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More than sixty years ago, while studying feather tracks on the shoulder of the chick embryo, Dr. John Saunders used Nile Blue dye to stain the tissue. There, he noticed a darkly stained line of cells that neatly rims the tip of the growing limb bud. Rather than ignoring this observation, he followed it up by removing this tissue and found that it led to a striking truncation of the limb skeletons. This landmark experiment marks the serendipitous discovery of the apical ectodermal ridge (AER), the quintessential embryonic structure that drives the outgrowth of the limb. Dr. Saunders continued to lead the limb field for the next fifty years, not just through his own work, but also by inspiring the next generation of researchers through his infectious love of science. Together, he and those who followed ushered in the discovery of fibroblast growth factor (FGF) as the AER molecule. The seamless marriage of embryology and molecular biology that led to the decoding of the AER serves as a shining example of how discoveries are made for the rest of the developmental biology field. [ABSTRACT FROM AUTHOR]

Vertebrate limb development relies on the activity of signaling centers that promote growth and control patterning along three orthogonal axes of the limb bud. The apical ectodermal ridge, at the distal rim of the limb bud ectoderm, produces WNT and FGF signals, which promote limb bud growth and progressive distalization. The zone of polarizing activity, a discrete postero-distal mesenchymal domain, produces SHH, which stimulates growth and organizes patterning along the antero-posterior axis. The dorsal and ventral ectoderms produce, respectively, WNT7A and BMPs, which induce dorso-ventral limb fates. Interestingly, these signaling centers and the mechanisms they instruct interact with each other to coordinate events along the three axes. We review here the main interactions described between the three axial systems of the developing limb and discuss their relevance to proper limb growth and patterning. [ABSTRACT FROM AUTHOR]

Objective: To observe the effect of sodium phenytoin on the development of the apical ectodermal ridges (AER) in chick wing buds. Study Design: Laboratory based case-control study. Place and Duration of Study: This study was carried out at the Department of Anatomy located at the Regional Center of College of Physicians and Surgeons Islamabad Pakistan, from Jan 2014 to Jan 2015. Material and Methods: Sixty fertilized chicken eggs of 'Egyptian fayoumi' breed were selected and divided into two subgroups, experimental and control, each having 30 eggs. A single dose of 3.5 mg sodium phenytoin was injected in each egg of the experimental subgroup while controls were administered same volume of normal saline. The embryos were extracted 96 hours (day 4) after incubation. Histological sections were cut at 5 µm thickness and stained with Feulgen Nuclear and Light Green to measure the maximum thickness of the apical ectodermal ridges. The number of cells and mitoses in each ridges were also counted. SPSS 16 was used for statistical analysis. Results: There was no significant difference between experimental and control subgroups regarding the maximum thickness and number of cells in the apical ectodermal ridge but there was a statistically significant reduction in the number of mitoses in the experimental ridges. Conclusion: The study shows that sodium phenytoin exerts an inhibitory effect on the apical ectodermal ridges of developing limbs evident by the decreased number of mitoses in the AER of wing buds of experimental chick embryos making it a potential target site for limb teratogenicity associated with its maternal intake during pregnancy. [ABSTRACT FROM AUTHOR]

Limb development is a very complex process, not completely understood yet. There are multiple genes implicated during the process and the complexity of limb development consists in the interaction of these genes through transcription factors. The responsible structures for limb development are the centres of signalling, areas of the limb bud where gene transcription has a high rate and transcription factors determine the pattern of the cells situated nearby. The Apical Ectodermal Ridge (AER) is the signalling centre responsible for the proximal-distal development of the limb; the Zone of Polarizing Activity (ZPA) is responsible for the patterning of the limb on the anterior-posterior axis. The non -AER ectoderm is responsible for the dorsal-ventral pattering. [ABSTRACT FROM AUTHOR]

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors ( FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein ( BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis. [ABSTRACT FROM AUTHOR]

Background: The 39 mammalian Hox genes show problematic patterns of functional overlap. In order to more fully define the developmental roles of Hox genes it is necessary to remove multiple combinations of paralogous and flanking genes. In addition, the downstream molecular pathways regulated by Hox genes during limb development remain incompletely delineated. Results: In this report we examine limb development in mice with frameshift mutations in six Hox genes, Hoxa9,10,11 and Hoxd9,10,11. The mice were made with a novel recombineering method that allows the simultaneous targeting of frameshift mutations into multiple flanking genes. The Hoxa9,10,11–/–/Hoxd9,10,11–/– mutant mice show a reduced ulna and radius that is more severe than seen in Hoxa11–/–/Hoxd11–/– mice, indicating a minor role for the flanking Hox9,10 genes in zeugopod development, as well as their primary function in stylopod development. The mutant mice also show severe reduction of Shh expression in the zone of polarizing activity, and decreased Fgf8 expression in the apical ectodermal ridge, thereby better defining the roles of these specific Hox genes in the regulation of critical signaling centers during limb development. Importantly, we also used laser capture microdissection coupled with RNA-Seq to characterize the gene expression programs in wild type and mutant limbs. Resting, proliferative and hypertrophic compartments of E15.5 forelimb zeugopods were examined. The results provide an RNA-Seq characterization of the progression of gene expression patterns during normal endochondral bone formation. In addition of the Hox mutants showed strongly altered expression of Pknox2, Zfp467, Gdf5, Bmpr1b, Dkk3, Igf1, Hand2, Shox2, Runx3, Bmp7 and Lef1, all of which have been previously shown to play important roles in bone formation. Conclusions: The recombineering based frameshift mutation of the six flanking and paralogous Hoxa9,10,11 and Hoxd9,10,11 genes provides a resource for the analysis of their overlapping functions. Analysis of the Hoxa9,10,11–/–/Hoxd9,10,11–/– mutant limbs confirms and extends the results of previous studies using mice with Hox mutations in single paralogous groups or with entire Hox cluster deletions. The RNA-Seq analysis of specific compartments of the normal and mutant limbs defines the multiple key perturbed pathways downstream of these Hox genes. [ABSTRACT FROM AUTHOR]

Background: The 39 mammalian Hox genes show problematic patterns of functional overlap. In order to more fully define the developmental roles of Hox genes it is necessary to remove multiple combinations of paralogous and flanking genes. In addition, the downstream molecular pathways regulated by Hox genes during limb development remain incompletely delineated. Results: In this report we examine limb development in mice with frameshift mutations in six Hox genes, Hoxa9,10,11 and Hoxd9,10,11. The mice were made with a novel recombineering method that allows the simultaneous targeting of frameshift mutations into multiple flanking genes. The Hoxa9,10,11−/−/Hoxd9,10,11−/− mutant mice show a reduced ulna and radius that is more severe than seen in Hoxa11−/−/Hoxd11−/− mice, indicating a minor role for the flanking Hox9,10 genes in zeugopod development, as well as their primary function in stylopod development. The mutant mice also show severe reduction of Shh expression in the zone of polarizing activity, and decreased Fgf8 expression in the apical ectodermal ridge, thereby better defining the roles of these specific Hox genes in the regulation of critical signaling centers during limb development. Importantly, we also used laser capture microdissection coupled with RNA-Seq to characterize the gene expression programs in wild type and mutant limbs. Resting, proliferative and hypertrophic compartments of E15.5 forelimb zeugopods were examined. The results provide an RNA-Seq characterization of the progression of gene expression patterns during normal endochondral bone formation. In addition of the Hox mutants showed strongly altered expression of Pknox2, Zfp467, Gdf5, Bmpr1b, Dkk3, Igf1, Hand2, Shox2, Runx3, Bmp7 and Lef1, all of which have been previously shown to play important roles in bone formation. Conclusions: The recombineering based frameshift mutation of the six flanking and paralogous Hoxa9,10,11 and Hoxd9,10,11 genes provides a resource for the analysis of their overlapping functions. Analysis of the Hoxa9,10,11−/−/Hoxd9,10,11−/− mutant limbs confirms and extends the results of previous studies using mice with Hox mutations in single paralogous groups or with entire Hox cluster deletions. The RNA-Seq analysis of specific compartments of the normal and mutant limbs defines the multiple key perturbed pathways downstream of these Hox genes. [ABSTRACT FROM AUTHOR]

We have developed an upper-level undergraduate laboratory exercise that enables students to replicate a key experiment in developmental biology. In this exercise, students have the opportunity to observe live chick embryos and stain the apical ectodermal ridge, a key tissue required for development of the vertebrate limb. Impressively, every student who has tried this protocol has been successful, making it a good introduction to the use of the chick model system in studying development. The array of materials about limb development, using chick embryos in teaching laboratories, and the history of this experiment provide a rich background for teachers and students. [ABSTRACT FROM AUTHOR]

The article discusses non-traditional models in understanding mechanisms that regulate limb development among mammals. Research in non-traditional mammals have been done on organisms including opossum Monodelphis domestica, bats Carollia perspicillata, and pigs Sus scrofa, to understand the modified evolution of their limb development, as well as generate adult morphologies. Knowledge of differing limb development among mammals will improve understanding of human development.

Abstract: The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. In the present work, we have investigated the role of Epiprofin (Epfn/Sp6), a member of the SP/KLF transcription factor family that is expressed in the limb ectoderm and the AER, during limb development. Epfn mutant mice have a defective autopod that shows mesoaxial syndactyly in the forelimb and synostosis (bony fusion) in the hindlimb and partial bidorsal digital tips. Epfn mutants also show a defect in the maturation of the AER that appears flat and broad, with a double ridge phenotype. By genetic analysis, we also show that Epfn is controlled by WNT/b-CATENIN signaling in the limb ectoderm. Since the less severe phenotypes of the conditional removal of b-catenin in the limb ectoderm strongly resemble the limb phenotype of Epfn mutants, we propose that EPFN very likely functions as a modulator of WNT signaling in the limb ectoderm. [Copyright &y& Elsevier]

Cognitive-behavioral practices such as meditation and yoga have long been viewed as methods of reaching states of peace and relaxation, but recent research has focused on the role of these practices in reducing endogenous mediators of stress and inflammation that would otherwise be harmful to our bodies. Further, these stress-related factors play major roles in inflammation, acting as barriers to wound healing and tissue regeneration. Fractures, denervation, tendon and ligament rupture, and cartilage degradation are morbidities associated with injury and often act as an impediment for healing. Studies of human fingertip regeneration exist; however, the underlying molecular and environmental changes have yet to be completely elucidated. Studying the regenerative capabilities of lower organisms and fetal wound healing has allowed scientists to understand the mechanisms behind regeneration, coming closer to a human application. Much research relies on the idea that the developing embryo shares a great deal in common with regenerating appendages of organisms such as the salamander. This review will cover historical perspectives of regeneration biology and current topics in limb regeneration, with particular interest given to the upper extremity, including the commonalities between human embryological development and amphibian regeneration, growth factors and pathways that show correlation with development and regeneration, recently discovered differences in fetal and adult wound healing, and current research and knowledge regarding human extremity tissue regeneration. With a greater understanding of the mechanisms and mediators involved in regeneration, the application of cognitive-behavioral practices may assist in seeing the future goals of regeneration come to fruition. [ABSTRACT FROM AUTHOR]

The apical ectodermal ridge (AER) in Neoceratodus develops after an initial period of mesenchymal proliferation and outgrowth of the fin bud and persists until chondrogenesis of the stylopod and zeugopod is initiated. At this time, the lateral margins of the AER convert to the fin fold leading to subsequent development of the dermal fin skeleton. Thorogood's (1991 ) fin fold model predicts that the AER should persist longer in Neoceratodus than it does in actinopterygians because of the comparatively extensive endochondral skeleton in lungfish. While the AER does persist into early chondrogenesis and is extended compared to actinopterygians (lost before fin radial chondrogenesis) it does not persist into further stages of chondrogenesis, providing partial support for Thorogood's model. Fgf8 appears in the lungfish fin epithelium during the initial period of fin outgrowth before a physical AER forms, when Fgf8 is restricted to the AER plus the preaxial and postaxial epithelium immediately adjacent to the AER. Fgf8 is no longer detected after the AER is replaced by a fin fold. Neoceratodus appears to provide a halfway point between ray fins and limbs during very early development as Thorogood proposed, but not precisely for the reasons his model suggests. [ABSTRACT FROM AUTHOR]

Patterning of the developing vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) via the expression of Sonic hedgehog ( Shh) and along the proximal-distal axis by the apical ectodermal ridge (AER) through the production of fibroblast growth factors (FGFs). ZPA grafting, as well as ectopic application of SHH to the anterior chick limb bud, demonstrate that digit patterning is largely influenced by these secreted factors. Although signal transduction pathways have been well characterized for SHH and for FGFs, little is known of how these signals are regulated extracellularly in the limb. The present study shows that alteration of the extracellular environment through trypsin treatment can have profound effects on digit patterning. These effects appear to be mediated by the induction of Shh in host tissues and by ectopic AER formation, implicating the extracellular matrix in regulating the signaling activities of key patterning genes in the limb. [ABSTRACT FROM AUTHOR]

Mouse R-spondin2 (Rspo2) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. R-spondin is a secreted molecule that activates Wnt/ β-catenin signaling. Rspo2-deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild-type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development. [ABSTRACT FROM AUTHOR]

Abstract: Cadmium chloride (CdCl2) and all-trans-retinoic acid (RA) induce postaxial forelimb ectrodactyly in C57BL/6N mice when administered during early limb development, and co-administration yields a synergistic response suggesting a common final pathway to the defect. In the current study, forelimb buds from embryos given high maternal teratogenic doses of CdCl2 or RA, or the combination of both agents at low doses were collected at various time points after treatment on GD 9.5 and examined for cellular apoptosis, proliferation, and patterning genes. Some cellular perturbations detected in the developing limb bud were similar for both teratogens, whereas other alterations were unique to each agent. For example, at 12 and 18 h, CdCl2 treatment increased apoptotic cells in the mesenchyme underneath the apical ectodermal ridge (AER), whereas RA caused apoptosis in the AER and proximal mesenchyme. Further, the combined low-dose treatment increased cell death synergistically in all three regions. CdCl2 and the low-dose combined treatment inhibited mesenchymal proliferation at 12 h, which was associated with induction of p21 cip1 and inhibition of phospho-c-Jun. In contrast, RA did not inhibit mesenchymal proliferation and did not induce p21 cip1 expression or change c-Jun phosphorylation. All three treatment groups showed a delay in the patterning of distal chondrogenesis centers as indicated by Sox9 expression. There was also common inhibition in the expression of AER markers, Fgf8 and Fgf4, and the mesenchymal marker Msx1 involved in the maintenance of epithelial–mesenchymal interactions. Collectively, a model is hypothesized where limb patterning can be perturbed by insults to both ectoderm and mesoderm. [Copyright &y& Elsevier]

Abstract: Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell–cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis. [Copyright &y& Elsevier]

Abstract: We have analysed the expression during mid-gestation mouse development of the four member LRRTM gene family which encodes type 1 transmembrane proteins containing 10 extracellular leucine rich repeats and a short intracellular tail. Each family member has a developmentally regulated pattern of expression distinct from all other members. LRRTM1 is expressed in the neural tube, otic vesicle, apical ectodermal ridge, forebrain and midbrain up to a sharp central boundary. LRRTM2 is expressed in a subset of progenitors in the neural tube. LRRTM3 is expressed in a half somite wide stripe in the presomitic mesoderm adjacent to the boundary with the most recently formed somite. Additional expression is seen in the neural tube, forebrain and hindbrain. LRRTM4 is expressed in the limb mesenchyme, neural tube, caudal mesoderm and in three distinct regions of the head. Later expression occurs in a subset of the developing sclerotome. Each family member has a unique expression domain within the neural tube. [Copyright &y& Elsevier]

Abstract: A central question in developmental biology is how cells interact to organize into tissues? In this paper, we study the role of mesenchyme-ectoderm interaction in the growing chick limb bud using Glazier and Graner''s cellular Potts model, a grid-based stochastic framework designed to simulate cell interactions and movement. We simulate cellular mechanisms including cell adhesion, growth, and division and diffusion of morphogens, to show that differential adhesion between the cells, diffusion of growth factors through the extracellular matrix, and the elastic properties of the apical ectodermal ridge together can produce the proper shape of the limb bud. [Copyright &y& Elsevier]

Abstract: Formation of paired limbs in vertebrate embryos has long been a particularly useful paradigm for the study of pattern formation. Here, we show that Blimp-1, a SET domain and zinc finger-containing transcriptional factor, plays an important role in the development of the pectoral fins of the zebrafish structures that are homologous to forelimbs of amniotes. The blimp-1 gene is expressed dynamically in the mesenchyme as well as the ectodermal cells of the early fin bud, and later, in the cells of the apical ectodermal ridge (AER) of the outgrowing fin. Consistent with this expression profile, loss of Blimp-1 activity severely impairs fin outgrowth and patterning. We present evidence that blimp-1 functions downstream of tbx5 and fgf24 and therefore is not required for the initial specification of the fin bud primordia. Subsequently, however, its function is necessary for the induction of fgf10 and sonic hedgehog in the mesenchyme. In addition, Blimp-1 activity is absolutely critical for the proper induction of gene expression in the ectoderm and establishment of the AER. Taken together, these results identify an additional layer of control in the genetic pathway that operates in the developing limb and provides novel insights into regulatory mechanisms that organize its pattern. [Copyright &y& Elsevier]

Abstract: Mulefoot disease (MFD) is an autosomal recessive disorder of phenotypically variable expression that causes syndactyly in certain strains of cows. MFD maps to a narrow interval on bovine chromosome 15 that is syntenic to human chromosome 11p12–p11.2. This region contains MEGF7/LRP4 (approved gene symbol LRP4), a gene that encodes a member of the multifunctional low-density lipoprotein receptor gene family. Targeted and naturally occurring mutations in the murine Megf7/Lrp4 gene, a putative coreceptor in the Wnt signaling pathway, cause polysyndactyly in the rodent. Thus, Megf7/Lrp4 is a strong candidate for the MFD mutation. Using PCR analysis of tissue samples and sperm from confirmed homozygous MFD carriers, we have identified a functional single base pair mutation in the affected animals. We show that a G → A transition at the first nucleotide in the splice donor site of intron 37 completely disables this splice site. The abnormal splicing that is caused by this mutation predicts the generation of a dysfunctional membrane-anchored receptor lacking the normal cytoplasmic domain. These findings confirm that autosomal recessive loss-of-function mutations in Megf7/Lrp4 result in phenotypically similar forms of syndactyly in different mammalian species and that such mutations are the cause of MFD in bovines. [Copyright &y& Elsevier]

Abstract: Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other’s expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA. [Copyright &y& Elsevier]

Initiation and maintenance of signaling centers is a key issue during embryonic development. The apical ectodermal ridge, a specialized epithelial structure and source of Fgf8, is a pivotal signaling center for limb outgrowth. We show that two closely related buttonhead-like zinc-finger transcription factors, Sp8 and Sp9, are expressed in the AER, and regulate Fgf8 expression and limb outgrowth. Embryological and genetic analyses have revealed that Sp8 and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. We also found that Wnt/β-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled their role as positive regulators of Fgf8 expression. Moreover, a dominant-negative approach in chick and knockdown analysis with morpholinos in zebrafish revealed their requirement for Fgf8 expression and limb outgrowth, and further indicate that they have a coordinated action on Fgf8 expression. Our study demonstrates that Sp8 and Sp9, via Fgf8, are involved in mediating the actions of Fgf10 and Wnt/β-catenin signaling during vertebrate limb outgrowth. [ABSTRACT FROM AUTHOR]

Although numerous molecules required for limb bud formation have recently been identified, the molecular pathways that initiate this process and ensure that limb formation occurs at specific axial positions have yet to be fully elucidated. Based on experiments in the chick, Fgf8 expression in the intermediate mesoderm (IM) has been proposed to play a critical role in the initiation of limb bud outgrowth via restriction of Fgf10 expression to the appropriate region of the lateral plate mesoderm. Contrary to the outcome predicted by this model, ablation of Fgf8 expression in the intermediate mesoderm before limb bud initiation had no effect on initial limb bud outgrowth or on the formation of normal limbs. When their expression patterns were first elucidated, both Fgf4 and Fgf8 were proposed to mediate critical functions of the apical ectodermal ridge (AER), which is required for proper limb bud outgrowth. Although mice lacking Fgf4 in the AER have normal limbs, limb development is severely affected in Fgf8 mutants and certain skeletal elements are not produced. By creating mice lacking both Fgf4 and Fgf8 function in the forelimb AER, we show that limb bud mesenchyme fails to survive in the absence of both FGF family members. Thus, Fgf4 is responsible for the partial compensation of distal limb development in the absence of Fgf8. A prolonged period of increased apoptosis, beginning at 10 days of gestation in a proximal–dorsal region of the limb bud, leads to the elimination of enough mesenchymal cells to preclude formation of distal limb structures. Expression of Shh and Fgf10 is nearly abolished in double mutant limb buds. By using a CRE driver expressed in both forelimb and hindlimb ectoderm to inactivate Fgf4 and Fgf8, we have produced mice lacking all limbs, allowing a direct comparison of FGF requirements in the two locations. [Copyright &y& Elsevier]

In developing limb buds, Msx2 transcripts are expressed in the apical ectodermal ridge (AER) and in various regions of the limb mesenchyme. To identify DNA sequences responsible for Msx2 expression in the AER, we characterized the expression of LacZ reporter constructs driven by chicken Msx2 regulatory sequences in transgenic mice. We have identified a 55-bp enhancer that can direct AER-specific reporter gene expression. This 55-bp enhancer contains three elements that are evolutionary conserved among five vertebrate Msx2 genomic sequences. AER expression of reporter constructs in transgenic mice is lost or reduced when mutations are introduced into each of these three regions. Moreover, changing the relative orientation by reverse complementing one of the three elements also results in loss of expression, suggesting that the relative orientations of transcription factor binding is important. To identify the transcription factor(s) binding to these elements, we conducted one-hybrid screening and identified Dlx5 and Sox11. Both Dlx5 and Sox11 are expressed in the AER, and the proteins encoded by these genes bind to separate conserved elements, supporting their possible roles in regulating Msx2 expression. [Copyright &y& Elsevier]

Split hand foot malformation (SHFM) is a congenital limb malformation presenting with a median cleft of the hand and/or foot, syndactyly and polydactyly. SHFM is genetically heterogeneous with four loci mapped to date. Murine Dactylaplasia (Dac ) is phenotypically similar, and it has been mapped to a syntenic region of 10q24, where SHFM3 has been localized. Structural alterations of the gene-encoding dactylin, a constituent of the ubiquitinization pathway, leading to reduced levels of transcript have been identified in Dac . Here, we report a significant decrease of Dactylin transcript in several individuals affected by SHFM. This observation supports a central role for dactylin in the pathogenesis of SHFM. [ABSTRACT FROM AUTHOR]

Blimp is a transcriptional repressor that has been identified in humans, mice and frogs. Here, we describe the cloning of the gene that encodes for the chick homolog of Blimp, cBlimp-1, and the tempo-spatial expression pattern of cBlimp-1, during chick development. cBlimp-1 whose gene product shares a high level of identity with its homologs from other organisms, was found to be expressed in the apical ectodermal ridge and posterior dorsal ectoderm of developing limb buds. In addition, cBlimp-1 is expressed in the developing eyes, branchial arches and otic placodes. [Copyright &y& Elsevier]

Six to eight copies of a transgene integrated into mouse chromosome 15 resulting in a new transgene insertional mutant, Footless, presenting with malformations of the limbs, kidney, and soft palate. Homozygotes possess a unique asymmetric pattern of limb truncations. Posterior structures from the autopod and zeugopod of the hindlimbs are missing with left usually more severely affected than right. In contrast, anterior structures are missing from the right forelimbs. The left forelimb is usually normal except for the absence of the distal telephalanges and nails. These structures are absent on all formed digits. In situ hybridization assays examined the expression of Shh, dHand, Msx2, Fgf8, En1, and Lmx1b in mutant limb buds and indicated normal establishment of the anterior/posterior and dorsal/ventral axes of the developing limbs. However, dysmorphology of the apical ectodermal ridge was observed in the mutant limb buds. [Copyright &y& Elsevier]

We have used whole-mount in situ hybridization to investigate the patterns of c-kit and Sl expression in stage 11–22 chicken embryos. Our analysis shows that c-kit and Sl are expressed quite differently in chicken embryos compared to the reported expression patterns of these genes in embryos of other taxa. Most notably, chicken c-kit is expressed in primordial germ cells as well as in the developing somite, the apical ectodermal ridge, and in the early foregut endoderm. Sl is expressed in the lateral and intermediate mesoderm and in extraembryonic membranes. These data suggest that chicken c-kit and Sl may play novel and unexpected roles in somitogenesis, limb development, and foregut development in avian embryos. [Copyright &y& Elsevier]

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5–E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds. [Copyright &y& Elsevier]

Congenital malformations of the extremities are conspicuous and have been described through the ages. Over the past decade, a wealth of knowledge has been generated regarding the genetic regulation of limb development and the underlying molecular mechanisms. Recent studies have identified several of the signaling molecules, growth factors, and transcriptional regulators involved in the initiation and maintenance of the apical ectodermal ridge (AER) as well as the molecular markers defining the three axes of the developing limb. Studies of abnormal murine phenotypes have uncovered the role played by genes such as p63 and Dactylin in the maintenance of AER activity. These phenotypes resemble human malformations and in this review we describe the underlying mechanisms and clinical associations of split hand/foot malformation and ectrodactyly–ectodermal dysplasia–cleft lip/palate syndrome, which have both been associated with mutations in the p63 gene. [ABSTRACT FROM AUTHOR]

An asymptomatic atypical U shaped cleft hand has been found in a 21-year-old lady attending OPD. On digital skiagram it was found that central digits were absent with remnants of bases of the metacarpals, which have fused with the carpal bones. Moreover, the scaphoid and trapezium had fused to form a single mass. There was no other anomaly in other limbs, so far searched for. An endeavor has been made to explain the anomaly with ontogenetic review. [ABSTRACT FROM AUTHOR]

Members of the CD44 protein family are engaged in various physiological and pathological processes such as lymphocyte activation, lymphocyte homing, wound healing, delayed type hypersensitivity, apoptosis, tumor progression and limb development. Here we summarize observations which lead to a molecular understanding of the function of CD44 in some of these physiological processes. The most striking function for a CD44 variant protein is its presentation of growth factors to high affinity receptors on mesenchymal cells in the developing limb. Furthermore we summarize the experiments that allowed the identification of CD44 variant exon encoded sequences which account for differentiation specific and inducible splice regulation. [ABSTRACT FROM AUTHOR]

The fork head domain-containing gene family (Fox) comprises over 20 members in mammals and is defined by a conserved 110 amino-acid motif containing a winged helix structure DNA-binding domain. The members of this gene family have been implicated as key regulators of embryogenesis, cell cycling, cell lineage restriction and cancer. The Foxn2 gene (Ches1) is expressed in postgastrulation embryos in multiple tissues that serve as important signaling centers as well as end-stage-differentiated cell types that arise from different germ layers of the developing embryo. The dynamic and specific expression of Foxn2 during embryonic development suggest multiple independent roles for Foxn2 function during gestation. [Copyright &y& Elsevier]

Ours is a tertiary care center dealing with a variety of congenital hand anomalies. Recently, a patient with central polydactyly arising mid-palm near the wrist joint and capable of independent movements was encountered. The case and its treatment are described, and polydactyly is discussed. [ABSTRACT FROM AUTHOR]

P-cadherin is a member of the classical cadherin family that forms the transmembrane core of adherens junctions. Recently, mutations in the P-cadherin gene (CDH3) have been shown to cause two inherited diseases in humans: hypotrichosis with juvenile macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly, macular dystrophy (EEM syndrome). The common features of both diseases are sparse hair and macular dystrophy of the retina, while only EEM syndrome shows the additional finding of split hand/foot malformation (SHFM). We identified five consanguineous Pakistani families with either HJMD or EEM syndrome, and detected pathogenic mutations in the CDH3 gene of all five families. In order to define the role of P-cadherin in hair follicle and limb development, we performed expression studies on P-cadherin in the mouse embryo, and demonstrated the predominant expression of P-cadherin not only in the hair follicle placode, but also at the apical ectodermal ridge (AER) of the limb bud. Based on the evidence that mutations in the p63 gene also result in hypotrichosis and SHFM, and that the expression patterns of p63 and P-cadherin overlap in the hair follicle placode and AER, we postulated that CDH3 could be a direct transcriptional target gene of p63. We performed promoter assays and ChIP, which revealed that p63 directly interacts with two distinct regions of the CDH3 promoter. We conclude that P-cadherin is a newly defined transcriptional target gene of p63, with a crucial role in hair follicle morphogenesis as well as the AER during limb bud outgrowth in humans, whereas it is not required for either in mice. [ABSTRACT FROM AUTHOR]

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5–E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds. [Copyright &y& Elsevier]

The fibroblast growth factor (FGF) and β-catenin-dependent Wnt signaling pathways are key regulators of vertebrate limb development. FGF10 induces expression of Wnt3a , which regulates the formation and FGF8 expression of the apical ectodermal ridge (AER). In amelic limbless limbs, an AER fails to form and FGF8 is not expressed, despite expression of FGF10 . It has been found that Wnt3a is initially expressed in limbless ectoderm, although subsequently is drastically reduced. In addition, changes in the expression pattern or level of several Frizzled receptors, Axin, Lef1/Tcf1 and β-catenin have been found in limbless limbs. Notably, while normal wing buds respond to LiCl-stimulated activation of β-catenin-dependent signaling by forming ectopic, FGF8 -expressing AER, LiCl was unable to induce an AER in limbless wing buds. The results of this study suggest that the limbless gene is required for β-catenin-dependent Wnt signaling in limb ectoderm leading to FGF8 expression and AER formation. [ABSTRACT FROM AUTHOR]