Premature regression of the leg apical ectodermal ridge in the Japanese chick wingless mutant

The autosomal dominant Japanese wingless mutant has varying degrees of wing and leg truncations. The wing defects range from complete loss to negligible defects, whereas leg abnormalities are usually restricted to loss of the phalanges. Further analyses of the mutant focusing on the leg, which has been relatively uncharacterized, were performed. The expression pattern of Fgf8, a marker gene for the apical ectodermal ridge (AER) that controls outgrowth of the limbs, revealed premature regression at stage 28. Electron microscopy study showed abnormalities in the basement membrane all through the AER in the same stage. In the mutant, cell death was observed in the mesenchyme underlying AER between stages 31 and 32, although in the wild-type leg, AER regression and cell death occurred almost simultaneously at stages 33–34. To know if the cell death and cessation of the outgrowth are common mechanisms of wild-type and the mutant, we removed the AER in wild-type embryos at stage 28 and followed the fate of the limb. This also resulted in premature cell death 48 h after AER removal (equivalent to stage 32) and limb truncations similar to those observed in mutant limbs. To confirm whether either AER or underlying mesenchyme is responsible for the truncation, transplantation of the AER between the wild-type and the mutant was performed. This revealed that AER is the defective tissue in this mutant.     

Retinoic acid is essential for Shh/Hoxd signaling during rat limb outgrowth but not for limb initiation.

Retinoids long have been implicated in limb development and their endogenous contributions to this process are finally being elucidated. Here we use an established model of retinoid depletion during specific gestational windows to investigate the role of endogenous retinoic acid (RA) in supporting limb outgrowth. Rat embryos were deprived of RA starting at days-postcoitum (dpc) 3.0, 5.5, or 7.0 and harvested at the 35-somite stage (dpc 12-12.5). Although embryos from all these windows possessed many characteristics of gestational retinoid deficiency (frontonasal hypoplasia, straight tail, reduced CRBPI and RAR beta), their limb buds emerged with only modest size reductions. Molecular analysis of RA-deficient limb buds revealed enhanced gli-3 and reduced hoxd-12, hoxd-13, shh, and fgf-4, while fgf-8, en-1, and wnt-7a expression remained unaltered. Occasional posterior truncations were observed at low incidence in the longest deficiency window; otherwise, the deficiency window length had no discernable impact on the severity of these changes. At the 45-somite stage, RA-deficient limbs had additional losses of hoxd-13 and fgf-8, accompanied by a flattened AER, suggestive of an ultimate failure in limb bud outgrowth. Results could not confirm a function for endogenous retinoids in limb initiation, but show they are required to maintain the signaling loops between the developing mesenchyme and AER that govern limb outgrowth after the initial emergence of limb bud.     

Antero-posterior skeletal patterning is not dependent on continuity of the apical ectodermal ridge in the chick wing bud

The mechanism of antero-posterior specification of limb skeletal pattern is still controversial. If, as proposed by the ZPA model, a diffusible morphogen does exist, its route of passage across the limb field has not been resolved. To investigate the contribution of the apical ectodermal ridge (AER) to the control of anteroposterior pattern formation, we examined the consequences of small wounds made to the AER. The wound response was investigated by means of resin histology and scanning electron microscopy; subsequent limb development and cartilage pattern were examined in wholemount preparations. Although regrowth of the bilaminate dorsal and ventral ectoderm over the wound occurred within 15 h, the more highly differentiated pseudostratified columnar epithelium of the AER did not reform, and there was subsequent retardation of limb outgrowth at the wound site. At 10–11 days of development, the appearance of the limbs allowed them to be placed into one of three categories; presence of supernumerary elements, accentuation of an inter-digital cleft, or normal. The first of these categories included limbs in which digit 3 had bifurcated such that the sum of the parts of the resultant digital skeleton was greater than that which forms in a normal limb. Since in all of the experimental limbs all skeletal elements were present, we propose that continuity of the AER is not a pre-requisite for anteroposterior skeletal pattern formation in the chick wing.     

Notch1 signals through Jagged2 to regulate apoptosis in the apical ectodermal ridge of the developing limb bud.

The Notch family of receptors is involved in a wide variety of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during cell differentiation and tissue morphogenesis. Notch1 and Notch ligands are expressed in the developing limbs, and Notch signalling has been implicated in the formation of a variety of tissues that comprise the limb, such as the skeleton, musculature, and vasculature. Notch signalling has also been implicated in regulating overall limb size. We have used a conditional allele of Notch1 in combination with two different Cre transgenic lines to delete Notch1 function either in the limb mesenchyme or in the apical ectodermal ridge (AER) and limb ectoderm. We demonstrate that Notch signalling, involving Notch1 and Jagged2, is required to regulate the number of Fgf8-expressing cells that comprise the AER and that regulation of the levels of fibroblast growth factor signalling is important for the freeing of the digits during normal limb formation. Regulation of the extent of the AER is achieved by Notch signalling positively regulating apoptosis in the AER. We also demonstrate that Notch1 is not required for proper formation of all the derivatives of the limb mesenchyme.     

Ectodermal Wnt3/beta-catenin signaling is required for the establishment and maintenance of the apical ectodermal ridge

The formation of the apical ectodermal ridge (AER) is critical for the distal outgrowth and patterning of the vertebrate limb. Recent work in the chick has demonstrated that interplay between the Wnt and Fgf signaling pathways is essential in the limb mesenchyme and ectoderm in the establishment and perhaps the maintenance of the AER. In the mouse, whereas a role for Fgfs for AER establishment and function has been clearly demonstrated, the role of Wnt/beta-catenin signaling, although known to be important, is obscure. In this study, we demonstrate that Wnt3, which is expressed ubiquitously throughout the limb ectoderm, is essential for normal limb development and plays a critical role in the establishment of the AER. We also show that the conditional removal of beta-catenin in the ventral ectodermal cells is sufficient to elicit the mutant limb phenotype. In addition, removing beta-catenin after the induction of the ridge results in the disappearance of the AER, demonstrating the requirement for continued beta-catenin signaling for the maintenance of this structure. Finally, we demonstrate that Wnt/beta-catenin signaling lies upstream of the Bmp signaling pathway in establishment of the AER and regulation of the dorsoventral polarity of the limb.     

Upper beak truncation in chicken embryos with the cleft primary palate mutation is due to an epithelial defect in the frontonasal mass.

In this study, we used the chicken mutant strain known as cleft primary palate (cpp) to study the mechanisms of beak outgrowth. cpp mutants have complete truncation of the upper beak with normal development of the lower beak. Based on structural analysis and grafts of facial prominences, we localized the defect to the frontonasal mass and its derivatives. Several explanations that would account for the outgrowth defect were investigated, including abnormal expression of genes in the frontonasal epithelium, intrinsic defects in epithelium and/or mesenchyme defects in epithelial-mesenchymal signalling, a localized decrease in cell proliferation or a localized increase in programmed cell death. One of the genes expressed in the frontonasal epithelial growth zone, Fgf8, failed to down-regulate and was maintained for at least 48 hr beyond the time when down-regulation normally occurs. Recombination experiments further illustrated that the frontonasal mass epithelium was abnormal in the cpp mutants, whereas mutant mesenchyme was capable of normal outgrowth when combined with wild-type epithelium. Cell proliferation was not decreased in mutant embryos nor was cell death initially increased. Later, at stages 31-32, when the prenasal cartilage begins directed outgrowth, there was an increase in cell death within the mutant upper but not lower beak cartilage. The cpp beak truncation, therefore, is due to an epithelial defect in the frontonasal mass that is coincident with a failure to down-regulate expression of Fgf8.     

In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic chick leg bud

The formation of the digits in amniote vertebrates is accompanied by a massive degeneration process that accounts for the disappearance of the interdigital mesenchyme. The establishment of these areas of interdigital cell death (INZs) is concomitant with the flattening of the apical ectodermal ridge (AER), but a possible causal relationship between these processes has not been demonstrated. Recent studies have shown that the function of the AER can be substituted for by implantation of beads bearing either FGF-2 or FGF-4 into the apical mesoderm of the early limb bud. According to these observations, if the onset of INZs is triggered by the cessation of the AER function, local administration of FGFs to the interdigital tissue prior to cell death should delay or inhibit interdigit degeneration. In the present study we have confirmed this prediction. Implanting Affi-gel blue or heparin beads pre-absorbed with either FGF-2 or FGF-4 into the interdigital tissue of the chick leg bud in the stages prior to cell death stimulates cell proliferation and causes the formation of webbed digits. Vital staining with neutral red confirmed an intense temporal inhibition of interdigital cell death after FGF treatment. This inhibition of interdigital cell death was not accompanied by modifications in the pattern of expression of Msx-1 or Msx-2 genes, which in normal development display a domain of expression in the interdigital tissue preceding the onset of degeneration.     

The Divergent Development of the Apical Ectodermal Ridge in the Marsupial Monodelphis domestica

Marsupials give birth after short gestation times to neonates that have an intriguing combination of precocial and altricial features, based on their functional necessity after birth. Perhaps most noticeably, marsupial newborns have highly developed forelimbs, which provide the propulsion necessary for the newborn's crawl to the teat. To achieve their advanced state at birth, the development of marsupial forelimbs is accelerated. The development of the newborn's hind limb, which plays no part in the crawl, is not accelerated, and is likely even delayed. Given the large differences in the rate of limb outgrowth among marsupials and placentals, we hypothesize that the pathways underlying the early development and outgrowth of marsupial limbs, especially that of their forelimbs, will also be divergent. As a first step toward testing this, we examine the development of one of the two major signaling centers of the developing limb, the apical ectodermal ridge (AER), in a marsupial, Monodelphis domestica. We found that, while both opossum limbs have reduced physical AER's, in the opossum forelimb this reduction has been taken to the extreme. Where the M. domestica forelimb should have an AER, it instead has only a few patches of disorganized cells. These results make the marsupial, M. domestica, the only known amniote (without reduced limbs) to exhibit no morphological AER. However, both M. domestica limbs normally express Fgf8, a molecular marker of the AER. Anat Rec 293:1325–1332, 2010. © 2010 Wiley‐Liss, Inc.     

Apical epithelial cap morphology and fibronectin gene expression in regenerating axolotl limbs.

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate limbs. The regenerated structure is often indistinguishable from the developmentally produced original. Thus, the two processes by which the limb is produced - development and regeneration - are likely to use many conserved biochemical and developmental pathways. Some of these limb features are also likely to be conserved across vertebrate families. The apical ectodermal ridge (AER) of the developing amniote limb and the larger apical epithelial cap (AEC) of the regenerating urodele limb are both found at the limb's distalmost tip and have been suggested to be functionally similar even though their morphology is quite different. Both structures are necessary for limb outgrowth. However, the AEC is uniformly smooth and thickly covers the entire limb-tip, unlike the AER, which is a protruding ridge covering only the dorsoventral boundary. Previous data from our laboratory suggest the multilayered AEC may be subdivided into separate functional compartments. We used hematoxylin and eosin (H+E) staining as well as in situ hybridization to examine the basal layer of the AEC, the layer that lies immediately over the distal limb mesenchyme. In late-stage regenerates, this basal layer expresses fibronectin (FN) message very strongly in a stripe of cells along the dorso-ventral boundary. H+E staining also reveals the unique shape of basal cells in this area. The stripe of cells in the basal AEC also contains the notch/groove structure previously seen in avian and reptilian AERs. In addition, AEC expression of FN message in the cells around the groove correlates with previous amniote AER localization of FN protein inside the groove. The structural and biochemical analyses presented here suggest that there is a specialized ridge-like compartment in the basal AEC in late-stage regenerates. The data also suggest that this compartment may be homologous to the AER of the developing amniote limb. Thus, the external differences between amniote limb development and urodele limb regeneration may be outweighed by internal similarities, which enable both processes to produce morphologically complete limbs. In addition, we propose that this basal layer of the AEC is uniquely responsible for AEC functions in regeneration, such as secreting molecules to promote mesenchymal cell cycling and dictating the direction of limb outgrowth. Finally, we include here a clarification of existing nomenclature to facilitate further discussion of the AEC and its basal layer.     

Expression patterns of Fgf-8 during development and limb regeneration of the axolotl.

Fgf-8 is one of the key signaling molecules implicated in the initiation, outgrowth, and patterning of vertebrate limbs. However, it is not clear whether FGF-8 plays similar role in development and regeneration of urodele limbs. We isolated a Fgf-8 cDNA from the Mexican axolotl (Ambystoma mexicanum) through the screening of an embryo cDNA library. The cloned 1.26-kb cDNA contained an open reading frame encoding 212 amino acid residues with 84%, 86%, and 80% amino acid identities to those of Xenopus, chick, and mouse, respectively. By using the above clone as a probe, we examined the temporal and spatial expression patterns of Fgf-8 in developing embryos and in regenerating larval limbs. In developing embryos, Fgf-8 was expressed in the neural fold, midbrain-hindbrain junction, tail and limb buds, pharyngeal clefts, and primordia of maxilla and mandible. In the developing axolotl limb, Fgf-8 began to be expressed in the prospective forelimb region at pre-limb-bud and limb bud stages. Interestingly, strong expression was detected in the mesenchymal tissue of the limb bud before digit forming stages. In the regenerating limb, Fgf-8 expression was noted in the basal layer of the apical epithelial cap (AEC) and the underlying thin layer of mesenchymal tissue during blastema formation stages. These data suggest that Fgf-8 is involved in the organogenesis of various craniofacial structures, the initiation and outgrowth of limb development, and the blastema formation and outgrowth of regenerating limbs. In the developing limb of axolotl, unlike in Xenopus or in amniotes such as chick and mouse, the Fgf-8 expression domain was localized mainly in the mesenchyme rather than epidermis. The unique expression pattern of Fgf-8 in axolotl suggests that the regulatory mechanism of Fgf-8 expression is different between urodeles and other higher species. The expression of Fgf-8 in the deep layer of the AEC and the thin layer of underlying mesenchymal tissue in the regenerating limbs support the previous notion that the amphibian AEC is a functional equivalent of the AER in amniotes.     

Connexin expression and gap junction communication compartments in the developing mouse limb.

Fundamental to the understanding of mouse limb morphogenesis and pattern formation is the need to elucidate the spatial and temporal distribution of gap junction proteins (connexins, Cx) and cell-cell communication compartments. To this end, we used immunofluorescence and confocal microscopy together with 3-dimensional reconstruction software to map the distribution of Cx43 and Cx32 in 11-14.5 days postcoitum (dpc) mouse limbs. Cx43 was strictly localized to the apical ectodermal ridge (AER) and nonridge ectoderm throughout all stages of mouse limb development studied. Cx32, on the other hand, was abundant in the mesenchyme with only low levels of expression in the 11-13.5 dpc ectoderm. However, at 14-14.5 dpc there was a clear increase in Cx32 expression in the ectoderm. Double labeling for connexins and confocal microscopy revealed Cx43 and Cx32 in the same optical section of the basal cells of the ectoderm but in separate plaques. Lucifer yellow dye injections showed that the cells of the AER were in direct communication with the nonridge ectoderm but dye was never observed to spread to the mesenchyme. Cells of the mesenchyme were coupled to each other but to a much lesser extent than cells of the ectoderm. Finally, although there was an increase in Cx32 expression in the ectoderm at 14-14.5 dpc, this was not correlated with any detectable change in communication compartments. Thus, the lack of dye transfer between the ectoderm and underlying mesenchyme from the peak of AER height through its decline suggests that bulk transfer of morphogens between these two layers is not necessary for mouse limb development.     

A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth

Signals from the apical ectodermal ridge (AER) of the developing vertebrate limb, including fibroblast growth factor-8 (FGF-8), can maintain limb mesenchymal cells in a proliferative state. We report here that a specific CD44 splice variant is crucial for the proliferation of these mesenchymal cells. Epitopes carried by this variant colocalize temporally and spatially with FGF-8 in the AER throughout early limb development. A splice variant containing the same sequences expressed on model cells binds both FGF-4 and FGF-8 and stimulates mesenchymal cells in vitro. When applied to the AER, an antibody against a specific CD44 epitope blocks FGF presentation and inhibits limb outgrowth. Therefore, CD44 is necessary for limb development and functions in a novel growth factor presentation mechanism likely relevant in other physiological and pathological situations where a cell surface protein presents a signaling molecule to a neighboring cell.     

Talpid2 mutant chick limb has anteroposterior polarity and altered patterns of programmed cell death.

The talpid2(ta2) chick mutant is of interest for the study of limb pattern formation. Talpid2 is a simple Mendelian recessive lethal mutation which affects the mesoderm and results in short, spade-like, polydactylous wings and legs. Here, we describe ta2 limb development with particular attention to those aspects of ta2 which may illuminate the process of normal limb development. From the onset of budding, ta2 limb buds are significantly wider than normal buds along the anteroposterior axis. They lack the normal anterior and posterior necrotic zones and have variable development of the central opaque patch. Interdigital programmed cell death is variable and may result in development of distal phalanges without more proximal ones. Talpid2 wing vasculature is similar to that of normal wings; but ta2 legs are supplied by four large blood vessels. Feathers form regular, parallel rows, similar to normal feathers, but ta2 embryos lack the loose mesenchyme which separates the feather buds. Finally, and most significantly, ta2 wings and legs display anteroposterior polarity. Anterior and posterior limb skeletal elements can be clearly distinguished from one another within the ta2 phenotype. Our observations suggest that the ta2 mutant may be useful in analyzing programmed embryonic cell death and anteroposterior limb pattern formation.     

Altered expression of insulin-like growth factor-1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity

Insulin-like growth factor-I (IGF-I) mediated signalling has been implicated to be of significant importance during vertebrate embryonic development. IGF-I signalling has also been shown to be modulated by a number of IGF binding proteins that are thought to act as either agonists or antagonists of IGF activity. IGF-I has been implicated in a number of cellular processes, including cell division and programmed cell death (apoptosis). We have used the mouse mutant Hypodactyly (Hd) as a tool to determine the role of IGF-I and two key IGF binding proteins (IGFBP-2 and IGFBP-5) during embryonic development. The Hd mutant is a good model with which to study developmental cascades, since it has a distinct phenotype in the limb where cellular and molecular circuits have been thoroughly investigated. The distinctive pointed limb buds observed in Hd mutant embryos have been shown to be the result of a massive increase in apoptosis. We show that all three genes, IGF-1, IGFBP-2 and IGFBP-5, display restricted expression patterns during limb development. Indeed, IGFBP-5 shows a remarkable similarity to the expression of Engrailed-1, which is the vertebrate homologue of the Drosophila selector gene Engrailed. We show that there is downregulation in the expression of IGFBP-2 in the entire apical ectodermal ridge (AER) in homozygous Hd/Hd limb buds, whereas IGFBP-5 is downregulated in specific regions in the mutant AER. IGF-I expression is downregulated in Hd limb buds in regions undergoing high levels of cell death, consistent with its proposed role as an anti-apoptotic factor, while IGFBP-5 is found at higher levels in regions of cell death, consistent with reports of its association with apoptosis in adult tissues. We propose that these three components of the IGF axis could be involved in the manifestation of the mutant phenotype in Hypodactyly, and that this is probably a result of their ability to regulate cell survival and cell death.     

Age-related behavioral,morphological and physiological changes in the hippocampus of zitter rats

Convulsive seizure is known to be associated with hippocampal abnormalities, such as hilar cell degeneration, abnormal mossy fiber sprouting in the dentate gyrus (DG) and abnormal expression of immediate early genes. However, whether these morphological changes are a cause or consequence of convulsive seizures has remained contentious. Zitter (zi/zi) rats carry a mutation of the attractin gene and display spongiform degeneration of the brain. Spontaneous convulsive seizures in zi/zi rats over 8 months (M) old were demonstrated using 24-h video monitoring. Spontaneous convulsive seizures did not occur before this age. The present study examined structural changes in the hippocampus of zi/zi rats at different ages. Fluoro-Jade B-positive cells first appeared in the hilus of 1-M zi/zi rats, indicating hilar cell degeneration. After 2 M, mossy fiber sprouting was observed in granular cell layers and in the inner molecular layer. After 10 M, granule cells showed Fos expression. In the hippocampal slices from 12-M zi/zi rats, abnormal population spikes in the DG were observed in the presence of bicuculline and strychnine. Conversely, Sprague-Dawley rats showed no aberrant zinc distribution, few Fos-positive cells, no Fluoro-Jade B-positive cells in the hippocampus and no abnormal population spikes in the DG. These data indicate that morphological changes in the hippocampus might contribute to epileptogenesis in this mutant rat.