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.     

Histogenesis in 11-day mouse embryo limb buds explanted in organ culture

Fore- and hindlimb buds from 11-day mouse embryos with 40 to 52 somites (including the four occipital pairs) were explanted in organ culture and submitted to systematic histological analysis. Chondrogenesis occurs normally in culture in all preskeletal rudiments which were already represented by condensed blastemas before explantation. In the proximal territories, the progress of cartilage differentiation occurs according to the normal pattern and can be revealed histologically much earlier than in bulk preparations. In all explanted hindlimbs as well as in forelimbs from embryos with less than 50 somites, a primary coalescence occurs between the IId and IIId digital rays, leading to various fusions from soft tissue syndactyly to oligosyndactyly. This is the result of two combined unfavorable effects of the culture conditions: the lack of simultaneous volumetric growth of the foot- or handplate, which normally would provide the necessary space for the laying down of a pentadactylous pattern, and a loss of cells resulting from abnormal cell death affecting selective mesodermal sites in the zeugopod and in the marginal subridge area, the latter being more severely affected in hindlimb buds. Several observations suggest that the preferential sensitivity of the marginal mesoderm might be related to early changes in the apical ectoderm, which itself becomes excessively necrotic and rapidly looses its pseudostratified configuration. The forelimb buds from embryos with 50 somites and more usually develop a pentadactylous pattern with a better individuation of digital structures. In all explants, the prospective mesoderm of digit I exhibits stronger regulatory tendencies.     

Regulation and Function of SF/HGF during Migration of Limb Muscle Precursor Cells in Chicken

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.     

Isolation of the avian homologue of the homeobox gene Mox2 and analysis of its expression pattern in developing somites and limbs

We have isolated the cDNA of avian Mox2 and analyzed its expression pattern during somitogenesis and limb bud formation. Mox2 plays an important role in limb muscle differentiation in the mouse. Mox2 is expressed in the somites of developing chick embryos and in presumptive migrating myoblasts from the dermomyotome to the limb buds. It is also expressed in the ventral and dorsal part of limb buds and is associated with non-proliferating myoblasts. Significant differences were observed in chick and mouse expression patterns, namely in the chick dermomyotome and limb.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

Difference in the expression of asymmetric acetylcholinesterase molecular forms during myogenesis in early avian dermomyotomes and limb buds in ovo and in vitro

The A12 (asymmetric) form of acetylcholinesterase (AChE) is generally considered to be synthesized in leg muscle tissues by myotubes under neural influence, but not by myoblasts. We have examined the expression of the different molecular forms of AChE in explants of developing limb buds and dermomyotomes (the myogenic part of the somites) obtained from 3-day-old chick and quail embryos, either directly after removal or during in vitro culture. We describe a muscular differentiation of both territories in vitro, leading to the formation of myotubes which are morphologically similar to the class of early muscle cells described by Bonner and Hauschka (1974). In vivo the A12 form is present in quail dermomyotomes which are almost entirely composed of mononucleated poorly differentiated cells; in contrast, it is absent from similar cells in chick dermomyotomes and from limb buds in both species. This shows that in the case of quail embryos the appearance of the A12 form precedes the fusion of myoblasts into myotubes. In both species, dermomyotome explants express asymmetric and globular forms of the enzyme during muscular differentiation in vitro, whereas limb buds synthesize only globular forms. After surgical removal of neural tube and/or neural crest at 2 days in ovo, the biosynthesis of the A forms in quail dermomyotomes is not suppressed and is consequently not dependent upon prior connection of the dermomyotomes to central neurons or upon the presence of autonomic precursors. Since limb bud muscle cells derive from somites our results raise questions concerning the differentiation of migrating somitic cells in this territory where a neural influence appears necessary to induce the biosynthesis of asymmetric AChE forms.     

Developmental origins of precocial forelimbs in marsupial neonates

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.     

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.     

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo

We have examined the expression pattern of the avian Meox1 homeobox gene during early development and up to late limb bud stages. Its expression pattern indicates that it is involved in somite specification and differentiation. The domains of expression are similar but different to those of Meox2. Meox1 is expressed from stage 6 in the pre-somitic mesoderm and as development proceeds, in the tail bud, the dermomyotome of the rostral somites and in the dermomyotome and sclerotome of the caudal somites, the lateral rectus muscle, truncus arteriosus of the heart and the limb buds. Unlike Meox1, Meox2 is not expressed in the pre-somitic mesoderm, but is expressed first in somites formed from stage 11 onwards. In the developing limb, both genes are expressed in the dorsal and ventral limb mesoderm in adjacent domains with a small region of overlap. In the limb bud, Meox1 is co-expressed with Meox2 but neither Meox gene is co-expressed with MyoD. These expression patterns suggest that these two genes have overlapping and distinct functions in development.     

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.     

Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2

Limb skeletal muscle is derived from cells of the dermomyotome that detach and migrate into the limb buds to form separate dorsal and ventral myogenic precursor domains. Myogenic precursor cell migration is dependent on limb bud mesenchymal expression of hepatocyte growth factor/scatter factor (Hgf), which encodes a secreted ligand that signals to dermomyotome through the membrane receptor tyrosine kinase Met. Here, we find that correct patterning of Hgf expression in forelimb buds is dependent on retinoic acid (RA) synthesized by retinaldehyde dehydrogenase 2 (Raldh2) expressed proximally. Raldh2(-/-) forelimb buds lack RA and display an anteroproximal shift in expression of Hgf such that its normally separate dorsal and ventral expression domains are joined into a single anterior-proximal domain. Met and MyoD are expressed in this abnormal domain, indicating that myogenic cell migration and differentiation are occurring in the absence of RA, but in an abnormal location. An RA-reporter transgene revealed that RA signaling in the forelimb bud normally exists in a gradient across the proximodistal axis, but uniformly across the anteroposterior axis, with all proximal limb bud cells exhibiting activity. Expression of Bmp4, an inhibitor of Hgf expression, is increased and shifted anteroproximally in Raldh2(-/-) limb buds, thus encroaching into the normal expression domain of Hgf. Our studies suggest that RA signaling provides proximodistal information for limb buds that counterbalances Bmp signaling, which in turn helps mediate proximodistal and anteroposterior patterning of Hgf expression to correctly direct migration of Met-expressing myogenic precursor cells.