Developmental Changes of Cell Process Meshwork and Extracellular Space in Subepidermal Mesenchyme in Mouse Forelimb Buds

This study was designed to reveal morphological changes in epithelial-mesenchymal interface during limb development. An electron microscopic and morphometric analysis was done on the cell process meshwork (CPM) and subepidermal extracellular space (SEECS) in mesenchyme in mouse forelimb buds at embryonic ages from day 9.5 to 12.5 (vaginal plug=day 0). At days 9.5 and 10.0, when the apical ectodermal ridge (AER) had not yet appeared, no spatial differences were noted in the CPM density and SEECS width. However, at days 10.5, 11.0 and 11.5, when the AER was distinct, the SEECS was wide and the number of mesenchymal cell processes was small beneath the AER as compared with those at days 9.5 and 10.0; they differed significantly from those beneath the dorsal or ventral non-ridge epidermis. Between days 10.0 and 10.5, the SEECS width decreased and the number of cell processes increased in the dorsal region. At days 12.0 and 12.5, when the AER was regressing, the spatial differences in the CPM density and the SEECS width became less obvious. These findings indicate that spatial and temporal differences of the SEECS width and CPM density exist in mouse limb buds, and these differences are closely associated with the AER development.     

Histological changes induced in developing limb buds of C57BL mouse embryos submitted in utero to the combined influence of acetazolamide and cadmium sulphate

Microscopic defects in limb buds of C57BL mouse embryos after the combined teratogenic action of acetazolamide plus cadmium sulphate administered on day 9 of gestation were studied in serial sections. Postaxial deficiencies observed in 12-15-day embryos and affecting preferentially the right forelimbs were classified in nine morphological types according to increasing amounts of missing parts. Type X defect consists of a nearly complete amelia in which all four limbs are represented only by the girdle and proximal end of the stylopod. Type XI abnormality appears as an intermediate reduction affecting the area of digit IV. In addition to modifications of the forelimb bud shape detected from the 10-day stage onwards, observations made 24 and 48 hr after treatment confirmed that the postaxial defects result from an absolute lack of postaxial mesoderm occurring without cell necrosis as a consequence of a postaxial shortening of the apical ectodermal ridge (aer). In 10-day embryos, the latter appears shortened and hypertrophied; it is later fragmented into alternate thick and thin portions in 11-day affected limb buds. These ectodermal changes might account for the genesis of all types of defects observed. Untreated 9-day embryos with 12-25 pairs of somites display a number of asymmetries between their right and left forelimb territories: Until the 19-somite stage, the vascular supply to that area is provided exclusively by the umbilical vein, which is larger on the right side; the initial amount of somatopleural limb mesoderm is greater in the right rudiment and the genesis of its aer is slightly protracted as compared to the left one. These asymmetries might contribute to the right side predominance of the forelimb defects induced by acetazolamide and cadmium.     

Retinoic-acid-induced limb malformations resulting from apical ectodermal ridge cell death

Pregnant C57Bl/6J mice were treated with single oral doses of 400 mg/kg 13-cis retinoic acid (RA, isotretinoin, Accutane) in sesame oil at 9 days, 12 hours postfertilization. Among the live 16-day fetuses from ten treated mothers, 46% (26/56) had limb malformations including small fifth digits, preaxial and/or postaxial oligodactyly, and preaxial or postaxial polydactyly. Fetuses with preaxial digit deficiencies also had absent or malformed radii. Scanning electron microscopic and light microscopic analyses of the sequence of developmental alterations leading to these malformations demonstrated abnormalities in the apical ectodermal ridge (AER). Excessive cell death in the AER of 27-30 somite embryos (12 hours after treatment) appears to play a major role in the pathogenesis of the limb malformations observed. Previous investigations of retinoid-induced limb malformations have concentrated on later exposure times. Evidence from this study in addition to that from previous teratologic and clinical investigations has led to the hypothesis that 13-cis RA results in excessive cell death in regions of programmed cell death and subsequent malformations of affected regions.     

The Embryonic Limb Bud of the Urodele:

Scanning electron microscopy and histological investigation of the embryonic fore-limb bud of Notophthalamus (Triturus) cristatus and N. vulgaris has revealed that an apical ectodermal ridge (AER) is not detectable in these species of newt (Urodela). The limb buds examined ranged from those just visible as a slight projection from the body surface through to buds possessing two prominent digits. These stages correspond to the stages in Anurans during which the AER first appears, reaches its maximum size and then regresses. The significance of this observation is discussed.     

Acetazolamide: maternal toxicity, pattern of malformations, and litter effect

Thirty litters of C57BL 6J mice were administered intraperitoneally one of four doses (0, 500, 750, and 1,000 mg/kg maternal weight) of acetazolamide on day 9 of gestation. The fetuses were removed on day 18 and fixed, stained, cleared, and examined for the pattern of malformations. The forelimb postaxial limb deficiency was the most common abnormality, but forelimb postaxial polydactyly and a postaxial deficiency in the hindlimb were also observed. Males were significantly more likely to be malformed than females at all doses, in contrast to the predominance of females observed in rat fetuses exposed to acetazolamide (Scott et al.: Teratology 6:239-240, '73). The occurrence of limb malformations did not correlate with maternal weight loss, the birth weight of the fetus, or the position of the fetus in the uterus. A "litter effect" was demonstrated in that there was a nonuniform distribution of litters with different proportions of malformed fetuses.     

A Scanning Electron Microscopic Observations of the Leaves in Some Conifers

Scanning electron microscopic observations of the leaves in some conifers are described. These are hyplocheilie stomata surrounded by different morphological subsidiary cells. In Keteleeria, the lateral wall of larger subsidiary cells possesses prom.inant eutieular globoides. Cuticular semigloboides between the sinuoms epidermal anticlinal walls are most obvious in other epidermis. However the single laver of cushion cells which looated underneath the guard ceils of the abaxial epidermis in Cathaya is discovered. The mesophyll ceils with incurved walls of Cathaya consist mainly of single layer of flat oubical cells flat longi-cubieal cells as of the polytriehaeeous lamcllae, thus it may be designated as mesolamellae. It not only supports the establishment of Cathaya Chun and Kuang as a new genus of the Pinaeeae but also may be another important biosystematical evidence between Cathaya and Pinus. The distribution and polymorphism of foliar solereids in Amentotaxus which are different from leaves of other gymnosperms described. The transfusion tracheids are also different in some ways from other genera.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.     

Gestational ethanol exposure disrupts the expression of FGF8 and Sonic hedgehog during limb patterning

BACKGROUND: Ethanol is known to induce a wide variety of gestational anomalies, including skeletal malformations. Gestational ethanol exposure in mice has been shown to induce postaxial digit loss (ectrodactyly). How ethanol induces limb malformations is not understood. To better understand how ethanol effects limb development, we have utilized a transgenic line of mice that expresses beta-galactosidase in the apical ectodermal ridge (AER) of the limbs throughout gestation. METHODS: Pregnant female mice were injected with 2.9, 3.4, or 3.9 gm/kg ethanol at E9.3 and E9.5; embryos were isolated at E11.25, stained for beta-galactosidase activity, and evaluated for AER defects. Based upon the pattern of defects seen, expression of FGF8 in the AER and Sonic hedgehog in the postaxial mesoderm was evaluated by in situ hybridization. RESULTS: Two distinct phenotypes were seen in response to ethanol that were dose dependent. At 2.9 gm/kg ethanol, the most prevalent phenotype was a mislocalization of the AER to regions both dorsal and ventral to the midline. A higher dosage of 3.4 gm/kg ethanol did not increase the mislocalization phenotype, but resulted in a higher frequency of postaxial loss of the AER and associated mesenchymal tissue. The highest dosage utilized (3.9 gm/kg) resulted in a high frequency of both preaxial and postaxial loss of the AER. Through in situ hybridization, we found that ethanol exposure resulted in a concomitant reduction in FGF8 expression in the AER and Sonic hedgehog expression from the zone of polarizing activity (ZPA). CONCLUSIONS: We propose a model where ethanol disrupts the AER/ZPA positive feedback loop to induce postaxial malformations. Preaxial malformations seen at higher ethanol dosage suggest FGF8 as a critical target of ethanol in producing limb defects.     

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.     

The effect of administration time on malformations induced by three anticonvulsant agents in C57BL/6J mice with emphasis on forelimb ectrodactyly.

Exposure of C57BL/6J mice to three anticonvulsant derivatives, namely, dimethadione, sodium valproate, and sodium diphenylhydantoin, each induced postaxial forelimb ectrodactyly. The agents were administered at gestational days 9, 9 1/3, 9 2/3, and 10. It was determined that administration at day 9 2/3 induced the highest percentage of forelimb ectrodactyly for each of the three agents. The forelimb ectrodactyly response in the C57BL/6J strain was compared with the A/J strain (Collins et al., Teratology, 41:61-70, 1990); it was found that the C57BL/6J strain was more sensitive to dimethadione and the A/J strain was more sensitive to diphenylhydantoin and sodium valproate. The position of vertebral defects induced by sodium valproate correlated with the time of drug administration. The overall syndrome of malformations induced by the three anticonvulsant agents was relatively similar in the two mouse strains and differed between each of the anticonvulsant agents.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Microarray analysis of murine limb bud ectoderm and mesoderm after exposure to cadmium or acetazolamide

BACKGROUND: A variety of drugs, environmental chemicals, and physical agents induce a common limb malformation in the offspring of pregnant mice exposed on day 9 of gestation. This malformation, postaxial, right‐sided forelimb ectrodactyly, is thought to arise via an alteration of hedgehog signaling. METHODS: We have studied two of these teratogens, acetazolamide and cadmium, using the technique of microarray analysis of limb bud ectoderm and mesoderm to search for changes in gene expression that could indicate a common pathway to postaxial limb reduction. RESULTS: Results indicated a generalized up‐regulation of gene expression after exposure to acetazolamide but a generalized down‐regulation due to cadmium exposure. An intriguing observation was a cadmium‐induced reduction of Mt1 and Mt2 expression in the limb bud mesoderm indicating a lowering of embryonic zinc. CONCLUSIONS: We propose that these two teratogens and others (valproic acid and ethanol) lower sonic hedgehog signaling by perturbation of zinc function in the sonic hedgehog protein. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Aplasia of tibia with split-hand/split-foot deformity. Report of six families with 35 cases and consideration about variability and penetrance

Summary Six families with a total of 34 affected persons with the syndrome of tibial aplasia and ectrodactyly are reported. The spectrum of malformations is compared to that of 99 familial cases from the literature. The full-blown syndrome consists of bilateral aplasia of tibiae and split-hand/split-foot deformity. Additional malformations may be distal hypoplasia or bifurcation of femora, hypo- or aplasia of ulnae, and minor anomalies such as aplasia of patellae, hypoplastic big toes, postaxial and intermediate polydactyly in connection with split-hand deformity, and cup-shaped ears. The mildest visible manifestation may be hypoplastic big toes, the severest is tetramonodactyly or transverse hemimelia. This disorder is autosomal dominantly inherited. The penetrance is markedly reduced.     

Contribution to the morphometry of limb bud structures in the normodactylous and polydactylous rat. II. Density of filopodia in the subectodermal zone

In hindlimb buds of normodactylous and polydactylous embryos in the stage of the 16th and 17th embryonal day the mesenchymal region, closely adjoining the site below the AER, was investigated. This space is called the subridge zone and is filled with a large amount of variously formed processes of mesenchymal cells, chiefly with filopodia. With the use of the morphometric point-couting method it was found that in normodactylous limb buds the density of filopodia in the given area of the subridge zone was 2.15% as compared with the 6.48% representation of filopodia in the same zone of polydactylous animals. Numerous filopodia localized right under the AER established connection between the mesenchyma and the intact basal membrane, and their higher density is undoubtedly related to the existence of the maintenance factor.     

Analysis of limb anomalies induced in vitro by vitamin A (retinol) in mice.

Forelimb buds of day 11 ICR-JCL mouse embryos were cultured on liquid medium consisting of 90% Eagle's MEM and 10% fetal calf serum. Experimental medium contained 10 iu/ml vitamin A alcohol (retinol). In controls four of five metacarpals chondrified and the epidermis began to keratinize after 3-4 days of culturing. In experimental explants many pycnotic cells were observed in the peripheral mesenchyme in the hand plate, especially in pre- and postaxial regions. Only 2-3 metacarpals chondrified, and keratinization was inhibited in the experimental explants. Uptake of labeled sulfate was suppresssed by vitamin A. Excess vitamin A is thus thought (1) to act directly on limb buds, and thereby to induce limb anomalies, (2) to induce cell death in the mesenchyme, (3) to suppres the formation of chondroitin sulfate, and (4) to inhibit keratinization.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Cadmium induced limb defects in mice: strain associated differences in sensitivity.

Cadmium (CdSO4) was given ip on day 9 at 12 or 24 mumol/kg to pregnant CD-1 (non-inbred) mice. Fetuses showed malformations of the limbs, face, trunk, and tail. There was a statistically significant relationship between the dose of cadmium and the malformation rate. Cadmium (12 mumol/kg ip on day 9) was then given to mice of six inbred strains three of which (A/J, BALB/cJ, and C57BL6J) carry a gene cdm for resistance to cadmium-induced testicular damage, and three strains (AKR/J, CBA/J, and DBA/2J) which do not. Paradoxically, the three strains resistant to cadmium induced testicular damage were significantly more sensitive to its teratogenic effects than were the other three strains. In all inbred strains most malformations involved the limbs. All forelimb defects found in inbred or non-inbred cadmium treated mice were postaxial and indistinguishable from those produced by acetazolamide in mice. The remarkable similarity of the cadmium- and acetazolamide-induced forelimb malformations may be a reflection of the limited number of ways that a rodent forelimb can react to a teratogenic insult. The hindlimb defects were all preaxial.     

Congenital kyphoscoliosis and spinal cord lesion produced in the rat by beta-aminopropionitrile

Pregnant rats received the lathyrogen beta-aminopropionitrile (1,500 mg/kg) intraperitoneally on day 16 (plug day = 0 day). Kyphoscoliosis was produced in a high incidence in the fetuses at the level of the upper thoracic spine as early as 24 hours after treatment. Although most of the affected newborns died within two weeks, survivors were studied until 20 weeks after birth. Survivors developed paraplegia in consequence of kyphoscoliosis. Both spinal deformity and motor disturbance were progressive. Biochemical and electron microscopic observations suggested that beta-aminopropionitrile treatment resulted in an inhibition of collagen formation in the spinal column and surrounding longitudinal ligaments of the fetuses six hours after the treatment. In addition, electron micrographs of vertebral bodies showed a decrease of proteoglycan granules in the extracellular matrix. Therefore, rupture and collapse of weakened ligaments and vertebral bodies might result in severe spinal deformity and spinal cord lesion.     

Genetic differences in the frequency of acetazolamide-induced ectrodactyly in the mouse exhibit directional dominance of relative embryonic resistance

Eleven of the common inbred strains of the mouse were surveyed for their teratogenic response to acetazolamide that was administered three times per os at 1,000 mg/kg (9 A.M. and 4 P.M. on day 9 and 9 A.M. on day 10). The products of conception were examined for gross malformations on day 15. One strain, SJL/J, exhibited maternal toxicity to the dosage regime and was excluded from the survey. Five strains exhibited significantly increased resorption rates after treatment. All strains responded with the expected malformation of postaxial forelimb ectrodactyly with a right-sided predominance. Nine of the strains could be assigned to one of four mutually exclusive classes of frequency of ectrodactyly and the tenth strain (BALB/cByJ) showed overlap between the two intermediate classes. The data suggest major genes determine the difference in sensitivity to ectrodactyly rather than a polygenic mode of inheritance. Induced cleft lip was found in four strains and one of these strains, SWR/J, exhibited a significantly higher frequency. The strain differences in sensitivity to induced resorption, forelimb ectrodactyly, and cleft lip were genetically independent. A reciprocal cross study was conducted with five of the strains from the four classes of frequency of ectrodactyly response in order to determine gene action. A significant maternal effect on the ectrodactyly response was found only with one of the strain pairs in the ten sets of reciprocal crosses with the five strains. When there was a significant difference between two strains, the F1 embryos exhibited dominance of relative resistance to ectrodactyly. The directional dominance of relative resistance to acetazolamide-induced ectrodactyly suggests that regulatory genes control the embryonic differences in frequency of ectrodactyly response to acetazolamide. By analogy with other metric traits of development that exhibit directional dominance, the genetic variation in ectrodactyly response that has been observed so far in the mouse embryo may not be involved with the primary target of acetazolamide teratogenesis.