The normal development of the anorectum in the pig

The development of the anorectum was studied in forty-four embryos and foetuses of pig varying in length from 9 mm total length to 210 mm crownrump length and in three newborn pigs. The presence of some features during critical stages in the development of the cloaca in the pig such as an epithelial mass protruding into the dorsal cloaca near its intestinal orifice and distinct differences in the type of epithelium in different regions of the cloacal system greatly facilitated the study of the developmental process. Thus it could be established that a change in position of the dorsal cloaca and adjacent structures such as the distal part of the gut and the urorectal septum via the dorsal part of the cloacal plate towards the tailgroove is of major importance for the partition of the cloaca into a separate intestinal and urogenital division. A subsequent disintegration of the dorsal part of the cloacal plate results in two separate openings for both the systems at the same time. Disintegration of the ventral part of the cloacal plate leads only to a further widening of the external opening of the urogenital system. In the cloacal system three distinct zones were discerned, a dorsal and ventral cloacal and a cloacal plate region. From the dorsal cloacal epithelium the whole anorectal segment between the intestinal mucosa and the anal epidermis develops. The epithelium of ventral cloacal origin seems to disappear completely . Cloacal plate epithelium forms the epithelial lining of distal parts of the urogenital system. The penile urethra in the male is formed by a ventralward movement of the urogenital opening by the growing perineum and not by fusion of genital folds.     

Spatiotemporal expression of proprotein convertase subtilisin/kexin type 5 in the development of anorectal malformations in fetal rats

This study examined the expression patterns of proprotein convertase subtilisin/kexin type 5 (Pcsk5) during anorectal development in normal and anorectal malformations (ARM) rat embryos, determine the possible role of Pcsk5 in the pathogenesis of ARM. An ARM rat model was developed by the administration of ethylenethiourea gestational day 10 (GD10). Embryos were harvested by surgical excision from GD13 to GD16, and the spatiotemporal expression of Pcsk5 was evaluated, using immunohistochemistry staining, Western blotting and real time RT-PCR. Immunohistochemistry staining in normal embryos revealed that Pcsk5 was abundantly expressed on the epithelium of the cloaca (CL) on GD13. On GD14 and GD15, positive cells were noted on the urorectal septum and the thin anal membrane. However, the epithelium of the CL of ARM embryos only faintly expressed Pcsk5 from GD13 to GD15. Western blotting and real time RT-PCR showed time-dependent increase of Pcsk5 expression in the developing hindgut. Pcsk5 expression levels were lower in the ARM group from GD14 to GD16 (p?≤?0.05). These results indicate that downregulation of Pcsk5 during cloaca development into the rectum and urethra might be related to the formation of ARMs.     

Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-β-catenin signaling and cell death of the cloaca endoderm,and anorectal malformations

In mammalian urorectal development, the urorectal septum (urs) descends from the ventral body wall to the cloaca membrane (cm) to partition the cloaca into urogenital sinus and rectum. Defective urs growth results in human congenital anorectal malformations (ARMs), and their pathogenic mechanisms are unclear. Recent studies only focused on the importance of urs mesenchyme proliferation, which is induced by endoderm-derived Sonic Hedgehog (Shh). Here, we showed that the programmed cell death of the apical urs and proximal cm endoderm is particularly crucial for the growth of urs during septation. The apoptotic endoderm was closely associated with the tempo-spatial expression of Wnt inhibitory factor 1 (Wif1), which is an inhibitor of Wnt-β-catenin signaling. In Wif1^lacZ/lacZ mutant mice and cultured urorectum with exogenous Wif1, cloaca septation was defective with undescended urs and hypospadias-like phenotypes, and such septation defects were also observed in Shh^−/− mutants and in endodermal β-catenin gain-of-function (GOF) mutants. In addition, Wif1 and Shh were expressed in a complementary manner in the cloaca endoderm, and Wif1 was ectopically expressed in the urs and cm associated with excessive endodermal apoptosis and septation defects in Shh^−/− mutants. Furthermore, apoptotic cells were markedly reduced in the endodermal β-catenin GOF mutant embryos, which counteracted the inhibitory effects of Wif1. Taken altogether, these data suggest that regulated expression of Wif1 is critical for the growth of the urs during cloaca septation. Hence, Wif1 governs cell apoptosis of urs endoderm by repressing β-catenin signal, which may facilitate the protrusion of the underlying proliferating mesenchymal cells towards the cm for cloaca septation. Dysregulation of this endodermal Shh-Wif1-β-catenin signaling axis contributes to ARM pathogenesis.Urorectal development starts at gestational week-4 in humans and at E10.5 in mice, and is divided into three phases: genital tubercle (GT) outgrowth, cloaca septation and urethra tubularization. GT outgrowth is initiated with a paired genital swellings on either side of the cloaca (caudal end of the rectum), which fuse medially to form the future external genitalia. Cloaca septation is the partitioning of the cloaca by urorectal septum (urs) into the rectum and urogenital sinuses (ugs). Urorectal development is indistinguishable in male and female embryos before urethra tubularization. Defective urorectal development results in a number of congenital anomalies frequently accompany with deficient excretory and copulatory functions, greatly influencing the patients'' quality of life. In particular, the anorectal malformations (ARMs) are the most common urorectal defects and affect boys and girls with an incidence of 2–5 in 10 000 live births,^{1, 2} but its pathogenic mechanisms are poorly understood. ARM phenotypes are also partly included in several diseases, such as Currarino syndromes, Townes Brocks Syndromes and VACTERL complex, and these patients display other anomalies including anal fistula, sacral malformations and renal malformations.^{3, 4, 5, 6}During cloaca septation, the urs, an endoderm-lined mesenchyme, elongates from the ventral body wall to the cloaca membrane (cm) (Figure 1a). The proximal cm is regressing when the apical urs endoderm approximates to the cm leading to the formation of the urethral duct (ud) and the anal opening, which signifies the completion of cloaca septation (Figure 1b). After septation, the cm regression associated with the proximal-to-distal urs elongation coupled with fusion of the preputial fold mesoderm along the ventral midline of the tubercle leads to the formation of urethral opening at the distal end of the GT (Figure 1c).Open in a separate windowFigure 1Descent of the urorectal septum and regression of the cloaca membrane during cloaca septation. Schematic diagram of cloaca development showing the descent of urs and regression of proximal cm leads to anal opening after cloaca septation (a–c). Normal cloaca development in rat embryos from E14.5 to E16.5 (a′–c′). Rat treated with ethylenethiourea displayed persistent cloaca at E14.5 (d; n=12), hypospadias-like phenotypes with incomplete cloaca septation at E15.5 (e; n=15) and E16.5 (f); n=9). Scale bar: 50 μmDeletion of Shh, Wnt5a or Bmp7 in mouse embryos resulted in reduced proliferation of the urs mesenchyme, incomplete urs elongation and septation defects.^{7, 8, 9, 10} In addition, the cm was either disintegrated or remained intact in these null embryos, giving rise to hypospadias-like or persistent cloaca phenotypes (Figure 1). Nonetheless, deletion of these genes not only leads to a decrease in cell proliferation but also affect cell apoptosis.^{11, 12}Shh and Wnt signaling are indispensable for the septation process. Shh is expressed in the cloaca endoderm and mediates the proliferation of urs mesenchyme through Gli2. Shh and Gli2 mutants displayed persistent cloaca. However, deletion of Shh eradicated the cm, exposing the unseptated cloaca exteriorly, whereas the cm remained intact in the loss of Gli2.^{7, 13} Wnt5a is crucial for urorectal development and regulates the outgrowth of GT.^{8, 10} Studies suggest that Wnt-β-catenin signaling activity is tightly regulated in urorectal development.^{14, 15} Furthermore, β-catenin expression was downregulated in Shh mutants.¹⁶ Constitutive activated β-catenin in Shh null background can partially rescue defective development of the GT and the cm.^{11, 16} All these indicated that Wnt-β-catenin signaling functions downstream of Shh. How Wnt signaling is regulated during cloaca septation has not been investigated.Wif1 (Wnt inhibitory factor 1) is a secreted Wnt inhibitor, which exerts its inhibitory effect on Wnt signaling by binding directly to Wnt ligands and by preventing the ligands from binding to their cell surface receptors. The presence of Wif1 leads to β-catenin degradation, thereby turning off Wnt-β-catenin signaling.¹⁷ Most of the recent studies focused on the impact of epigenetic silencing of Wif1 in various carcinomas,¹⁸ and restoration of Wif1 activity in cancer cells induced apoptosis of malignant cancers.¹⁹ By contrast, reports on the regulatory functions of Wif1 in embryonic development are limited. Previous study suggested that Wif1 has high affinity to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11;²⁰ and Wif1 regulated chondrogenesis in cartilage-mesenchyme interfaces via the inhibition of Wnt3a-mediated mesenchyme growth in embryos.²¹ A recent study revealed that expression of Wif1 is androgen responsive in prostate bud formation. Loss of Wif1 in prostate glands induced ectopic expression of other secretory Wnt inhibitors to compensate for the loss of Wif1 activity in these mutants.²² Furthermore, Smad1 directly targets the Wif1 promoter and controls Wif1 gene expression in lung epithelial cell development.²³ Taken all these indicated that Wnt-β-catenin signaling is precisely regulated in a number of embryonic developmental processes, and Wif1 modulates the Wnt-β-catenin signaling activity.In this study, we discovered that the finely regulated expression of Wif1 is crucial for cloaca septation, and dysregulated Wif1 expression caused septation defects. Shh and Wif1 were expressed in a complementary manner at the cloaca endoderm, and deletion of Shh induced ectopic Wif1 expression, associated with excessive endoderm apoptosis and septation defects. Comparable septation defects were observed in Wif1^lacZ/lacZ mutant mice, in cultured urorectum with exogenous Wif1, in Shh^−/− mutants and in endodermal β-catenin GOF mutants. In conclusion, this study suggests that Wif1 regulates endodermal cell apoptosis by mediating and regulating Shh-Wnt-β-catenin signaling, thus having an essential role during urorectal development.     

Development of aortic and mitral valve continuity in the human embryonic heart

The anatomic relationship of the aortic and mitral valves is a useful landmark in assessing congenital heart malformations. The atrioventricular and semilunar valve regions originate in widely separated parts of the early embryonic heart tube, and the process by which the normal fibrous continuity between the aortic and mitral valves is acquired has not been clearly defined. The development of the aortic and mitral valve relationship was studied in normal human embryos in the Carnegie Embryological Collection, and specimens of Carnegie stages 13, 15, 17, 19, and 23, prepared as serial histologic sections cut in the sagittal plane, were selected for reconstruction. In stage 13, the atrioventricular valve area is separated from the semilunar valve area by the large bend between the atrioventricular and outflow-tract components of the single lumen heart tube created by the left interventricular sulcus. In stages 15 and 17, the aortic valve rotates into a position near the atrioventricular valves with development of four chambers and a double circulation. In stage 19, there is fusion of aortic and mitral endocardial cushion material along the endocardial surface of the interventricular flange, and this relationship is maintained in subsequent stages. Determination of three-dimensional Cartesian coordinates of the midpoints of valve positions shows that, while there is growth of intervalvular distances up to stage 17, the aortic to mitral distance is essentially unchanged thereafter. During the period studied, the left ventricle increases in length over threefold. The relative lack of growth in the saddle-shaped fold between the atrioventricular and outflow tract components of the heart, contrasting with the rapid growth of the outwardly convex components of most of the atrial and ventricular walls, may be attributed to the different mechanical properties of the two configurations. It is postulated that the pathogenesis of congenital heart malformations, which characteristically have failure of development of aortic and mitral valve continuity, may involve abnormalities of rotation of the aortic region or malpositioning of the fold in the heart tube.     

Liver development in rats during the embryonic period (Carnegie stages 11-14).

The hepatic structures appearing during Carnegie stages 11-14 were analyzed in a series of 61 OFA rat embryos. The group of embryos (crown-rump length 2-7 mm, 10th-12th days after coitus) was composed of 52 specimens of the somite period (stages 11 and 12) and of 9 specimens of the postsomite period (stages 13 and 14). The embryos were submitted to serial histological sectioning with graphic reconstruction. Stage 11 was characterized by the development of the hepatic diverticulum induced by differential growth of the endodermal plate and fixed contact between the entoderm and endothelium lining the heart. Stage 12 presented obvious signs of cellular differentiation, the septum transversum giving the liver stroma and the hepatic diverticulum the epithelial trabecula. At stage 13 the epithelial cords enmeshed the stromal capillaries, while hepatocardiac veins drained the hepatic flow into the sinus venosus. Stage 14 was recognized by the presence and development of the hepatic lobes and the enlargement of vascular channels. The cystic bud was never observed. The identification of these features permits to precise the hepatic developmental staging in rats and to obtain accurate criteria for the characterization of the end of the somite period and the beginning of the postsomite period.     

Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.     

An analysis of the age and size of 483 human embryos

The crown-rump length of 483 fixed human embryos of Carnegie stages 6-23 was analyzed and median and predicted mean lengths were calculated. The results were compared with those of other series and confirmed that early human growth rates are different from those of macaques with which human embryo growth has previously been compared. The study indicated that it is possible to predict: 1. the median size of an embryo of given Streeter horizon or Carnegie stage; 2. the age of a fresh embryo, or one of undisputed Streeter staging, by comparison with mean figures of other authorities; and 3. the corrected age of an embryo of known length or known Streeter staging or both in terms of postovulation age. Since teratogens may reduce the embryonic growth rate this information is relevant in the analysis of teratogenic factors in human development.     

Histological study of the cloacal region and associated structures in the hedgehog tenrec Echinops telfairi

The present investigation is based on several careful dissections and on extensive series of histological sections. It has led us to the conclusion that adult male and female Echinops telfairi are in the possession of a cloaca which represents a primitive feature among mammals. This cloaca is a small, bowl-shaped pouch at the ventro-posterior end of the body. Intestinal, genital and urinary tract open into this cloaca. The opening of the intestinal tract into the cloaca is regulated by a sphincter muscle. In the female the genital and the urinary tract open into the urogenital sinus, a subcompartment of the cloaca. The cloaca of the lesser hedgehog tenrec is lined by a multilayered, non-keratinized squamous epithelium without skin glands. In a small transitory zone between the cloaca and the outer skin the epithelium changes into the keratinized, multilayered squamous epithelium of the epidermis with eccrine and holocrine glands as well as hairs. In addition, there is a distinct circular cloacal sphincter muscle, built up by cross-striated skeletal muscle tissue.

In the terminal parts of intestinal, urinary and genital tracts of male animals the following glandular structures were observed: prostate gland, Cowper's glands and strongly pigmented seminal vesicles; in female animals: the urethral and the Bartholin glands. Both males and females, in addition, possess (a) a cloacal gland, the excretory ducts of which open into the cloaca and (b) a pericloacal gland which is located in the adipose tissue on both sides of the cloaca; it presumably also opens into the cloaca.     

Phenotypic variability in human embryonic holoprosencephaly in the Kyoto Collection

BACKGROUND: Holoprosencephaly (HPE) is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Although numerous postnatal cases have been reported, early phases of its pathogenesis are not well understood. We examined over 200 cases of HPE human embryos both grossly and histologically, and studied their phenotypic variability and stage-specific characteristics. METHODS: Among over 44,000 human embryos in the Kyoto Collection of Human Embryos, 221 embryos have been diagnosed as HPE. Their developmental stages ranged from Carnegie stage (CS) 13 to CS 23. They were examined grossly and were also serially sectioned for detailed histological analysis. RESULTS: HPE embryos after CS 18 were classified into complete (true) cyclopia, synophthalmia (partially fused eyes in a single eye fissure), closely apposed separate eyes (possible forerunners of ethmocephaly and cebocephaly), and milder HPE with median cleft lip (premaxillary agenesis). At CS 13-17, when facial morphogenesis is not completed, HPE embryos had some facial characteristics that are specific to these stages and different from those in older HPE embryos. The midline structures of the brain, including the pituitary gland, were lacking or seriously hypoplastic in HPE embryos. Complete cyclopia was found in two cases after CS 18 but none at earlier stages. CONCLUSIONS: The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.     

Defining the molecular pathologies in cloaca malformation: similarities between mouse and human

Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.KEY WORDS: Anorectal malformation, Cloaca, Patterning, Epithelial differentiation, Sonic hedgehog     

High prevalence of defective human embryos at the early postimplantation period

Thirty-seven cases of human embryos at the early postimplantation period were procured after induced abortion and examined histologically. Their developmental stages ranged from Carnegie stages 6 to 11, and their standard ages ranged from 14 to 24 days after fertilization. Five cases (13.5%) were grossly abnormal, and seven (18.9%) were degenerating partially or in toto. Gross abnormalities included distorted embryonic disc, disorganized neural groove or tube, and neural tube dysraphism. The high prevalence rate of defective embryos at the early postimplantation period supports the clinical finding that a substantial proportion of human conceptions are eliminated from an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated pathological embryos is uncertain and remains to be determined.     

Fourth ventricular floor in human embryos: scanning electron microscopic observations

The ultrastructural surface features of the normal fourth ventricular floor of seven human embryos ranging from Carnegie stage 14 to stage 19 (crown-rump length: 7.6-16.2 mm) were examined by using scanning electron microscopy (SEM). Low-power SEM views showed the median sulcus, sulcus limitans, and neuromeres, transient structures characteristic of the earlier embryonic period. High-power SEM observation revealed supraependymal cells (SE cells) and supraependymal fibers (SE fibers) which exhibited a characteristic localization, as well as generalized surface-membrane modifications such as microvilli and cilia. SE cells could be classified into two major groups. The type 1 SE cells seem to possess neuronal functions, as deduced from morphological similarities to their counterparts in adults and the specialized distribution closely related to neuromeres. The type 2 SE cell morphologically resembled the phagocytic SE cell described in related literature. SE fibers ran a course either rostrocaudally in the median sulcus or mediolaterally on the neuromeres, most frequently near the interneuromeric cleft; they made contact with type 1 SE cells and ependymal surface modifications and then penetrated the ependymal layer.     

The pathogenesis of hereditary congenital malformations of the anorectum in the pig

The pathogenesis of anorectal malformations was studied in 41 abnormal embryos, foetuses and newborn pigs descended from a herd in which this anomaly was caused by a hereditary trait. The principal development error was found to be situated in the cloacal plate, of which a dorsal part of variable size was missing. This defect impeded the normal migration of the dorsal cloaca and adjacent structures along the dorsal border of the plate to the body surface of the tailgroove. Correlated to the size of the cloacal plate defect diverse types of anorectal malformations may develop ranging from slight abnormalities such as anal stenosis and perineal or vulvar ectopic anus to more serious anomalies such as imperforate anus of low, intermediate and high types. In the cases with imperforate anus a communication with the urogenital system is always formed, although it may disappear later in some animals. This communication which represents a persistent cloaca, has to be considered as an ectopic anorectal canal according to its origin and structure. The abnormalities of the internal and external sphincter ani muscles and puborectal muscles are secondary although distinctly correlated to the malformations of the epithelial structures.     

Developmental plasticity of glandular trichomes into somatic embryogenesis in Tilia amurensis

BACKGROUND AND AIMS: In Tilia amurensis, two types of trichomes (hairy and glandular) develop from epidermal surfaces of cotyledons and hypocotyls of zygotic embryos soon after germination. Here, it is demonstrated that glandular trichome initials develop directly into somatic embryos when treated in vitro with 2,4-dichlorophenoxyacetic acid (2,4-D). METHODS: Zygotic embryos of Tilia amurensis were cultured on Murashige and Skoog medium with 3 % sucrose and various concentrations (0, 2.2, 4.4 and 8.8 microm) of 2,4-D. Morphological development of trichomes and somatic embryos was analysed by scanning electron microscope and light microscope after histological sectioning. KEY RESULTS: In zygotic embryos cultured on medium with 4.4 microM 2,4-D, formation of hairy trichomes was completely suppressed but formation of glandular trichome initials increased. That some filamentous trichome initials developed directly into somatic embryos was confirmed by histological and scanning electron microscope observation. When explants with different stages of trichome initials (two-, four- and eight-celled filamentous and fully mature trichomes) were temporally pre-treated with 4.4 microM 2,4-D for 24 h and transferred into hormone-free medium, two-celled and four-celled filamentous trichome initials were the effective stage of trichomes for somatic embryo induction. CONCLUSIONS: It is suggested that early developing filamentous trichome initials have developmental plasticity and that with 2,4-D treatment these trichome initials develop directly into somatic embryos.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.     

富钟瘰螈和广西瘰螈泄殖系统研究

用解剖学技术研究和比较了富钟瘰螈Paramesotriton fuzhongensis和广西瘰螈Paramesotriton guangxiensis的泄殖系统。结果显示两种瘰螈的泄殖系统基本相同,仅生殖腺在颜色和形状上稍有差异。它们具有后位肾,雄螈肾脏由副睾肾和尾肾组成。但雄螈尾肾发出的多条集尿管并不通往后位肾管而是向后汇合成副尿管通往泄殖腔。后位肾管主要起输精作用,而副尿管则负责输尿。结果揭示雄性泄殖管道有功能分离的趋势,这在两栖类中处于较高进化水平。