and growth of facial primor-
arches, which is less invasive. After
dia. Genes Dev 18:937 951.
Republic) to Miloa
Grim, and by NIH
injection, embryos were incubated for
Kolesova H, Lametschwandtner A, Rocek
grant 1R01HD042307 to Henk Ro-
1 to 3 days and isolated for analysis.
Z. 2007. The evolution of amphibian
elink.
Embryos were either injected at
metamorphosis: insights based on the
transformation of the aortic arches of Pe-
stages 10 12 and re-incubated either
lobates fuscus (Anura). J Anat 210:379
until they reached stage 18 or 21 23,
393.
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DEVELOPMENTAL BIOLOGY 180, 35 40 (1996)
ARTICLE NO. 0282
Surgical Removal of Limb Bud Sonic hedgehog
Results in Posterior Skeletal Defects
Sylvia M. Pagan,*,1 Maria A. Ros, ,1 Cliff Tabin,* and John F. Fallon! ,2
*Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
Departamento de Anatomia y Biologia Celular, Universidad de Cantabria, 39011 Santander,
Spain; and ! Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706
Using Sonic Hedgehog (Shh) as a marker for polarizing region cells we have repeated the experiments of MacCabe et al.
(1973) and Fallon and Crosby (1975) in an attempt to reexamine the question of a continuous role for the polarizing region
during limb development. We report that the earlier experiments probably left Shh-expressing cells after surgery. Our
results show that Shh-expressing cells do not regenerate and complete removal of the polarizing region results in truncations
along the anteroposterior (A P) axis; further, A P patterning cannot be restored when a bead soaked in FGF is implanted
in the limb bud mesenchyme to maintain outgrowth after extirpation of the polarizing region. However, in order to
reproducibly remove all Shh-positive cells, it is possible that cells with posterior limb skeletal fate also must be removed.
Therefore, microsurgical approaches do not permit an unequivocal answer to the question raised in this and the earlier
papers and it remains a reasonable possibility that at least up to stage 20 21 the polarizing region plays a continuous role
in patterning of the limb bud during its development. 1996 Academic Press, Inc.
INTRODUCTION of the limb. Arguing that these data could also reflect the
regeneration of polarizing activity after the surgeries, Fallon
and Crosby (1975) took posterior tissue from wing buds 24
The polarizing region (zone of polarizing activity or ZPA)
and 48 hr after polarizing region removal and assayed it for
was operationally defined more than 25 years ago as a re-
polarizing activity in a host limb bud. Negative results for
stricted area of posterior limb mesoderm that caused mirror
the presence of polarizing activity in the operated buds al-
image duplications of digit patterns along the anteroposter-
lowed them to conclude that the polarizing region was not
ior (A P) axis when grafted into the anterior margin of a
regenerated following removal. They also suggested that,
host wing bud (Saunders and Gasseling, 1968; Tickle et al.,
given the fact that normal wing development still occurred
1975). It was assumed that the polarizing region played an
in about 30% of the cases, this indicated that   if the polariz-
important role in the anteroposterior patterning of the de-
ing zone had any role during limb development, it must be
veloping limb. One way to test this hypothesis was to surgi-
at an early stage, as during limb induction, and any informa-
cally remove it, expecting either truncations along the A
tion from the zone is further acted upon throughout the
P axis or uniform skeletal elements along the A P axis if
morphogenetic field.  Nevertheless, the presence of the po-
the polarizing region had an essential role in A P specifica-
larizing region until nearly the end of limb development
tion during limb development. Results published by Mac-
has been taken as a strong suggestion of it playing an active
Cabe et al. (1973) argued against this conclusion: when the
role in patterning as the limb bud elongates (Tickle et al.,
polarizing region was removed from early limb buds, normal
1975). However, there is no conclusive evidence arguing
wings developed in about half of the cases, suggesting that
against the hypothesis that it exclusively acts early.
it was not involved in normal anteroposterior patterning
Three possibilities could explain why MacCabe et al.
(1973) and Fallon and Crosby (1975) obtained a high percent-
1 age of normal wings after polarizing region removals. First,
The first two authors contributed equally to this work.
2 all of the polarizing region was removed after the surgeries
To whom correspondence should be addressed at Department
and it is indeed dispensable for A P patterning after limb
of Anatomy, University of Wisconsin, 1300 University Avenue,
induction stages, either because it has already fixed the posi-
Madison WI 53706. Fax: [phone]. E-mail: jffallon@facstaff.
wisc.edu. tional identity of the future wing elements or because it
0012-1606/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved. 35
36 Pagan et al.
has already set the cascade of events that ultimately result tissue in each successive experiment until we obtained no
in A P patterning. Second, all of the polarizing region was Shh signal in the limbs as tested by in situ hybridizations.
removed but it eventually regenerates, resulting in restora- We scored the amount of remaining Shh in the limbs and
tion of anteroposterior pattern. This alternative would im- compared these results with the wing patterns of Day 11
ply that the negative results for polarizing activity obtained embryos from the same batch.
by Fallon and Crosby (1975) were due to assays that were When we performed more conservative surgeries, we ob-
not sensitive enough. Third, perhaps not all of the polarizing tained results strikingly similar to those reported by Mac-
region was removed with the surgeries and the remaining Cabe et al. (1973) and Fallon and Crosby (1975): at Day 11
amounts were sufficient to maintain normal A P pat- normal wings were obtained in about 30% of the cases, with
terning of the developing bud, implying again that this re- the rest exhibiting various postaxial defects, one of the most
sidual activity must have been missed by the less sensitive common being the presence of only a humerus, a radius,
polarizing activity assays. Discerning among these three and digits 2 and 3, as was also the case for Fallon and Crosby
possibilities requires a molecular marker for the polarizing (Table 1A, Fig. 1B). However, in situ hybridization of the
region cells, which did not exist at the time that the earlier stage 20 21 limbs revealed that only a small percentage
studies were carried out. had no Shh left, and a large fraction exhibited a considerable
Several lines of evidence suggest that Sonic Hedgehog degree of remaining Shh signal in the operated limb (Table
(Shh), a vertebrate homologue of the Drosophila hedgehog 1A, Fig. 1A). When we modified our surgeries such that
gene, is a crucial component of the polarizing region signal- 64% of the limbs had no Shh left and the remnant had very
ing pathway. Transcripts of Shh strongly localize to the small amounts, the wing skeletal patterns of the Day 11
polarizing region and its expression pattern strikingly corre- wings looked noticeably different: 100% exhibited extreme
lates with maps of the polarizing region throughout devel- truncations along the A P axis, most of them having only
opment (Riddle et al., 1993). Grafts of Shh-expressing cells a humerus, a radius, and a digit 2 (Table 1B, Fig. 1C).
(Riddle et al., 1993; Chang et al., 1994) as well as SHH To investigate whether Shh regenerated after the polariz-
protein in beads (López-Mart1
B nez et al., 1995) have been ing region removals we performed in situ hybridizations of
shown to induce polarized digit duplications along the ante- limbs from embryos harvested 24 hr after the surgeries. Our
roposterior axis, which are indistinguishable from the clas- results show that the amount of Shh signal and the percent-
sical polarizing region grafts. It thus seems likely that Shh age of Shh-positive limbs after 24 hr is correlated with the
is the molecule responsible for the patterning role of the amount of Shh and percentage of positive limbs at 0 hr. In
polarizing region in the developing limb and that it would an experiment where we analyzed limbs at several times
serve as an excellent marker for identifying the cells that after the surgery, it was clear that the Shh signal in the 24-
belong to the polarizing region. We have reinvestigated the hr limbs came from Shh-expressing cells that had not been
requirement for polarizing region tissue during limb devel- removed during the surgery (data not shown). Table 1C
opment using Shh expression as a marker. summarizes an experiment where we removed a more distal
portion of the polarizing region, purposefully leaving proxi-
mal Shh-expressing cells. Almost 90% of the limbs har-
MATERIALS AND METHODS vested at 0 hr after the surgeries had high remaining
amounts of proximal Shh, close to the junction of the limb
White Leghorn chick embryos of stages 20 21 (Hamburger and bud and body wall (Fig. 2). After 24 hr, 38% of the limb
Hamilton, 1951) were used for surgeries. The polarizing region was buds still showed high amounts of Shh, 50% had low levels
excised with tungsten wire needles and embryos were either har-
of the signal, in each case confined to the proximal regions
vested immediately or allowed to develop for 24 hr or until the
of the limb buds (Fig. 2, n Å 8). In similar specimens, the
11th day of embryonic development. Whenever indicated, a bead
skeletal patterns of the Day 11 wings showed a relatively
(Affi-Gel Blue Gel, Bio-Rad) soaked in FGF-4 (0.85 mg/ml, a gift
high percentage (33%) of normal limbs; those remaining
from the Genetics Institute) was inserted in a cut made in the limb
formed a humerus, radius, ulna, and digit 2. When we com-
bud mesenchyme after the polarizing region removal. Embryos har-
pared these skeletal patterns with those obtained from our
vested 0 and 24 hr after the surgeries were fixed in 4% paraformal-
previous experiments, the most obvious difference was the
dehyde, dehydrated in a graded methanol series, and used for in situ
addition of an ulna in all limbs (compare Tables 1A and 1B
hybridization using the Shh probe as previously described (Riddle et
al., 1993). Embryos harvested at Day 11 were washed in PBS, fixed with Table 1C). This is consistent with the in situ analysis
in 4% paraformaldehyde, and stained for cartilage with Alcian blue. of the 0- and 24-hr limbs, where the Shh signal was always
proximal, i.e., not near the digit area, and correlated with
the A P pattern restoration at the zeugopod level. In con-
RESULTS AND DISCUSSION trast, we never observed an ulna in truncated limbs at Day
11 from surgeries done that were similar to those where no
Table 1 summarizes the results from surgeries performed Shh was left proximally.
on stage 20 21 chicken embryos. Our approach was to at- We conclude from these experiments that Shh, and thus
tempt to achieve a constant extent of cutting during each the polarizing region, does not regenerate following polariz-
individual experiment, progressively trying to remove more ing region removal and that the degree of A P patterning
Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Shh and Limb Patterning 37
TABLE 1
Remaining Shh Signal after Polarizing Region Removal and Corresponding Wing Structures Present at Day 11
Goal of surgery: A (Leave Shh) B (Leave no Shh) C (Proximal Shh) D (No Shh / FGF)
Shh expressions
0 hr
No Shh 17% 64% 0% 100%
Low levels Shh 33% 29% 0% 0%
Some Shh 25% 7% 11% 0%
High levels Shh 25% 0% 89% 0%
n Å 12 n Å 14 n Å 9 n Å 6
Skeletal elements
11 days
R 0% 10% 0% 57%
R, 2 43% 90% 0% 0%
R, 2, 3 28.5% 0% 0% 0%
R, U, 2 0% 0% 66% 43%
R, U, 2, 3 0% 0% 0% 0%
R, U, 2, 3, 4 28.5% 0% 33% 0%
n Å 7 n Å 5 n Å 3 n Å 6
Note. During each surgery, great care was taken to be consistent in the amount of tissue removed in embryos of each batch (A, B, C,
and D). However, some variation is likely between surgeries done on different days; therefore, the phenotypic outcomes are only analyzed
relative to the amount of Shh left in representative limbs done in parallel, and numbers are not combined between experiments. We
performed a total of 203 polarizing region removals; a subset of representative experiments is shown here. The results obtained with those
experiments not shown are consistent with the data presented in the table. R, radius; U, ulna; 2, digit 2; 3, digit 3; 4, digit 4.
in the Day 11 wings is correlated with the amount of Shh al., 1993). Shh maintains FGF-4 expression necessary for
left in the operated buds. We reason that MacCabe et al. limb outgrowth and FGF-4, in turn, maintains Shh in the
(1973) and Fallon and Crosby (1975) did not remove all of posterior wing mesoderm. Hence, after limb induction
the polarizing region in their experiments, such that enough stages the positional fates along the A P axis could have
Shh-expressing cells were left to maintain patterning of the already been established, and the role of Shh and the po-
limbs. Negative assays for polarizing activity that allowed larizing region at that point would be to support limb
Fallon and Crosby (1975) to conclude that the polarizing outgrowth through its interaction with FGF-4 in the overly-
region was not regenerated seem to contradict our result ing ectoderm. These hypotheses were tested by attempting
that Shh is found in some limbs 24 hr after polarizing region to maintain limb outgrowth by implanting a bead loaded
removals. This inconsistency can be reconciled by assum- with FGF in the limb mesoderm after polarizing region re-
ing that at the time, the assay for polarizing activity was moval. Table 1D summarizes the results of one such experi-
not sensitive enough. Tickle (1981) has shown that placing ment, where a bead of FGF-4 was implanted in the meso-
a tissue graft beneath an intact apical ridge constitutes a derm of stage 20 21 limb buds after the polarizing region
significantly more sensitive test for polarizing activity, as had been removed. As is shown in the table, no Shh signal
opposed to placing the graft in a notch, the procedure that was detectable in 100% of the limbs harvested right after
Fallon and Crosby followed. We speculate that assaying the the surgeries, and at Day 11 all of the wings showed extreme
24-hr postsurgery limb buds using the more sensitive assay A P truncations (Fig. 3). Interestingly, the ulna seemed to
would have resulted in duplications along the A P axis be the only structure that was to some extent restored with
indicative of the residual polarizing activity. these experiments, since 43% of the limbs now had an ulna
We envisioned two possible explanations of why remov- in addition to the humerus, the radius, and digit 2 (compare
ing all of Shh resulted in severe pattern defects along the to 0% in experiment of Table 1B).
A P axis of the limbs. One was that Shh could be necessary When we analyzed the limbs harvested 24 hr after polariz-
to actively pattern structures along the A P axis as the ing region removal and FGF bead implants, we found that
wing grows and develops, in which case removing it at early 100% showed Shh signal to various extents and always in
stages results in postaxial defects. Alternatively, it was pos- a proximal position, away from the digit region (Fig. 3, n Å
sible that Shh has completed its A P patterning role after 6). Given that we removed all of Shh at 0 hr and that the
early limb induction stages and the reason truncations oc- position of the Shh signal at 24 hr is always proximal, we
cur is simply because of the disruption of a feedback loop assume that these Shh-expressing cells belonged to the
between Shh and FGF-4 (Laufer et al., 1994; Niswander et flank at the time of polarizing region removal and that they
Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
38 Pagan et al.
FIG. 1. (A) Tissue from the polarizing region was surgically removed and remaining polarizing region cells were visualized by in situ
hybridizations with the Shh probe. Limbs in the figure show examples of what were categorized as low or high levels of remaining Shh
as classified in Table 1. Control: Shh expression in an unoperated normal limb at stage 20 21. Arrows indicate remaining Shh signal in
the limb buds. (B) Representative Day 11 (Table 1A) limbs show the range of wing skeletal patterns obtained after incomplete polarizing
region removals. (C) When all or most of the polarizing region was removed the resulting Day 11 (Table 1B) wing skeletal patterns exhibited
extreme truncations along the A P axis.
are brought into proximity with the FGF bead as the limb We have shown that after complete polarizing region re-
bud grows and heals, inducing expression of Shh in a poste- moval, posterior truncations along the anteroposterior axis
rior region of the limb bud where it normally is not ex- result. The cause of truncations occurring in the absence of
pressed at that stage of development. Similar results have Shh remains to be addressed. First, it is possible that the
been reported by Yang and Niswander (1995), where a bead surgery to remove all of Shh-expressing cells is radical
of FGF-4 placed close to the limb flank enables cells in this enough to result in the observed deletions. Second, cells
region to express Shh when they normally would not. We may no longer realize their fate without the signal from the
propose that the proximal Shh expression permits regula- polarizing region. Third, cell death may be induced in these
tion of patterning at the level of the zeugopod and the pres- cells after polarizing region removal, with the loss of poste-
ence of an ulna in a high percentage of the wings. These rior structures being a direct result of this cell death. This
results parallel those from our previous experiments (com- is the least likely since it would be expected that all the
pare Tables 1C and 1D), where remaining proximal Shh digits would be affected (Todt and Fallon, 1987). In sum-
after polarizing region removal equally resulted in regula- mary, we have resolved a discrepancy in the literature by
tion of the ulna. In conclusion, our FGF experiments indi- showing that the results previously obtained by MacCabe
cate that Shh is probably needed to actively pattern the et al. (1973) and Fallon and Crosby (1975) probably were
A P axis throughout development of the bud. When we achieved after incomplete polarizing region removals. In
provided an FGF bead to maintain outgrowth, we did not order to consistently remove all Shh-positive cells it is pos-
observe any restoration of A P pattern other than the ap- sible that cells with posterior skeletal fates are also re-
pearance of an ulna, which can be explained by an induction moved. Our data indicate that microsurgery cannot unam-
of Shh expression in cells close to the limb flank. biguously answer the question of a continuous role for po-
Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Shh and Limb Patterning 39
FIG. 2. When we performed polarizing region removals such that Shh-expressing cells were left proximally when examined on the day
of surgery (0 D; D, day), the skeletal patterns were normal in a considerable number of wings, and the remaining wings showed extreme
A P truncations at the autopod level, but restoration of the ulna at the zeugopod level (11 D). This phenotype correlated with continued
proximal Shh expression in a high degree of limbs analyzed 1 day after surgery (1 D). Control: Shh expression in unoperated limbs at 0
time and 1 day postsurgery. Arrows indicate remaining Shh signal in the limb buds.
FIG. 3. Implanting an FGF-soaked bead in the operated limbs to maintain outgrowth did not restore A P pattern at the autopod level
(11 D). Regulation of the ulna in almost half of the cases correlates with induction of proximal Shh by FGF, as analyzed in limbs 1 day
after surgery (1 D). Arrows indicate induced Shh signal.
Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
40 Pagan et al.
M. A., Simandl, B. K., Beachy, P. A., and Fallon, J. F. (1995). Limb-
larizing region during limb development posed by MacCabe
patterning activity and restricted posterior localization of the
et al. and Fallon and Crosby. Therefore, it remains a reason-
amino-terminal product of Sonic hedgehog cleavage. Curr. Biol.
able possibility that there is a continuous role for the polar-
5, 791  796.
izing region and Shh in A P patterning of the developing
MacCabe, A. B., Gasseling, M. T., and Saunders Jr., J. W. (1973).
limb at least up to stage 20 21 of development.
Spatio-temporal distribution of mechanisms that control out-
growth and antero-posterior polarization of the limb bud in the
chick embryo. Mech. Ageing Dev. 2, 1 12.
ACKNOWLEDGMENTS
Niswander, L., Tickle, C., Vogel, A., Booth, I., and Martin, G. R.
(1993). FGF-4 replaces the apical ectodermal ridge and directs
outgrowth and patterning of the limb. Cell 75, 579 587.
This work was supported by Grant 95/0576 from FISS to M.A.R;
Riddle, R. D., Johnson, R. L., Laufer, E., and Tabin, C. (1993). Sonic
NIH Grant HD32443 to C.T; and NIH Grant HD32551 to J.F.F. We
hedgehog mediates the polarizing activity of the ZPA. Cell 75,
thank B. Kay Simandl and Won-Sun Kim for comments on the
manuscript. 1401 1416.
Saunders, J. W., and Gasseling, M. T. (1968). Ectodermal-mesenchy-
mal interactions in the origin of limb symmetry. In   Epithelial
and Mesenchymal Interactions  (R. Fleischmajer and R. E. Bill-
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