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American Journal of Medical Genetics 87:115–127 (1999)
Caudal Dysgenesis in Staged Human Embryos:
Carnegie Stages 16–23
Rengasamy Padmanabhan,1* Ichiro Naruse,2 and Kohei Shiota2
1
Department of Anatomy, Faculty of Medicine, UAE University, Al Ain, United Arab Emirates
Congenital Anomaly Research Center and Department of Anatomy and Developmental Biology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan
2
The severity of expression of malformations
of the median axis in the caudal region of
human embryos is highly variable and
ranges from caudal dysgenesis and sirenomelia to simple sacral hypoplasia. Several
forms of sacral dysgenesis may be discovered later in life. This shows that caudal
malformations of relatively lesser severity
should occur at a greater frequency than actually reported. In the present study we
looked at the morphology and histology of
some human embryos with caudal dysgenesis. Several developmental alterations of
the median axis were observed. These included significant reduction in the craniofacial mesenchyme characterized by hypoplasia of the pharyngeal arches, palatal
shelves, and agenesis or hypoplasia of the
auricular hillocks at the rostral end, absence of the caudal trunk from midsacral to
all coccygeal segments, vertebral fusion or
agenesis, defective development of the primary and secondary neural tubes, rectal
and urinary tract dysgenesis, and deficiency, malrotation, and deficiency of the
limbs at the caudal end. Hindlimb malformations included bilateral agenesis (one
case), meromelia, and various forms of abnormal rotation, but no instances of sirenomelia were present. Radial dysgenesis has
been reported to be associated with caudal
dyplasia in the literature, however, we observed agenesis of the ulna in one and of the
fibula in another embryo. There was an impressive association between limb malfor-
Contract grant sponsor: Japanese Ministry of Education, Science, Sports and Culture; Contract grant sponsor: Ministry of
Health and Welfare; Contract grant sponsor: Heiwa Nakajima
Foundation (Tokyo).
*Correspondence to: Dr. R. Padmanabhan, Department of
Anatomy, Faculty of Medicine and Health Sciences, United Arab
Emirates University, PO Box 17666, Al Ain, United Arab Emirates. E-mail: [email protected]
Received 6 April 1999; Accepted 14 July 1999
© 1999 Wiley-Liss, Inc.
mations and body wall defects. The histological studies demonstrated caudal vascular deficiency and hemorrhagic lesions in
the limbs of the dysplastic embryos. The
data suggest that these polytopic field defects arise very early in development possibly as result of disturbances to fundamental
developmental events that share common
molecular and cellular mechanisms. Am. J.
Med. Genet. 87:115–127, 1999.
© 1999 Wiley-Liss, Inc.
KEY WORDS: human embryos; caudal dysgenesis; limb-body wall defects; morphological and histological study
INTRODUCTION
The “syndrome” of caudal dysgenesis comprises a
complex group of malformations of the caudal embryonic axis including abnormalities of the lumbosacral
spine, imperforate anus, rectovesical and rectourethral
fistulae, agenesis of the kidneys, internal genitalia
with the exception of the gonads, and flexion, inversion, and external rotation of the lower limbs [Duhamel, 1961]. Although Duhamel’s name is most commonly cited in the literature on caudal dysgenesis, the
condition was first described by Rocheus in 1542 [cited
by Kampmeier, 1927]. This anomaly has been variously and often synonymously (although incorrectly)
referred to as caudal dysgenesis, caudal dysplasia, sacral agenesis, sacral dysplasia, caudal spinal aplasia/
agenesis, lumbosacral agenesis, etc. [Pang and Hoffman, 1980; Welch and Aterman, 1984; Pappas et al.,
1989; Alles and Sulik, 1993]. The severity of expression
of the anomaly is variable [Pappas et al., 1989]. In extreme forms the lower extremities are represented by a
median limb with toes turned backward (symmelia/
sirenomelia) giving the appearance of the mythological
mermaid, hence the fanciful term mermaid anomaly.
The severe visceral malformations associated with caudal dysgenesis are incompatible with extrauterine life,
whereas symmelia is the least grave and most variable
abnormality. It has an earlier onset than lumbosacral
116
Padmanabhan et al.
agenesis. The incidence is reported to be 1 in 60,000
births. The frequency is 8 to 15% higher in twinning
and approximately 100 to 150 times higher among
monozygotic twins than among dyzygotic twins or
singletons. Concordance for sex is almost a rule although concordance for sirenomelia is unusual [Young
et al., 1986]. Familial occurrence has been reported,
but no Mendelian pattern of inheritance has been established. A recent study provides evidence for the homeobox gene HLXB9 involvement in dominantly inherited sacral agenesis, but the possible role of this gene in
caudal dysgenesis remains to be established [Ross et
al., 1998]. The fact that sacral dysgenesis may be discovered later in life indicates that caudal malformations of relatively lesser severity, which have no apparent clinical manifestations, occur at a greater frequency than actually reported.
Clinical data indicate that there may be many causes
for the defective differentiation of the caudal end of the
embryonic axis during gestation days 13 to 22 leading
to caudal deficiencies [Hoyme, 1988; Young et al.,
1986]. Cadmium, lead, trypan blue, retinoic acid, and
ochratoxin A have been shown to induce caudal dysgenesis in experimental animals [Rajala and Kaplan,
1980; Padmanabhan and Hameed, 1990; Alles and Sulik, 1993; Wei and Sulik, 1996; Padmanabhan, 1998].
The facts that insulin may cause rumplessness in chick
embryos, that sacral dysgenesis occurs when pregnant
rats are experimentally made diabetic, and that there
exist occasional clinical observations of sacral hypoplasia in infants of diabetic mothers, suggest that maternal diabetes may contribute to sacral hypoplasia in the
offspring [Landauer, 1945; Duraiswamy, 1950; Wilson
and Vallance-Owen, 1966; Pedersen et al., 1971;
Deuchar, 1977; Young et al., 1986; Welch and Aterman,
1984; Perrot et al., 1987; Padmanabhan and Al Zuhair,
1989]. However, the incidence in the babies of established diabetics is surprisingly as low as 1% or < 1%
[Sarnath et al., 1976; Pappas et al., 1989; Mills, 1982;
Kalter, 1993]. In addition to diabetes, embryonal
trauma, maternal fever, nutritional deficiency, toxic
substances, and genetic factors have been considered
causes of caudal malformations [Pang and Hoffman,
1980]. Kampmeier [1927] thought that an abnormal
umbilical artery of vitelline origin could deprive the
caudal region of the embryo of essential nutrients resulting in caudal dysgenesis. This abnormal artery was
found in a number of sirenomelic fetuses [Kapur et al.,
1991; Stocker and Heifetz, 1987]. These reports were
based on observations on late-stage fetuses and newborn infants, and there was no evidence that the vascular abnormality preceded the observed dysgenesis
sequence. O’Rahilly and Müller [1989] examined over
100 normal embryos of Carnegie stage 8–18 and four
synophthalmic embryos of stages 16–20 and commented on the pathogenesis of several median anomalies including sirenomelia. Studies on younger human
embryos with caudal dysgenesis are of great importance in delineating the pathogenetic mechanisms, but
published data are lacking. Recent studies have shown
that vascular disruption precede caudal dysgenesis in
the mouse [Seller and Wallace, 1993; Padmanabhan,
1998]. The objectives of the present study were to ex-
amine human embryos with caudal dysgenesis and
evaluate the morphological and histological alterations
that might subsequently contribute to caudal dysgenesis. Here we report on the gross and histological abnormalities of Carnegie stages 16–23 embryos with
caudal dysgenesis and discuss their possible pathogenetic mechanisms.
MATERIALS AND METHODS
The embryos used in this study were from a large
collection of conceptuses obtained mostly from apparently normal pregnancies terminated by dilation and
curettage for economic reasons and preserved at the
Congenital Anomaly Research Centre of the Kyoto University Faculty of Medicine. A few cases were also obtained from threatened abortions. The collection was
begun in 1961 by Prof. Hideo Nishimura and assisted
by many of his experienced colleagues and obstetricians. Details of these embryos have been described
earlier [Nishimura, 1975]. They were staged and preserved in formalin, Bouins or Lillies fluid. They were
first examined thoroughly under a stereomicroscope
and external anomalies were photographed meticulously from several aspects. Both age-matched normal
and abnormal embryos were sectioned and stained
with hematoxylin and eosin (H&E) and Mallory azan
trichrome. Included in the present report are 10 embryos with caudal dysgenesis ranging in age from Carnegie stages 16 to 23. Two of them (one of stage 21 and
one of stage 22) had been sectioned serially for histological examination. Alternate sections were stained
with H&E and trichrome. They were examined with an
Olympus BH-2 microscope. Both gross and histological
specimens were compared and contrasted with agematched normal embryos [Nishimura, 1983; O’Rahilly
and Muller, 1987].
RESULTS
Macroscopic Observations
All embryos shown in Table I lacked or had an extremely small trunk distal to the postaxial border of
the hindlimbs. The absent segments included midsacral to all coccygeal vertebrae. Virtually all of them had
one or more craniofacial malformations such as holoprosencephaly (1 case), hypoplasia of pharyngeal
arches, nasal processes and premaxilla, and reduction
in number, malpositioning, and hypoplasia of the auricular hillocks (Fig. 1 B–D,F). One of the embryos had
severe mandibular retrognathism and unfused lateral
nasal and maxillary processes (Fig. 2D). Meromelia of
the upper and lower limbs, unilateral cleft hand and
cleft foot (case no 50641 stage 18), and retardation of
the development of digits were also observed. The
lower limbs were commonly oriented horizontally with
the postaxial borders facing medially (inversion) (Fig.
2B). The foot plates adjoining each other in one case
(case 215052, stage 19) gave the appearance as if the
perineum was covered by the hindlimbs. One of the
embryos of stage 16 (case 37430) with a remarkable
deficiency in the caudal segment of the trunk, had
failed to develop lower limb buds; it had a fish-like
Caudal Dysgenesis in Human Embryos
117
TABLE I. Anomalies Associated With Caudal Dysgenesis in 10 Human Embryos of Carnegie Stage 16–23
Embryo
number
Photographic
examination
Gross
examination
Histological
examination
14535
+
+
–
37430
+
–
–
17
38638
+
+
–
18
382666
+
+
–
18
50641
+
+
–
19
21502
+
–
–
19
28410
+
–
–
21
3191
+
–
+
22
21743
+
–
+
23
33511
+
–
–
Stage
16
appearance (Fig. 1B). The genital tubercle was located
on the summit of a median genital swelling. The
anococcygeal distance appeared to be reduced in conformity to the caudal narrowing of the trunk. The external genitalia of most embryos were hypoplastic and
situated very close to the umbilicus possibly because of
underdevelopment of the infraumbilical portion of the
body wall (Fig. 2 E,F). The ventral abdominal wall and
the lower chest wall appeared thin and translucent
through which the liver, heart, and pericardium could
be easily seen.
Microscopic Observations
Embryo of Carnegie Stage 21 (Case 3191). The
cerebral ventricles of this embryo were rather hypoplastic and slightly collapsed. Structurally the neuro-
Abnormalities
Holoprosencephaly, two auricular hillocks in pharyngeal arch 1
and one in arch 2 - all hypoplastic; fore- and hindlimb
meromelia; narrow hand plate and absence of digital rays in
hand plate and foot plate.
Rudimentary first and second pharyngeal arches with no
auricular hillocks; hand plate as broad as the arm, which is
also hypoplastic; absence of both hindlimbs giving rise to
fish-like appearance; the trunk ends at the genital tubercle;
thin transparent ventral abdominal wall, through which the
liver could be seen.
Hypoplastic pharyngeal arch 1 and 2; arch 1 has no auricular
hillocks and arch 2 has two rudimentary hillocks; medial and
lateral nasal processes hypoplastic; ventral body wall
comprises a transparent membrane; tail consists of two
somites whereas the normal embryos of this stage contains
about 10 somites; the chorionic villi appeared withered.
Four auricular hillocks surround the first pharyngeal cleft;
forelimb buds are hypoplastic; left hand plate is pointed like
a tongue, with 1/4 of the postaxial portion missing; right
hand plate is like a narrow spade and corresponds in width
to two digital rays; leg portion of the right hind limb is
absent and foot plate is represented by a nipple-like stump at
apex of the thigh; the left hindlimb lacks leg segment and
has a second limb-like appendage on the preaxial aspect.
Left side split hand and split foot plate; the preaxial digit
precociously marked out in the left hand; postaxial borders of
hindlimbs are medial and preaxial borders are lateral in
orientation; the trunk ends at the level of the root of
hindlimbs; lower lumbar and all distal segments are absent;
genital tubercle and umbilicus adjoin each other; all
umbilical vessels are present although small in caliber.
Perineum is covered by hypoplastic hindlimbs postaxial borders
directed medially, separated only by the genital tubercle,
which lies at the center of the perineum; sacral and all
coccygeal segments of the trunk are absent; there is a
median dorsal elevation in the lower trunk.
Medial and lateral nasal processes are hypoplastic and widely
separated (bilateral cleft upper lip?); preaxial polydactyly of
right lower limb (extra digital ray; deep interdigital notches
are seen; mid-sacral to all coccygeal segments of the trunk
are missing. There are four nipple primordia (polythelia).
Hypoplastic medial and lateral nasal processes; cleft left hand;
ventral abdominal wall deficient; more details in the text.
Lower sacral and all coccygeal segments of the trunk absent;
lower limb meromelia; more details in the text.
Narrow perineum; lower limb meromelia; midsacral to all distal
segments of the trunk absent.
epithelium of the brain was just as mature as that of
the normal embryos of stage 21. The medial and lateral
nasal processes were severely hypoplastic indicative of
the possibility of giving rise to facial clefts later in development. The vomeronasal organs were present. The
palatal shelves were on either side of the tongue pointing to the floor of the oral cavity. The primordia of the
pituitary, thyroid, parathyroid, heart, digestive organs,
kidneys, suprarenals, and gonads (testes) appeared to
be normal and appropriate for the stage of development. The clavicles showed evidence of ossification in
the lateral and midshaft regions. The cartilaginous
models of the humerus and scapula were normally developed. However, the radii and ulnae were remarkably short. There were six carpal primordia and five
metacarpals. The 4th and 5th metacarpals distally
118
Padmanabhan et al.
Fig. 1. Human embryos of Carnegie stage 16 (A and B), 17(C), and 18(D–F). A: A normal embryo. Observe in the caudal dysgenesis embryos marked
hypoplasia of the pharyngeal arches and absence of auricular hillocks (B–F), retarded development of forelimbs (C,F), absence of hind limbs (B), absence
of caudal trunk (B,C,D,F), and split hand (D). E: Limbs of embryo F. It has an accessory preaxial digit (arrow in lower picture) and no digital rays in the
left foot plate; there is a single median digit in the right hand plate (upper picture).
reached the ring finger. Metacarpals 1 and 2 entered
the middle finger. The thumb consisted of only a skin
appendage. The normal right hand possessed no first
metacarpal and all fingers including the thumb had
one phalanx each. The sternal bars were absent except
most proximally. Starting from a little lateral to the
midclavicular plane, the ventral abdominal wall was
deficient in musculature. The liver and the gut with the
mesentery protruded through a median defect of considerable size. The right edge of this defect in its caudal
aspect showed a clear skin covering, although the left
edge appeared to have been damaged during curettage.
The viscera were found to project outside the lower
abdomen. The umbilical cord contained two arteries
and a vein. The mesonephric ducts and ureters entered
the vesicourethral portion of the urogenital sinus (the
bladder). The rectum ended abruptly at the level between the first and second sacral segments. Pubic
bones were absent. The genital tubercle was small; the
glandular urethra consisted of an epithelial plate. In
the lower limbs, the primordia of the femur and tibia
were present but not that of the fibula (Fig. 3 A,B).
Hemorrhage was extensive in the thigh and leg but
most prominent at the knee (Fig. 3C). The spinal cord
(primary neural tube) had no skeletal protection below
the midsacral level and became continuous with the
short, kinky secondary neural tube at an angle. Irregular vascular proliferation and hemorrhage were promi-
Caudal Dysgenesis in Human Embryos
119
Fig. 2. Embryos of Carnegie stage 19 (A–C), 21 (D), and 22 (E: lateral view; F: ventral view) showing caudal dysgenesis. The hypoplastic hindlimbs
of the embryo A are attached to the caudal tip of the trunk with their roots closely positioned in the narrow perineum (B) and postaxial borders facing
each other. The genital tubercle is either absent (B) or adjoins the caudal tip (arrow in C). Varying degrees of malrotation and hypoplasia of hindlimbs,
deficiency of lower abdominal wall (E,F), oblique facial cleft (nonfusion of maxillary and lateral nasal processes), and extreme mandibular hypoplasia (D)
are also obvious. (B) Perineum of embryo A. (F) Ventral view of embryo E.
nent in the area of union between the primary and
secondary neural tubes (Fig. 3 D,E). There was no evidence of a tail.
Embryo of Carnegie Stage 22 (Case 21743). The
histological structure of the head and neck of this embryo appeared normal except for the fact that the palatal shelves were asymmetric. The sternal bars were
hypoplastic, widely separated, and gradually disappeared more distally (Fig. 4 C,D), whereas the normal
embryos of this stage had well-formed primordia of costal cartilages, ribs, and sternal bars, and well-differ-
entiated abdominal musculature (Figs. 4 and 5). The
ventral abdominal wall was open at the midlumbar region where the parietal peritoneal lining was found to
be continuous with the epidermis. As a result, the abdominal contents were outside the abdomen (Fig. 5
A,B). The abdominal wall ventral to the midaxillary
plane lacked in differentiation and organization of the
musculature, and as a result, the lower pericardium,
heart, and liver remained unprotected below the costal
margin (Fig. 5 C,D).
The gonads (testes), kidneys, spleen, and digestive
120
Padmanabhan et al.
Fig. 3. (A–C) Transverse sections of an embryo of Carnegie stage 21 with caudal dysgenesis. Primordia of femur (F) and tibia (T) are present but not
that of fibula. Note the presence of prominent hematoma (arrow in A and B) at knees. (C) A high power view of hemorrhage (arrows) seen in A. The caudal
end of the same embryo (D) shows kinky union between the primary (arrow) and secondary (arrowheads) neural tubes. The neuroepithelial infoldings
of secondary neural tube seen in higher magnification (E) are possibly the result of kinking. Also note the patchy hemorrhages (hm in D and E) around
the neural tube. The neural tube lacks skeletal protection. Bar ⳱ 450 ␮m (A,B); 90 ␮m (C,E); 225 ␮m (D).
organs appeared to have been normally formed. The
separation of the bladder and rectum was complete although the rectum ended blindly. No ureters could be
traced to the bladder. The Wolffian ducts appeared to
be rather dilated. The phallus was rudimentary.
There was a single umbilical artery directly continuous with the dorsal aorta on the left of the median
plane (Fig. 6A). It was almost as large in size as the
dorsal aorta itself. Distal to this site of continuity the
dorsal aorta was absent. The internal iliac vessels were
also lacking. The third, fourth, and fifth sacral vertebral centra were fused with reduced disc space, which
was confined to the periphery. There were no coccygeal
vertebrae. In the normal embryos of this stage, there
were five sacral vertebrae and as many as seven coccygeal vertebrae (Fig. 6B); situated on the pelvic floor
were abundant autonomic elements extending from the
sympathetic chains towards the cloaca. The affected
embryo presented a caudal tissue mass comprising
cells similar to those of the ganglion impar but with no
definitive organization (Fig. 6A). The caudal portion of
the primary neural tube was kinky and dilated with
vacuolated neuroepithelium, and the secondary neural
tube was rudimentary at the beginning and partially
duplicated distally. Here both the primary and secondary neural tubes lacked skeletal protection (Fig. 6 D,E).
The clavicles showed signs of ossification in the mid
and lateral segments of the shaft. The primordia of the
scapula, humerus, and radius were well defined, but
the ulnae were absent. The humeri were relatively
short and curved. The distal end of the humerus and
proximal end of the radius were broad. The lower limbs
were short (meromelic) with inadequately developed
muscles. The femora were short and fused with the hip
bone without a proper cavity of the hip joint (Fig. 6F).
The pubic bones were absent.
Caudal Dysgenesis in Human Embryos
121
Fig. 4. Transverse sections of a normal embryo (A,C) and an embryo with caudal dysgenesis (B,D) at stage 22. The sternal bars (open arrows in C,D)
are well formed in the normal embryo in contrast to the hypoplastic and widely separated sternal bars of the caudal dysgenesis embryo (B,D). O,
oesophagus; T, trachea; AO, aorta; CV, cardinal vein; H, heart. Bar ⳱ 450 ␮m (A,B); 225 ␮m (C); 360 ␮m (D).
122
Padmanabhan et al.
Fig. 5. Transverse sections of normal (A,C) and caudal dysplastic (B,D) embryos of stage 22. The ventral abdominal wall of caudal dysgenesis embryo
is deficient allowing viscera to protrude externally (B). The musculature of the abdominal wall of the normal embryo has differentiated and organized
well into the external oblique (EO), internal oblique (IO), and transversus (TA) abdominis. In sharp contrast, the caudal dysgenesis embryo shows a gross
deficiency and lack of differentiation of this musculature (open arrow in D). Bar ⳱ 450 ␮m (A,B); 90 ␮m (C); 225 ␮m (D).
Caudal Dysgenesis in Human Embryos
123
Fig. 6. Sections of embryos of Carnegie stage 22. The embryo with caudal dysgenesis shows a gross reduction in number and fusion of caudal vertebral
primordia (S in A), disorganized caudal tissue mass (CTM in A), a single umbilical artery (UA in A), kinky and dilated central canal of the caudal portion
of the neural tube (CC in D), duplication of the secondary neural tube (SNT in D), and vacuolated ventricular zone of the neuroepithelium (NE in E).
The femur (F) remains fused with the ilium (IL) in the caudal dysgenesis embryo (F). (B,C) Normal embryos of stage 22 showing prominent caudal
vertebrae (B) and hip joint (C). B, bladder; R, rectum; U, urethra. Bar ⳱ 225 ␮m (A, C, D–F); 360 ␮m (B).
DISCUSSION
Several theories have been postulated to explain the
pathogenesis of caudal dysgenesis. The “lateral compression” theory originally proposed by Dareste [1891,
cited by Stevenson et al., 1986], holds that abnormal
amniotic folds compress the caudal end of the embryo
and suppress the development of the pelvic structures
and cause abnormal rotation of the limbs. This theory
is supported by observations on the mutant sirenomelic
mice [Hornbeek, 1970; Orr et al., 1982]. Matsunaga
and Shiota [1980] reported a high incidence of unilateral limb defects in tubal pregnancies and caudal dys-
genesis in pregnancies with uterine myomata possibly
as result of spatial restriction and associated vascular
compression. According to Kallen and Winberg [1974]
deficiency of the caudal mesoderm and/or irregularities
of the notochord and somites might lead to duplication
of the notochord and neural tube, chordoma of the sacrococcygeal region, anorectal dysgenesis (atresia, stenosis, and ectopia), and urogenital and vertebral malformations. Narrow defects of the caudal mesoderm
would result in median anomalies such as sacral agenesis and more extensive defects lead to lateralized malformations such as limb defects. Extreme median tissue deficiency will result in failure of fission of the limb
124
Padmanabhan et al.
fields resulting in symmelia [O’Rahilly and Müller,
1989]. Failure of the caudal somites to develop and the
lack of midline mesoderm promote fusion of the hindlimb buds. Whether it is merging, fusion of hindlimb
buds, or failure of fission of limb fields that leads to
sympodia is controversial [Hoyme, 1988; Barr, 1988].
The hindlimb buds of human embryos do not appear
until stage 13 (postconception day 28), whereas the
critical period for caudal dysgenesis is between day 13
and 22 [O’Rahilly and Müller, 1988]. Therefore, the
probability for fusion of formed limb buds is rather low.
Our recent study in which retinoic acid (RA)-treated
mouse embryos were examined at early stages did not
show any evidence for fusion of limb buds at all, although symmelia was observed in a number of embryos
at term [Padmanabhan, 1998].
The vascular steal theory originally proposed by
Weigert [1886] was supported by many subsequent investigators [Kampmeier, 1927; Stevenson et al., 1986;
Kapur et al., 1991; Murphy et al., 1992]. These authors
observed in babies with caudal dysgenesis a single umbilical artery that arose from the abdominal aorta
slightly proximal to its bifurcation. Distal to this origin,
the aorta was extremely hypoplastic. The caudal
anomalies were therefore interpreted as the result of
arrested development because of hypoperfusion. It is
important to point out here that none of these studies
provide evidence to show that the vascular abnormality
preceded caudal regression. This assumption cannot
also explain adequately the more cranial anomalies
(e.g., anencephaly, limb-body wall defects, VATER, or
VACTERL abnormalities) observed in many of the sirenomelic fetuses [Rodriguez et al., 1991; Tang et al.,
1991; Murphy et al., 1992; McCoy et al., 1994].
The possible role of vascular steal in the pathogenetic mechanism has earned attention possibly because of the frequently reported association of a single
umbilical artery of vitelline origin with sirenomelia
[Kampmeier, 1927; Stocker and Heifetz, 1987; Kapur
et al., 1991; McCoy et al., 1994]. This has also initiated
lively discussions on the role of vascular disruption and
hypoperfusion as a teratogenic mechanism [Van Allen,
1981; Hoyme et al., 1981; Hoyme, 1988; Barr, 1988;
Chandebois and Brunet, 1987; 1988; Alles and Sulik,
1993; McCoy et al., 1994; Padmanabhan, 1998]. Chandebois [Chandebois and Brunet, 1988] did not think
that hypoperfusion secondary to vascular disruption
contributed to caudal dysplasia possibly because the
fetus whose pelvic histology he examined was already
at an advanced stage of development. Although trypan
blue-induced hematomas in chick embryos disappear
quickly, rumplessness (the chicken equivalent of caudal dysgenesis) is generally observed at hatching.
Therefore, Barr [1988] argued that hematoma in caudal median axis was an appropriate conceptual model
to explain the diversity of malformations observed in
sirenomelic fetuses. The two early embryos we studied
histologically provided evidence that some vascular
anomalies were associated and that hemorrhage occurred in the malformed (or deformed) tissues including the limbs. Two more recent studies in mouse embryos [Seller and Wallace, 1993; Padmanabhan, 1998]
also provide particular evidence that hemorrhage does
occur prior to regression of the median axis. However,
it is important to understand that vascular disruption
and subsequent hemorrhage are possibly secondary
pathogenetic mechanisms and that what initiates the
cascade is not clear from these studies. While the debate continues, more work needs to be done to identify
the factors that contribute to the vulnerability of the
blood vessels of the median axis.
The hypothesis of overdistention of neural tube advocated by Gardner [1980] holds that the reopening of
a closed neural tube from which neural tube fluid escapes can impair a number of developmentally immature structures such as the primordia of the vertebral
column, neural tube, gut, limb buds, and metanephros.
Thus, he explained cranioschisis, anenencephalus, and
Klippel-Feil syndrome at the cranial end, VATER or
VACTERL anomalies in the mid-body, and caudal dysgenesis including sirenomelia at the caudal end as consequences of reopening of the closed neural tube. According to Gardner [1980], overdistention of the caudal
neural tube would move the hindlimb buds dorsally
and allow their fusion leading to sirenomelia. Our previous studies in rat embryos [Padmanabhan, 1984,
1988, 1991] provided experimental evidence for reopening of cranial neural tube and for several non-neural
malformations, but there was no limb bud fusion. Neither Shenefelt’s [1972] hamster embryos nor our mouse
embryos [Padmanabhan, 1998] that were exposed to
RA after caudal neural tube closure had any manifestation of sirenomelia, suggesting that Gardner’s theory
could not be proved experimentally. McCoy et al. [1994]
proposed that a combination of defective cell proliferation, cell migration or differentiation, and excessive cell
death could be triggered by a transient hypoperfusion;
the consequent mesodermal tissue deficiency and a hypoplastic vasculature might additionally contribute to
the malformation.
A close look at the anatomy of the structures affected
in our embryos shows that dysgenesis is not confined to
the caudal end of the embryonic axis but includes several median and paramedian structures such as the
body wall and limbs, observations confirmed by
O’Rahilly and Muller [1989] in human embryos and our
studies on RA-treated mouse embryos [Padmanabhan,
1998]. It is also apparent that no single theory could
accommodate the pathogenesis of all these malformations.
The midline of the embryo is considered to be an
important part of the primary field characterized by
active cell proliferation, migration, and differentiation
and tissue organization and induction [Opitz and Gilbert, 1982a; Opitz, 1993]. These developmental processes at the rostral end contribute precursor cell populations involved in the formation of the pharyngeal
arches and craniofacial development. At the caudal end
is another area of similarly intense morphogenetic activity called the caudal eminence, which is first identified at stage 9 [O’Rahilly and Müller, 1989; Opitz,
1993]. It is different from the end bud of avian embryos.
It extends from the neurenteric canal to the cloacal
membrane and provides precursor mesenchyme for the
formation of the notochord, somites, caudal vertebrae,
blood vessels, hindgut, neural tube, and hindlimbs.
Caudal Dysgenesis in Human Embryos
Blood vessels enter the caudal eminence at stages 14–
16, and the tissues formed in excess begin to regress at
stage 17. The portion of the trunk derived from the
caudal eminence and that derived from the primitive
streak meet at the upper sacral level at the site of closure of the caudal neuropore [Müller and O’Rahilly,
1987; O’Rahilly and Müller, 1989]. These developmental activities occurring along the median plane are spatially and temporally coordinated, delicately balanced,
and possibly regulated by several genes [Kessel and
Gruss, 1991; Kessel, 1992; Gruss and Walther, 1992;
Holland and Hogan, 1988], which may be interfered
with by physical or chemical agents or by vascular accidents. Clinical data show that the primary field is
uniquely susceptible to complex malformations [Opitz
and Gilbert, 1982a, 1982b; Lubinsky and Moeschler,
1987].
The results of this study have clearly established the
early onset of several developmental alterations associated with caudal dysgenesis in the human embryos.
These include deficiency of the craniofacial mesenchyme characterized by hypoplasia of the pharyngeal
arches, medial and lateral nasal processes, palatal
shelves, agenesis or hypoplasia of the auricular hillocks at the rostral end, reduction or absence of the
caudal trunk from midsacral to all coccygeal segments,
vertebral agenesis and/or fusion, defective development of the primary and secondary neural tubes, rectal
and urinary tract dysgenesis, and deficiency, malrotation, and malformations of the limbs at the caudal end
of the embryonic axis. Total absence (one case), meromelia, and various forms of abnormal rotation of hindlimbs were also observed, and no instance of sirenomelia was present. Whereas radial dysgenesis has been
reported to be associated with caudal dysgenesis [Quan
and Smith, 1973; Young et al., 1986; Alles and Sulik,
1993], we observed agenesis of the ulna in one of our
embryos. The fibula was absent in the other embryo.
The most common associated defect below the knee is
reported to involve an absence of the fibula and comparable abnormalities of the upper limb are relatively
uncommon [Passarge and Lenz, 1966; Welch and Aterman, 1984], and according to the filed theory, this homology (radius and tibia or ulna and fibula) is one of
the attributes of developmental field defects [MartínezFrías et al., 1998]. The occurrence of limb malformations in combination with body wall defects in our
study is impressive and consistent. The histological
studies have also demonstrated caudal vascular deficiency and hemorrhagic lesions in the limbs of the dysplastic embryos.
Basically the embryonic origin of precursors of these
seemingly diverse structures are to be found in the median and paramedian planes during blastogenesis
[Opitz and Gilbert, 1982a; O’Rahilly and Müller, 1989;
Opitz, 1993; Martínez-Frías et al., 1998]. These include
the neural plate, neural tube, notochord, gut, prechordal plate, the primitive streak, caudal eminence,
connecting stalk, etc., in the median plane and the bilaterally paired structures such as the somites, limb
fields, heart tubes, etc. At this stage, the whole embryo
forms a primary field and responds to intrinsic or extrinsic noxious stimuli largely as a pleuripotent single
125
unit. Pattern formation establishes upstream domains
of specific transcription, signaling, and growth factor
gene expression resulting in gradual subdivision of the
primary field into several progenitor fields, the primordia of all final structures [Opitz, 1993; Martínez-Frías
et al., 1998]. The HOX, PAX, T-Box, and sonic hedgehog
(SHH) genes and the RA molecule, which emanates
from the organizer, play major roles in regionalization,
positional information, and anteropoterior and dorsoventral axis formation [Kessel and Grus, 1991; Kessel,
1992; Gruss and Walther, 1992; Conlan, 1995; Smith,
1999; Mansouri et al., 1999]. After the initiation of gastrulation, HOX genes and other homeobox-containing
genes begin to be expressed in specific anteriorposterior domains [Conlan, 1995]. In response to RA
signals generated from midline structures, cells ingressing from the primitive streak, sequentially activate Hox genes leading to overlapping nonidentical expression domains of Hox genes along the anteroposterior axis. The combination of functionally active Hox
genes, known as the Hox code, specifies the identity of
body region [Kessel, 1992]. Exogenous RA alters Hox
codes resulting in homeotic transformations [Kessel
and Grus, 1991]. Misexpression of the Hox genes or
lack of morphogens such as RA during this critical period of development may result in segmentation defects
such as fusion or absence of vertebrae. There is evidence for involvement of HLXB9 gene in hereditary
sacral agenesis [Ross et al., 1998] The expression of
Shh is both temporally and spatially restricted and in
developing vertebrate body axis is tightly correlated
with expression of HNF3. Targeted mutation of HNF3
in the mouse results in defective development of the
notochord and loss of Shh expression. Exposure of
chicken wing bud to RA leads to activation of Shh indicating that both HNF3 and RA receptors regulate
Shh gene [Chang et al., 1997]. Shh emanates from the
axial midline structures such as the notochord and
floor plate, and influences dorsal-ventral specification
of somite derivatives [Johnson et al., 1994]. Targeted
gene disruption in the mouse has demonstrated that
the Shh plays a crucial role in patterning of embryonic
structures such as the brain, spinal cord, spinal column, and the limbs [Chiang et al., 1996]. Abnormalities are found to occur first in the establishment and
maintenance of midline structures such as the notochord and floor plate, and later, defects of the neural
tube and limbs and cyclopia develop. Mutations in the
C-terminal domain of SHH cause holoprosencephaly
[Roessler et al., 1997]. These findings suggest that the
polytopic abnormalities observed in our embryos arose
very early in development and represent defective signaling and pattern formation.
The literature on caudal regression indicates the frequent association of VATER, VACTERL (vertebral
malformations, anal atresia, tracheoesophageal fistula,
cardiac defects, and renal/radial agenesis), OEIS complex (omphalocele, extrophy of the bladder, imperforate
anus, and spinal defects), and Potter sequence with
caudal dysgenesis [Gardner, 1980; Young et al., 1986].
It is important to point out here that such combinations of multisystemic, often severe and complex
anomalies that affect predominantly the midline, arise
126
Padmanabhan et al.
as a response to a dysmorphogenetic stimulus during
blastogenesis or at a time when progenitor fields are
being established and share common molecular and
cellular mechanisms [Duncan and Shapiro, 1993].
Therefore, they should logically be called primary polytopic developmental field defects [Martínez-Frías et
al., 1998] rather than multiple idiopathic anomalies of
blastogenesis [Opitz, 1993]. This concept receives credence from epidemiological [Martínez-Frías, 1994] and
experimental [Alles and Sulik, 1993; Rutledge et al.,
1994; Padmanabhan, 1998] studies. In our mouse
model of caudal dysgenesis [Padmanabhan, 1998], administration of single doses of RA during blastogenesis
resulted not only in sirenomelia but also in defects of a
host of structures that could be grouped together as
primary polytopic field defects. The severity differed
according to the dose and the developmental stage at
the time of RA-administration. When embryos of early
somite stage were exposed to a low dose of RA, hematomas developed in the tail bud within 24 hr of treatment. This was accompanied by pronounced cell death,
edema, and tissue disruption resulting in total regression of the tail, complete in the following 3 to 4 days of
gestation. Most of the embryos of the moderately highdose group failed to develop a tail bud, whereas others
developed hemorrhagic or avascular tail buds that subsequently degenerated. In the high-dose group, extensive cell death was observed in the caudal median axis
as early as 6 hr post-treatment. Subsequently, cell
death also affected the hindgut, neural tube, and
sclerotome-myotome junction area, but not the notochord. The profound tissue loss in the median area appears to have led to agenesis of the tail and caudal
vertebrae. The dilated vertebral canals of the spina bifida fetuses was also associated with vertebral arch
fusion, hemivertebrae, reduction in number of ribs and
or fusion of lower ribs indicating defective segmentation, and/or loss of precursor tissues because of cell
death. One of the important new findings of this study
was the role of vascular disruption in caudal regression
resembling the vascular disruption sequence described
in the tail short variable [Seller and Wallace, 1993].
Teratogenic exposure-related tissue damage and the
consequent repair and regenerative events might also
contribute to abnormalities [Snow, 1984; Van Allen,
1981]. The concept of a combination of developmental
vulnerability inherent in the primary field and teratogen interaction with consequent alterations in molecular and cellular mechanisms of development may account for the variable expression of the malformations
that occur at the rostral and caudal ends of the embryo.
This view will also provide an explanation for the diversity and combinations of anomalies with caudal dysgenesis observed in our series of early embryos and
reported by earlier investigators in late fetuses and
newborn infants.
gratefully appreciated. Dr. Padmanabhan’s visit to the
Center was supported by a fellowship from the Heiwa
Nakajima Foundation (Tokyo). This work was supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture and from the Ministry of Health and Welfare.
ACKNOWLEDGMENTS
Kessel M. 1992. Respecification of vertebral identities by retinoic acid.
Development 115:487–501.
We thank the collaborating obstetricians and the
previous and present members of staff of the Congenital Anomaly Research Center of Kyoto University. The
excellent technical support of Ms. Chigako Uwabe is
Kessel M, Gruss P. 1991. Homeotic transformations of murine vertebrae
and concomitant alterations of Hox codes induced by retinoic acid. Cell
67:89–104.
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