Fjx1: a notch-inducible secreted ligand with specific binding sites in developing mouse embryos and adult brain.

The mouse fjx1 gene was identified as a homologue to the Drosophila gene four-jointed (fj). Fj encodes a transmembrane type II glycoprotein that is partially secreted. The gene was found to be a downstream target of the Notch signaling pathway in leg segmentation and planar cell polarity processes during eye development of Drosophila. Here, we show that fjx1 is not only conserved in vertebrates, but we also identified the murine fjx1 gene as a direct target of Notch signaling. In addition to the previously described expression of fjx1 in mouse brain, we show here that fjx1 is expressed in the peripheral nervous system, epithelial cells of multiple organs, and during limb development. The protein is processed and secreted as a presumptive ligand. Through the use of an fjx1-AP fusion protein, we could visualize fjx1 binding sites at complementary locations, supporting the notion that fjx1 may function as a novel signaling molecule.     

Expression of mouse dchs1, fjx1, and fat-j suggests conservation of the planar cell polarity pathway identified in Drosophila.

The dachsous (ds), fat (ft), and four-jointed (fj) genes have been identified in Drosophila as part of a signaling pathway that regulates planar cell polarity (PCP). A homologous PCP signaling pathway has also been identified in vertebrates, but nothing is known thus far about the conservation of Ds/Ft/Fj signaling. Here we analyzed and compared for the first time the expression patterns of all ds, ft and fj homologs in the mouse. During embryogenesis, expression analysis was performed by RNA in situ hybridization and in adult organs by real time PCR. As in Drosophila, we detected a complementary expression of fjx1 and dchs1 in organs like kidney, lung, and intestine. The ubiquitous expression of ft in several tissues in Drosophila appears to be split into an epithelial expression of fat1/fat3 and a mesenchymal expression of fat-j. These data are compatible with a conservation and sub-functionalization of the Drosophila Ds, Fj, and Fat signaling in higher vertebrates.     

Expression of slit-2 and slit-3 during chick development.

The Drosophila and vertebrate slit proteins have been characterized as secreted chemorepellents recognized by the robo receptor proteins that function principally for the guidance of neuronal axons and neurons. slit genes are also expressed in the limb. To provide a basis for the determination of slit functions in the limb we have isolated and characterized the expression of chick slit-2 and slit-3 in the developing limb and other tissues of the chick embryo. Both genes share similar expression profiles in the chick embryo when compared to that of their mammalian homologues, particularly in the neural tube. In the limb, their expression patterns suggest their involvement in many aspects of limb development. In the early limb bud, slit-2 is expressed in the peripheral mesenchyme and invading muscle precursors, while slit-3 expression is restricted to the future chondrogenic core of the limb bud. At later stages, both slit genes are expressed in interdigital mesenchyme, in inner periosteal cells, and in mesenchyme immediately radial to the periosteum and under the epidermis. slit-3 is also expressed in proliferating chondrocytes during cartilage development, while slit-2 is expressed in later muscle masses and peripherally to joints in the autopod.     

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.     

The chicken limb deformity gene encodes nuclear proteins expressed in specific cell types during morphogenesis.

The chicken limb deformity (ld) mutation affects morphogenesis of both limbs and kidneys and is one of few murine mutations for which the affected gene has been isolated. Analysis of the chicken homolog reveals evolutionary conservation of large parts of the encoded ld gene products. This is the first study of these proteins, their intracellular localization, and their temporal and spatial distribution during embryogenesis. A major 180-kD protein is expressed in chicken embryos and certain adult tissues. The proteins are localized in the nuclei of different embryonic cell types in a characteristic punctate pattern. In the developing chicken limb bud, they are expressed in the newly differentiated apical ectodermal ridge and the mesenchymal compartment, where an unequal distribution along the anteroposterior and, subsequently, the dorsoventral axes, is observed. During kidney morphogenesis, expression is initially restricted to the epithelial compartment of the pronephros and mesonephros. These results correlate well with the previous analysis of the murine ld phenotype and imply determinative roles for ld gene products during the morphogenesis of limbs and kidneys. Unexpected expression in the notochord, floor plate, and ventral horns suggests an involvement of the ld gene products in establishment of the dorsoventral polarity of the neural tube.     

Talpid2 limb bud mesoderm does not express GHox-8 and has an altered expression pattern of GHox-7.

We have studied the expression patterns of the chick homeobox-containing genes, GHox-7 and GHox-8, in the talpid2 (ta2) chick mutant whose limbs have abnormal pattern. These studies provide new insight into how homeobox gene expression and limb patterning may be related. This is the first study demonstrating a natural change in GHox-7 and GHox-8 along the anteroposterior axis. While GHox-7 is expressed asymmetrically in normal limb buds, it is expressed at a uniform level across the anteroposterior axis of ta2 limb buds. GHox-8 is expressed in anterior mesoderm of normal limb buds, but is undetectable in ta2 limb bud mesoderm. These data are consistent with the subtle anteroposterior polarity in ta2 limbs, and allow us to propose that ta2 limb buds lack anterior positional information, but have a narrow range of posterior positional values. We suggest that in normal limb buds GHox-8 may establish the anterior limb bud boundary. Furthermore, we point out that coexpression of GHox-7 and GHox-8 in normal anterior limb bud mesoderm can be correlated with the reduced apical ridge maintenance activity of this tissue, while the lack of coexpression in ta2 limb buds is correlated with the strong ridge maintenance activity in the mutant's anterior limb bud mesoderm. Last, ta2 limbs contain no dying cells in their anterior and posterior border mesoderm; nevertheless, they express GHox-7 in these regions. These data challenge the proposal that this gene determines cell death.     

Patterning the limb before and after SHH signalling

The vertebrate limb is one of the most relevant experimental models for analysing cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions co-ordinating limb bud outgrowth and patterning. Some of these studies have shown that there is a need to revise some of the textbook views of vertebrate limb development. In this review, we discuss how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. We also discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region.     

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.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4 , a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8 , which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh , nor of its receptor and downstream target gene Ptc , suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.     

Differential regional expression of multiple ADAMs during feather bud formation

The expression of seven members of the ADAM family was investigated by in situ hybridization in the developing feather buds of chicken. The expression profiles of the ADAMs in the cells and tissues of the feather buds differ from each other. ADAM9, ADAM10, and ADAM17 are expressed in the epidermis of the feather bud, whereas ADAM23 expression is restricted to the bud crest, with a distribution similar to that of sonic hedgehog. ADAM13 is not only expressed in the epidermis, but also in restricted regions of the dermis. Both ADAM12 and ADAM22 are expressed in the dermis of the feather bud, with an opposite mediolateral and anteroposterior polarity. Furthermore, the mRNAs of all investigated ADAMs show regional differences in their expression, for example, in the neck and in the roots of the leg and wing. These results suggest that ADAMs play a variety of roles during avian feather bud formation. Developmental Dynamics 240:2142–2152, 2011. © 2011 Wiley‐Liss, Inc.     

Modification of the phalangeal pattern of the digits in the chick embryo leg bud by local microinjection of RA,staurosporin and TGF β's

Many experimental studies show that in the avian chick limb the digits are specified at early stages of development by characteristic concentrations within the limb mesoderm of a still unidentified morphogen diffusing from the posterior margin of the bud, linked with a specifie pattern of homeobox gene expression. In ail these studies, digits are distinguished by their size, morpholgy and phalangeal pattern rather than by their position within the autopodium. In this work we report the induction of digits that have otherwise normal morphology but lack an interphalangeal joint. This suggests that the patterning of these joints is not necessarily linked to the control of the outgrowth of the digital rays. Missing interphalangeal joints were induced by microinjection into the third interdigital space of the leg bud of stage 28 to 31 chick embryos of retinoic Acid (RA), staurosporine and TGF β1 and β2, but not by microinjection of FGF or EGF. Our results also suggest that the pattern of insertion of the long tendons and the formation of the flexor cutaneous pad at the plantar surface of the digits are both linked to the establishment of the interphalangeal joints.     

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.     

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod

Retinoic acid, when administered to pregnant mice on d 11·0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4, a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8, which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh, nor of its receptor and downstream target gene Ptc, suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4‐induced skeletogenic mesenchymal cell proliferation.