The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch

The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.     

Comparative analysis of 3D expression patterns of transcription factor genes and digit fate maps in the developing chick wing

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.     

Hensen's node,but not other biological signallers,can induce supernumerary digits in the developing chick limb bud

Summary The purpose of this study was to determine whether the organizer regions of early avian and amphibian embryos could induce supernumerary (SN) wing structures to develop when they were grafted to a slit in the anterior side of stage 19–23 chick wing buds. Supernumerary digits developed in 43% of the wings that received anterior grafts of Hensen's node from stage 4–6 quail or chick embryos; in addition, 16% of the wings had rods of SN cartilage, but not recognizable SN digits. The grafted quail tissue did not contribute to the SN structures. When tissue anterior or lateral to Hensen's node or lateral pieces of the area pellucida caudal to Hensen's node were grafted to anterior slits, the wings usually developed normally. No SN structures developed when Hensen's nodes were grafted to posterior slits in chick wing buds. Wings developed normally when pieces of the dorsal lip of the blastopore from stage 10–11.5 frog (Xenopus laevis and Rana pipiens) embryos were grafted to anterior slits. No SN digits developed when other tissues that have limb-inducing activity in adult urodele amphibians [chick otic vesicle, frog (Rana pipiens) lung and kidney] or that can act as heteroinductors in neural induction (rat kidney, lung, submaxillary gland and urinary bladder; mouse liver and submaxillary gland) were grafted to anterior slits in chick wing buds. SN digits also failed to develop following preaxial grafts of chick optic vesicles. These results suggest that although the anteroposterior polarity of the chick wing bud can be influenced by factors other than the ZPA (e.g., Hensen's node, retinoids), the wing is not so labile that it can respond to a wide variety of inductively-active tissues.     

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.     

Expression of a homeobox gene in the chick wing bud following application of retinoic acid and grafts of polarizing region tissue.

Homeobox gene XlHbox 1 is expressed in a mesodermal gradient in vertebrate forelimbs with maximal expression anteriorly and proximally and may encode positional values. In chick wing buds, anterior cells can be reprogrammed to form posterior structures by grafts of polarizing region tissue and by beads soaked in retinoic acid (RA), which is a good candidate for an endogenous morphogen. Applications of RA anteriorly or at the bud apex, treatments which produce duplicated digits or truncations respectively, substantially increase the extent of mesodermal XlHbox 1 expression. Polarizing region grafts that also produce additional digits lead to a moderate increase. The effects of RA application and the behaviour of transplanted tissue show that only anterior cells are competent to express XlHbox 1 and that expression is cell autonomous. Ectodermal expression in wing buds is enhanced by RA but not by polarizing region grafts and ectoderm/mesoderm recombinations show that the mesoderm is irreversibly affected. The changes in mesodermal expression do not fit the predictions of the simple model that XlHbox 1 encodes anterior positional values but are correlated with a series of novel malformations of the shoulder girdle which, in normal wing buds, is derived from cells expressing XlHbox 1.     

qPCR array检测分析 C57BL/6小鼠胚胎后肢发育基因表达谱

目的:胚胎生育过程中因肢体发育异常造成的出生缺陷比率不低,其相关基因表达模式尚不明确。本实验通过建立实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究C57BL/6品系小鼠后肢发育相关基因的表达谱。方法:以同源异形盒基因家族(Hox)、Wnt5a、配对同源结构域基因(Pitx1)、成纤维生长因子(Fgf8)、音猬因子(Shh)等小鼠肢体发育相关的重要基因制作基因检测表达谱,以C57BL/6品系怀孕雌鼠为材料,取胚胎肢芽发育的四个关键时期(E10.5,E11.5,E12.5,E13.5)的胎鼠后肢,利用qPCR array方案检测表达谱中基因的相对表达水平差异。结果:通过已建立的qPCR array检测了C57BL/6品系小鼠胚胎后肢发育时期Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因的表达差异。以E10.5为对照,检测出在后肢发育时期基因呈三种表达模式,即Hoxb6、Hoxb8、Hoxc8、Hoxc9、Hoxc10、Hoxd9和Shh基因的表达水平呈上调;Hoxa11、Hoxa13、Hoxc12、Hoxc13、Hoxd13等基因表达出现下调;Hoxc9、Hoxc10、Hoxc11、Hoxd9、Hoxd12、Fgf8和Pitx1等基因的相对表达量呈先上调后下调的曲线表达模式,且有少部分基因在小鼠后肢发育时期表达水平无明显变化。结论:Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因在小鼠后肢发育时期表达,并且表达模式存在明显差异。     

Bambi and Sp8 Expression Mark Digit Tips and Their Absence Shows That Chick Wing Digits 2 and 3 Are Truncated

An often overlooked aspect of digit development is the special nature of the terminal phalanx, a specialized structure with characteristics distinct from other phalanges, for example the presence of ectodermal derivatives such as nails and claws. Here, we describe the unique ossification pattern of distal phalanges and characteristic gene expression in the digit tips of chick and duck embryos. Our results show that the distal phalanx of chick wing digit 1 is a genuine tip with a characteristic ossification pattern and expression of Bambi and Sp8; however, the terminal phalanx of digits 2* and 3 is not a genuine tip, and these are therefore truncated digits. Bambi and Sp8 expression in the chick wing provides a direct molecular assessment of digit identity changes after experimental manipulations of digit primordia. In contrast, digits 1 and 2 of the duck wing both possess true tips. Although chick wing-tip development was not rescued by application of Fgf8, this treatment induced the development of extra phalanges. Grafting experiments show that competence for tip formation, including nails, is latent in the interdigital tissue. Our results deepen understanding of the mechanisms of digit tip formation, highlighting its developmental autonomy and modular nature, with implications for digit reduction or loss during evolution. * Numbering of wing digits is 1, 2, 3 from anterior to posterior.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

The formation of supernumerary structures after grafting anterior quail wing bud mesoderm of various ages into chick wing buds

Wedges of anterior quail mesoderm grafted into posterior slits in the wing buds of chick embryo hosts result in the formation of rods and nodules of supernumerary cartilage in a high percentage of cases. Identifiable digits do not form unless the ectoderm is allowed to remain on the grafts. Control experiments have shown that wedges of anterior or posterior wing mesoderm placed into homologous locations of host wing buds produce few or no supernumerary skeletal structures. Anterior-to-posterior grafts of stage 17 mesoderm evoke a 71.4% incidence of supernumerary cartilage. This percentage increases to 100% with stage 22 donor mesoderm. The percentage of supernumerary structures formed declines markedly with donor mesoderm of stages 24-30. By stages 35-36, only 10% of the grafts result in the formation of supernumerary structures. The period of decline coincides with the onset of overt cytodifferentiation within the donor mesoderm.     

How the pterosaur got its wings

Throughout the evolutionary history of life, only three vertebrate lineages took to the air by acquiring a body plan suitable for powered flight: birds, bats, and pterosaurs. Because pterosaurs were the earliest vertebrate lineage capable of powered flight and included the largest volant animal in the history of the earth, understanding how they evolved their flight apparatus, the wing, is an important issue in evolutionary biology. Herein, I speculate on the potential basis of pterosaur wing evolution using recent advances in the developmental biology of flying and non‐flying vertebrates. The most significant morphological features of pterosaur wings are: (i) a disproportionately elongated fourth finger, and (ii) a wing membrane called the brachiopatagium, which stretches from the posterior surface of the arm and elongated fourth finger to the anterior surface of the leg. At limb‐forming stages of pterosaur embryos, the zone of polarizing activity (ZPA) cells, from which the fourth finger eventually differentiates, could up‐regulate, restrict, and prolong expression of 5′‐located Homeobox D (Hoxd) genes (e.g. Hoxd11, Hoxd12, and Hoxd13) around the ZPA through pterosaur‐specific exploitation of sonic hedgehog (SHH) signalling. 5′Hoxd genes could then influence downstream bone morphogenetic protein (BMP) signalling to facilitate chondrocyte proliferation in long bones. Potential expression of Fgf10 and Tbx3 in the primordium of the brachiopatagium formed posterior to the forelimb bud might also facilitate elongation of the phalanges of the fourth finger. To establish the flight‐adapted musculoskeletal morphology shared by all volant vertebrates, pterosaurs probably underwent regulatory changes in the expression of genes controlling forelimb and pectoral girdle musculoskeletal development (e.g. Tbx5), as well as certain changes in the mode of cell–cell interactions between muscular and connective tissues in the early phase of their evolution. Developmental data now accumulating for extant vertebrate taxa could be helpful in understanding the cellular and molecular mechanisms of body‐plan evolution in extinct vertebrates as well as extant vertebrates with unique morphology whose embryonic materials are hard to obtain.     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiative response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.