Talk:Musculoskeletal System - Limb Development

From Embryology
About Discussion Pages  
Mark Hill.jpg
On this website the Discussion Tab or "talk pages" for a topic has been used for several purposes:
  1. References - recent and historic that relates to the topic
  2. Additional topic information - currently prepared in draft format
  3. Links - to related webpages
  4. Topic page - an edit history as used on other Wiki sites
  5. Lecture/Practical - student feedback
  6. Student Projects - online project discussions.
Links: Pubmed Most Recent | Reference Tutorial | Journal Searches

Glossary Links

Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | Numbers | Symbols | Term Link

Cite this page: Hill, M.A. (2021, September 24) Embryology Musculoskeletal System - Limb Development. Retrieved from


Bastida MF, Pérez-Gómez R, Trofka A, Zhu J, Rada-Iglesias A, Sheth R, Stadler HS, Mackem S & Ros MA. (2020). The formation of the thumb requires direct modulation of Gli3 transcription by Hoxa13. Proc. Natl. Acad. Sci. U.S.A. , 117, 1090-1096. PMID: 31896583 DOI.

The formation of the thumb requires direct modulation of Gli3 transcription by Hoxa13.

Abstract In the tetrapod limb, the digits (fingers or toes) are the elements most subject to morphological diversification in response to functional adaptations. However, despite their functional importance, the mechanisms controlling digit morphology remain poorly understood. Here we have focused on understanding the special morphology of the thumb (digit 1), the acquisition of which was an important adaptation of the human hand. To this end, we have studied the limbs of the Hoxa13 mouse mutant that specifically fail to form digit 1. We show that, consistent with the role of Hoxa13 in Hoxd transcriptional regulation, the expression of Hoxd13 in Hoxa13 mutant limbs does not extend into the presumptive digit 1 territory, which is therefore devoid of distal Hox transcripts, a circumstance that can explain its agenesis. The loss of Hoxd13 expression, exclusively in digit 1 territory, correlates with increased Gli3 repressor activity, a Hoxd negative regulator, resulting from increased Gli3 transcription that, in turn, is due to the release from the negative modulation exerted by Hox13 paralogs on Gli3 regulatory sequences. Our results indicate that Hoxa13 acts hierarchically to initiate the formation of digit 1 by reducing Gli3 transcription and by enabling expansion of the 5'Hoxd second expression phase, thereby establishing anterior-posterior asymmetry in the handplate. Our work uncovers a mutual antagonism between Gli3 and Hox13 paralogs that has important implications for Hox and Gli3 gene regulation in the context of development and evolution. KEYWORDS: Gli3; Hox genes; limb development; thumb PMID: 31896583 DOI: 10.1073/pnas.1919470117


A point mutation in the pre-ZRS disrupts sonic hedgehog expression in the limb bud and results in triphalangeal thumb-polysyndactyly syndrome

Genet Med. 2018 Mar 15. doi: 10.1038/gim.2018.18. [Epub ahead of print]

Potuijt JWP1, Baas M1, Sukenik-Halevy R2,3,4, Douben H5, Nguyen P1, Venter DJ6,7,8, Gallagher R6, Swagemakers SM9,10, Hovius SER1, van Nieuwenhoven CA1, Galjaard RH5, van der Spek PJ9,10, Ahituv N2,3, de Klein A5.


PurposeThe zone of polarizing activity regulatory sequence (ZRS) is an enhancer that regulates sonic hedgehog during embryonic limb development. Recently, mutations in a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS, termed the pre-ZRS (pZRS), have been associated with polydactyly in dogs and humans. Here, we report the first case of triphalangeal thumb-polysyndactyly syndrome (TPT-PS) to be associated with mutations in this region and show via mouse enhancer assays how this mutation leads to ectopic expression throughout the developing limb bud.MethodsWe used linkage analysis, whole-exome sequencing, Sanger sequencing, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, single-nucleotide polymorphism array, and a mouse transgenic enhancer assay.ResultsTen members of a TPT-PS family were included in this study. The mutation was linked to chromosome 7q36 (LOD score 3.0). No aberrations in the ZRS could be identified. A point mutation in the pZRS (chr7:156585476G>C; GRCh37/hg19) was detected in all affected family members. Functional characterization using a mouse transgenic enhancer essay showed extended ectopic expression dispersed throughout the entire limb bud (E11.5).ConclusionOur work describes the first mutation in the pZRS to be associated with TPT-PS and provides functional evidence that this mutation leads to ectopic expression of this enhancer within the developing limb.GENETICS in MEDICINE advance online publication, 15 March 2018; doi:10.1038/gim.2018.18. PMID: 29543231 DOI: 10.1038/gim.2018.18

The chick limb: embryology, genetics and teratology

Int J Dev Biol. 2018;62(1-2-3):85-95. doi: 10.1387/ijdb.170315CT.

Davey MG1, Towers M, Vargesson N, Tickle C. Author information Abstract The chick embryo has a long history in investigations of vertebrate limb development because of the ease with which its limbs can be experimentally manipulated. Early studies elucidated the fundamental embryology of the limb and identified the key signalling regions that govern its development. The chick limb became a leading model for exploring the concept of positional information and understanding how patterns of differentiated cells and tissues develop in vertebrate embryos. When developmentally important molecules began to be identified, experiments in chick limbs were crucial for bridging embryology and molecular biology. The embryological mechanisms and molecular basis of limb development are largely conserved in mammals, including humans, and uncovering these molecular networks provides links to clinical genetics. We emphasise the important contributions of naturally occurring chick mutants to elucidating limb embryology and identifying novel developmentally important genes. In addition, we consider how the chick limb has been used to study mechanisms involved in teratogenesis with a focus on thalidomide. These studies on chick embryos have given insights into how limb defects can be caused by both genetic changes and chemical insults and therefore are of great medical significance. PMID: 29616743 DOI: 10.1387/ijdb.170315CT


Digits and fin rays share common developmental histories

Nakamura T, et al. Nature. 2016.


Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide a functional analysis, using CRISPR/Cas9 and fate mapping, of 5' hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods. We show that cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, we find that a marked reduction and loss of fin rays is associated with an increased number of endochondral distal radials. These discoveries reveal a cellular and genetic connection between the fin rays of fish and the digits of tetrapods and suggest that digits originated via the transition of distal cellular fates.

PMID 27533041

A somitic contribution to the apical ectodermal ridge is essential for fin formation

Nature. 2016 Jul 28;535(7613):542-6.

Masselink W, Cole NJ, Fenyes F, Berger S, Sonntag C, Wood A, Nguyen PD, Cohen N, Knopf F, Weidinger G, Hall TE, Currie PD.

Abstract The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

PMID 27437584


RA Acts in a Coherent Feed-Forward Mechanism with Tbx5 to Control Limb Bud Induction and Initiation

Cell Rep. 2015 Aug 4;12(5):879-91. doi: 10.1016/j.celrep.2015.06.068. Epub 2015 Jul 23.

Nishimoto S1, Wilde SM1, Wood S2, Logan MP3.


The retinoic acid (RA)- and β-catenin-signaling pathways regulate limb bud induction and initiation; however, their mechanisms of action are not understood and have been disputed. We demonstrate that both pathways are essential and that RA and β-catenin/TCF/LEF signaling act cooperatively with Hox gene inputs to directly regulate Tbx5 expression. Furthermore, in contrast to previous models, we show that Tbx5 and Tbx4 expression in forelimb and hindlimb, respectively, are not sufficient for limb outgrowth and that input from RA is required. Collectively, our data indicate that RA signaling and Tbx genes act in a coherent feed-forward loop to regulate Fgf10 expression and, as a result, establish a positive feedback loop of FGF signaling between the limb mesenchyme and ectoderm. Our results incorporate RA-, β-catenin/TCF/LEF-, and FGF-signaling pathways into a regulatory network acting to recruit cells of the embryo flank to become limb precursors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

PMID 26212321

Engrailed 1 mediates correct formation of limb innervation through two distinct mechanisms

PLoS One. 2015 Feb 24;10(2):e0118505. doi: 10.1371/journal.pone.0118505. eCollection 2015.

Huettl RE1, Luxenhofer G1, Bianchi E1, Haupt C2, Joshi R3, Prochiantz A3, Huber AB1.


Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms.

PMID 25710467

How the embryo makes a limb: determination, polarity and identity

J Anat. 2015 Aug 7. doi: 10.1111/joa.12361. [Epub ahead of print]

Tickle C.


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. © 2015 Anatomical Society. KEYWORDS: Hox genes; Pitx1; Sonic hedgehog; Tbx4/5; Wnts; antero-posterior polarity; apical ectodermal ridge; bone morphogenetic proteins; dorso-ventral polarity; embryo; fibroblast growth factors; lateral plate mesoderm; limb; limb bud; polarizing region; retinoic acid

PMID 26249743


Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development

PLoS Genet. 2014 Aug 28;10(8):e1004468. doi: 10.1371/journal.pgen.1004468. eCollection 2014.

Haro E1, Delgado I1, Junco M1, Yamada Y2, Mansouri A3, Oberg KC4, Ros MA5.


The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/βcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.

PMID 25166858

HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme

Dev Cell. 2014 Nov 10;31(3):345-57. doi: 10.1016/j.devcel.2014.09.018. Epub 2014 Nov 10.

Osterwalder M1, Speziale D1, Shoukry M2, Mohan R3, Ivanek R1, Kohler M4, Beisel C4, Wen X5, Scales SJ5, Christoffels VM3, Visel A6, Lopez-Rios J7, Zeller R8.


The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

PMID 25453830

Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb

Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):8844-9. doi: 10.1073/pnas.1407606111. Epub 2014 Jun 3.

Mayeuf-Louchart A1, Lagha M1, Danckaert A2, Rocancourt D1, Relaix F1, Vincent SD1, Buckingham M3.

Abstract Multipotent Pax3-positive (Pax3(+)) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. Overactivating the Notch pathway specifically in Pax3(+) progenitors, via a conditional Pax3(NICD) allele, results in an increase of the number of smooth muscle and endothelial cells contributing to the aorta. At limb level, Pax3(+) cells in the somite give rise to skeletal muscles and to a subpopulation of endothelial cells in blood vessels of the limb. We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3(+) cells in the somite of the mouse embryo.

PMID 24927569


Tbx2 terminates shh/fgf signaling in the developing mouse limb bud by direct repression of gremlin1

PLoS Genet. 2013;9(4):e1003467. doi: 10.1371/journal.pgen.1003467. Epub 2013 Apr 25.

Farin HF1, Lüdtke TH, Schmidt MK, Placzko S, Schuster-Gossler K, Petry M, Christoffels VM, Kispert A.


Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.

PMID 23633963

The evolution of lineage-specific regulatory activities in the human embryonic limb

Cell. 2013 Jul 3;154(1):185-96. doi: 10.1016/j.cell.2013.05.056.

Cotney J1, Leng J, Yin J, Reilly SK, DeMare LE, Emera D, Ayoub AE, Rakic P, Noonan JP.

Abstract The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution. Copyright © 2013 Elsevier Inc. All rights reserved. Comment in Evolutionary biology: The handiwork of tinkering. [Nature. 2013] PMID 23827682

How is digit identity determined during limb development?

Dev Growth Differ. 2013 Jan;55(1):130-8. doi: 10.1111/dgd.12022. Epub 2012 Dec 12.

Suzuki T.


Digit identity has been studied using the chick embryo as a model system for more than 40 years. Using this model system, several milestone findings have been reported, such as the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), the Shh gene, and the theory of morphogen and positional information. These experimental results and models provided context for understanding pattern formation in developmental biology. The focus of this review is on the determination of digit identity during limb development. First, the history of studies on digit identity determination is described, followed by descriptions of the molecular mechanisms and current models for determination of digit identity. Finally, future questions and remarkable points will be discussed. © 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

PMID 23230964


Development and morphogenesis of human wrist joint during embryonic and early feral period

J Anat. 2012 Jun;220(6):580-90. doi: 10.1111/j.1469-7580.2012.01496.x. Epub 2012 Mar 19.

Hita-Contreras F1, Martínez-Amat A, Ortiz R, Caba O, Alvarez P, Prados JC, Lomas-Vega R, Aránega A, Sánchez-Montesinos I, Mérida-Velasco JA. Author information


The development of the human wrist joint has been studied widely, with the main focus on carpal chondrogenesis, ligaments and triangular fibrocartilage. However, there are some discrepancies concerning the origin and morphogenetic time-table of these structures, including nerves, muscles and vascular elements. For this study we used serial sections of 57 human embryonic (n = 30) and fetal (n = 27) specimens from O'Rahilly stages 17-23 and 9-14 weeks, respectively. The following phases in carpal morphogenesis have been established: undifferentiated mesenchyme (stage 17), condensated mesenchyme (stages 18 and 19), pre-chondrogenic (stages 19 and 20) and chondrogenic (stages 21 and over). Carpal chondrification and osteogenic processes are similar, starting with capitate and hamate (stage 19) and ending with pisiform (stage 22). In week 14, a vascular bud penetrates into the lunate cartilaginous mold, early sign of the osteogenic process that will be completed after birth. In stage 18, median, ulnar and radial nerves and thenar eminence appear in the hand plate. In stage 21, there are indications of the interosseous muscles, and in stage 22 flexor digitorum superficialis, flexor digitorum profundus and lumbrical muscles, transverse carpal ligament and collateral ligaments emerge. In stage 23, the articular disc, radiocarpal and ulnocarpal ligaments and deep palmar arterial arch become visible. Radiate carpal and interosseous ligaments appear in week 9, and in week 10, dorsal radiocarpal ligament and articular capsule are evident. Finally, synovial membrane is observed in week 13. We have performed a complete analysis of the morphogenesis of the structures of the human wrist joint. Our results present new data on nervous and arterial elements and provide the basis for further investigations on anatomical pathology, comparative morphology and evolutionary anthropology. © 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.

PMID 22428933

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1 expression in mouse limb buds

Aimée Zuniga, Frédéric Laurent, Javier Lopez-Rios, Christian Klasen, Nicolas Matt and Rolf Zeller

BMC Developmental Biology 2012, 12:23 doi:10.1186/1471-213X-12-23

Published: 13 August 2012

Transient downregulation of Bmp signalling induces extra limbs in vertebrates

Development. 2012 Jun 6. [Epub ahead of print]

Christen B, Cavaco Rodrigues AM, Barragán Monasterio M, Fabregat Roig C, Izpisua Belmonte JC. Abstract Bone morphogenetic protein (Bmp) signalling has been implicated in setting up dorsoventral patterning of the vertebrate limb and in its outgrowth. Here, we present evidence that Bmp signalling or, more precisely, its inhibition also plays a role in limb and fin bud initiation. Temporary inhibition of Bmp signalling either by overexpression of noggin or using a synthetic Bmp inhibitor is sufficient to induce extra limbs in the Xenopus tadpole or exogenous fins in the Danio rerio embryo, respectively. We further show that Bmp signalling acts in parallel with retinoic acid signalling, possibly by inhibiting the known limb-inducing gene wnt2ba.

PMID 22675213


Global Gene Expression Analysis of Murine Limb Development

Detailed information about stage-specific changes in gene expression is crucial for understanding the gene regulatory networks underlying development and the various signal transduction pathways contributing to morphogenesis. Here we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature and nerves are specified and the musculoskeletal system of limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore- and hind-limb patterning. We found that the onset of limb formation is characterized by an up-regulation of transcription factors, which is followed by a massive activation of genes during E10.5 and E11.5 which levels off at later time points. Among the 3520 genes identified as significantly up-regulated in the limb, we find ~30% to be novel, dramatically expanding the repertoire of candidate genes likely to function in the limb. Hierarchical and stage-specific clustering identified expression profiles that are likely to correlate with functional programs during limb development and further characterization of these transcripts will provide new insights into specific tissue patterning processes. Here, we provide for the first time a comprehensive analysis of developmentally regulated genes during murine limb development, and provide some novel insights into the expression dynamics governing limb morphogenesis.

PMID 22174793

Special Issue: Special Issue on Limb Development

Developmental Dynamics - Special Issue: Special Issue on Limb Development May 2011 Volume 240, Issue 5

Origin of vertebrate limb muscle: the role of progenitor and myoblast populations

Curr Top Dev Biol. 2011;96:1-32.

Murphy M, Kardon G. Source Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA. Abstract Muscle development, growth, and regeneration take place throughout vertebrate life. In amniotes, myogenesis takes place in four successive, temporally distinct, although overlapping phases. Understanding how embryonic, fetal, neonatal, and adult muscle are formed from muscle progenitors and committed myoblasts is an area of active research. In this review we examine recent expression, genetic loss-of-function, and genetic lineage studies that have been conducted in the mouse, with a particular focus on limb myogenesis. We synthesize these studies to present a current model of how embryonic, fetal, neonatal, and adult muscle are formed in the limb.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID 21621065

Keeping up with the zone of polarizing activity: New roles for an old signalling center

Dev Dyn. 2011 May;240(5):915-9. doi: 10.1002/dvdy.22597. Epub 2011 Feb 28.

Harfe BD. Source Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College of Medicine, Gainesville, Florida, USA. Abstract The vertebrate limb is an excellent model organ system to investigate how signaling pathways interact. Over the last half-century, experiments investigating patterning in the vertebrate limb have led directly to the discovery of many of the molecules and molecular pathways that are not only responsible for limb patterning but the patterning of many different organ systems. In the limb bud, the zone of polarizing activity (ZPA) has long been known to produce factor(s) that are essential for normal limb formation. Recently, one of these factors, Sonic Hedgehog (SHH) was shown to function not only in the limb bud mesenchyme but also in the ectoderm overlying the ZPA. This review discusses the role and potential implications hedgehog signaling in the limb bud ectoderm plays in patterning the vertebrate limb.

Copyright © 2011 Wiley-Liss, Inc.

PMID 21360795

3D reconstructions of quail-chick chimeras provide a new fate map of the avian scapula

Dev Biol. 2011 Apr 16. [Epub ahead of print]

Shearman RM, Tulenko FJ, Burke AC.


Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID: 21527257

Axial Hox9 activity establishes the posterior field in the developing forelimb

Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4888-91. Epub 2011 Mar 7.

Xu B, Wellik DM.

Department of Internal Medicine, Division of Molecular Medicine and Genetics and Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2200.


Current models hold that the early limb field becomes polarized into anterior and posterior domains by the opposing activities of Hand2 and Gli3. This polarization is essential for the initiation of Shh expression in the posterior margin of the limb bud, but how this polarity is established is not clear. Here we show that initial anteroposterior polarization of the early forelimb field requires the function of all four Hox9 paralogs (Hoxa9, Hoxb9, Hoxc9, and Hoxd9). This is unexpected, given that only HoxA and HoxD AbdB group genes have been shown to play a role in forelimb patterning, regulating the activation and maintenance of Shh expression and subsequent proximal-distal patterning of the forelimb. Our analysis of Hox9 quadruple mutants demonstrates that Hox9 function is required for the expression of Hand2 in the posterior limb field. Subsequently, Gli3 expression is not repressed posteriorly, Shh expression is not initiated, and collinear expression of HoxA/D10-13 is not established, resulting in severely malformed forelimbs lacking all posterior, Shh-regulated elements. This Hox9 mutant phenotype is restricted to the forelimbs; mutant hindlimbs are normal, revealing fundamental differences in the patterning mechanisms governing the establishment of forelimb and hindlimb fields.

PMID: 21383175


Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development

PLoS Genet. 2010 Apr 8;6(4):e1000901.

Galli A, Robay D, Osterwalder M, Bao X, Bénazet JD, Tariq M, Paro R, Mackem S, Zeller R.

Developmental Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland. Abstract The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

PMID: 20386744

Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning

Hasson P, DeLaurier A, Bennett M, Grigorieva E, Naiche LA, Papaioannou VE, Mohun TJ, Logan MP. Dev Cell. 2010 Jan 19;18(1):148-56. PMID: 20152185

Genetic evidence that SOST inhibits WNT signaling in the limb

Dev Biol. 2010 Jun 15;342(2):169-79. Epub 2010 Mar 30.

Collette NM, Genetos DC, Murugesh D, Harland RM, Loots GG.

Biology and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.

Abstract SOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior-posterior and proximal-distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost(-/-) significantly rescues the Lrp6(-/-) skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6.

"Our findings are also consistent with recent reports that BMP signaling negatively regulates bone mass by induction of sclerostin which then inhibits the canonical WNT pathway (Kamiya et al., 2008)."

Copyright 2010 Elsevier Inc. All rights reserved.

PMID: 20359476

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

Geetha-Loganathan P, Nimmagadda S, Christ B, Huang R, Scaal M. BMC Dev Biol. 2010 Mar 25;10:32. PMID: 20334703

BACKGROUND: Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis.

RESULTS: In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II.

CONCLUSION: Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

The role of spatially controlled cell proliferation in limb bud morphogenesis

Boehm B, Westerberg H, Lesnicar-Pucko G, Raja S, Rautschka M, Cotterell J, Swoger J, Sharpe J. PLoS Biol. 2010 Jul 13;8(7):e1000420. PMID: 20644711

EMBL-CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), UPF, Barcelona, Spain.


Although the vertebrate limb bud has been studied for decades as a model system for spatial pattern formation and cell specification, the cellular basis of its distally oriented elongation has been a relatively neglected topic by comparison. The conventional view is that a gradient of isotropic proliferation exists along the limb, with high proliferation rates at the distal tip and lower rates towards the body, and that this gradient is the driving force behind outgrowth. Here we test this hypothesis by combining quantitative empirical data sets with computer modelling to assess the potential role of spatially controlled proliferation rates in the process of directional limb bud outgrowth. In particular, we generate two new empirical data sets for the mouse hind limb--a numerical description of shape change and a quantitative 3D map of cell cycle times--and combine these with a new 3D finite element model of tissue growth. By developing a parameter optimization approach (which explores spatial patterns of tissue growth) our computer simulations reveal that the observed distribution of proliferation rates plays no significant role in controlling the distally extending limb shape, and suggests that directional cell activities are likely to be the driving force behind limb bud outgrowth. This theoretical prediction prompted us to search for evidence of directional cell orientations in the limb bud mesenchyme, and we thus discovered a striking highly branched and extended cell shape composed of dynamically extending and retracting filopodia, a distally oriented bias in Golgi position, and also a bias in the orientation of cell division. We therefore provide both theoretical and empirical evidence that limb bud elongation is achieved by directional cell activities, rather than a PD gradient of proliferation rates.

PMID: 20644711 <pubmed>20644711</pubmed>| PMC2903592 | PLoS

Citation: Boehm B, Westerberg H, Lesnicar-Pucko G, Raja S, Rautschka M, et al. (2010) The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis. PLoS Biol 8(7): e1000420. doi:10.1371/journal.pbio.1000420

Copyright: © 2010 Boehm et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Limbs made to measure.

Kicheva A, Briscoe J. PLoS Biol. 2010 Jul 13;8(7):e1000421. No abstract available. PMID: 20644713

<pubmed>20644713</pubmed>| PMC2903596 | PLoS

Citation: Kicheva A, Briscoe J (2010) Limbs Made to Measure. PLoS Biol 8(7): e1000421. doi:10.1371/journal.pbio.1000421

Published: July 13, 2010

Copyright: © 2010 Kicheva, Briscoe. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The temporal dynamics of vertebrate limb development, teratogenesis and evolution

Curr Opin Genet Dev. 2010 Aug;20(4):384-90. Epub 2010 May 27.

Zeller R.

Developmental Genetics, Department of Biomedicine, University of Basel Medical Faculty, Mattenstrasse 28, Basel, Switzerland. Abstract Recent genetic and functional analysis of vertebrate limb development begins to reveal how the functions of particular genes and regulatory hierarchies can drastically change over time. The temporal and spatial interplay of the two instructive signalling centres are part of a larger signalling system that orchestrates limb bud morphogenesis in a rather self-regulatory manner. It appears that mesenchymal cells are specified early and subsequently, the progenitors for the different skeletal elements are expanded and determined progressively during outgrowth. Mutations and teratogens that disrupt distal progression of limb development most often cause death of the early-specified progenitors rather than altering their fates. The proliferative expansion and distal progression of paired appendage development was one of the main driving forces behind the transition from fin to limb buds during paired appendage evolution. Finally, the adaptive diversification or loss of modern tetrapod limbs in particular phyla or species appear to be a consequence of evolutionary tampering with the regulatory systems that control distal progression of limb development.

PMID: 20537528


Avian pelvis originates from lateral plate mesoderm and its development requires signals from both ectoderm and paraxial mesoderm

Cell Tissue Res. 2008 Mar;331(3):595-604. Epub 2007 Dec 18.

Malashichev Y, Christ B, Pröls F.

Department of Vertebrate Zoology, Faculty of Biology and Soil Sciences, St Petersburg State University, Universitetskaya nab, St Petersburg, Russia. Abstract The pelvic girdle is composed of three skeletal elements: ilium, pubis, and ischium. In comparison with other parts of the postcranial skeleton, its development is not well known to date. To elucidate the embryonic origin of the avian pelvic girdle and the signaling centers that control its development, we have performed extirpation and quail-to-chick grafting experiments. The results reveal that the entire pelvic girdle originates from the somatopleure at somite levels 26 to 35. No somitic cell contribution to skeletal elements of the pelvis has been detected. Removal of the surface ectoderm covering the lateral plate mesoderm has revealed that ectodermal signals control the development of the pelvic girdle, especially the formation of the pubis and ischium. The impaired development of the ischium and pubis correlates with the downregulation of Pax1 and Alx4, two transcription factors that control the normal development of the ischium and pubis. Although of somatopleural origin, the development of the ilium depends on somitic signals. Insertion of a barrier between somites and somatopleure disrupts the expression of Emx2 and prevents normal development of the ilium but does not affect the expression of Pax1 or Alx4 and the development of the pubis and ischium. Thus, the development of the ilium, but not of the pubis and ischium, depends on somitic and ectodermal signals.

PMID: 18087724


Six proteins regulate the activation of Myf5 expression in embryonic mouse limbs

Giordani J, Bajard L, Demignon J, Daubas P, Buckingham M, Maire P. Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11310-5. Epub 2007 Jun 25. PMID: 17592144

Département de Génétique et Développement, Institut Cochin, Université Paris Descartes, Paris, France.

Abstract Myf5, a member of the myogenic regulatory factor family, plays a major role in determining myogenic cell fate at the onset of skeletal muscle formation in the embryo. Spatiotemporal control of its expression during development requires multiple enhancer elements spread over >100 kb at the Myf5 locus. Transcription in embryonic limbs is regulated by a 145-bp element located at -57.5 kb from the Myf5 gene. In the present study we show that Myf5 expression is severely impaired in the limb buds of Six1(-/-) and Six1(-/-)Six4(-/+) mouse mutants despite the presence of myogenic progenitor cells. The 145-bp regulatory element contains a sequence that binds Six1 and Six4 in electromobility shift assays in vitro and in chromatin immunoprecipitation assays with embryonic extracts. We further show that Six1 is able to transactivate a reporter gene under the control of this sequence. In vivo functionality of the Six binding site is demonstrated by transgenic analysis. Mutation of this site impairs reporter gene expression in the limbs and in mature somites where the 145-bp regulatory element is also active. Six1/4 therefore regulate Myf5 transcription, together with Pax3, which was previously shown to be required for the activity of the 145-bp element. Six homeoproteins, which also directly regulate the myogenic differentiation gene Myogenin and lie genetically upstream of Pax3, thus control hypaxial myogenesis at multiple levels.

PMID 17592144

Hedgehog signaling regulates the amount of hypaxial muscle development during Xenopus myogenesis

Martin BL, Peyrot SM, Harland RM. Dev Biol. 2007 Apr 15;304(2):722-34. Epub 2007 Feb 7.

PMID 17320852

Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo

Grifone R, Demignon J, Giordani J, Niro C, Souil E, Bertin F, Laclef C, Xu PX, Maire P. Dev Biol. 2007 Feb 15;302(2):602-16. Epub 2006 Sep 1.

PMID 17098221

Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

BMC Evol Biol. 2007 Oct 1;7:182.

Bininda-Emonds OR, Jeffery JE, Sánchez-Villagra MR, Hanken J, Colbert M, Pieau C, Selwood L, Ten Cate C, Raynaud A, Osabutey CK, Richardson MK.

Institute of Biology, University of Leiden, Kaiserstraat 63, 2311GP, Leiden, The Netherlands.

Abstract BACKGROUND: Tetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony) to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb.

RESULTS: An overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons) and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs.

CONCLUSION: Major heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. Our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology.

PMID 17908305


Fetal development of the hand, digits and digit ratio (2D:4D)

Early Hum Dev. 2006 Jul;82(7):469-75. Epub 2006 Feb 13.

Malas MA, Dogan S, Evcil EH, Desdicioglu K. Source Medical Faculty, Department of Anatomy, Süleyman Demirel University, 32260 Isparta, Turkey.


OBJECTIVE: The purpose of this study was to investigate growth patterns in human hands, digits and digit ratio (2D:4D) during the fetal period. METHODS: The study is carried out on 161 human fetuses (83 males, 78 females) free from external pathology or malformation with ages ranging between 9 and 40 weeks of gestation. Following general external measurements, length and width of the hand, digit lengths separate for each hand was measured, hand index and the ratio of the lengths of the 2nd finger to the 4th finger (2D:4D index) was computed. RESULTS: Means and standard deviations of the parameters with respect to gestational weeks, months and trimesters were calculated. There was a significant correlation between all parameters and gestational age (p<0.001). No significant differences were observed between sexes or sides for any of the parameters (p>0.05). 2D:4D ratio was significantly higher in females compared to males (p<0.05) and mean 2D:4D did not change with gestational age. CONCLUSION: Detailed information of hand and digit parameters related to the fetal period will reveal the extent of biological variations of hand and digit parameters to be used in future studies. We hope that data acquired in this study will facilitate other studies on hand and digit anomalies, pathologies and variations as well as diagnoses and treatments of such conditions conducted in obstetrics, perinatology, forensic medicine and fetal pathology departments.

PMID 16473482


A two-step mechanism for myotome formation in chick

Dev Cell. 2004 Jun;6(6):875-82.

Gros J, Scaal M, Marcelle C.

Laboratoire de Génétique et Physiologie du Développement, Developmental Biology Institute of Marseille, CNRS/INSERM, Université de la Méditerranée, 13288 Marseille, France. Abstract The study of the morphogenetic cell movements underlying myotome formation in the chick embryo has led to the emergence of highly controversial models. Here we report a real-time cell lineage analysis of myotome development using electroporation of a GFP reporter in newly formed chick somites. Confocal analysis of cell movements demonstrates that myotome formation involves two sequential steps. In a first phase, incremental myotome growth results from a contribution of myocytes derived solely from the medial border of the dermomyotome. In a second phase, myocytes are produced from all four borders of the dermomyotome. The relative distribution of myocytes demonstrates that the medial and the lateral borders of the somite generate exclusively epaxial and hypaxial muscles. This analysis also identified five myotomal regions, characterized by the origin of the myocytes that constitute them. Together, our results provide a comprehensive model describing the morphogenesis of the early myotome in higher vertebrates.

PMID: 15177035


Developmental mechanisms of vertebrate limb evolution

Novartis Found Symp. 2001;232:47-57; discussion 57-62.

Cohn MJ.

Division of Zoology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK. Abstract Over the past few years, our understanding of the evolution of limbs has been improved by important new discoveries in the fossil record. Additionally, rapid progress has been made in identifying the molecular basis of vertebrate limb development. It is now possible to integrate these two areas of research in order to identify the molecular developmental mechanisms underlying the evolution of paired appendages in vertebrates. After the origin of paired appendages, several vertebrate lineages reduced or eliminated fins and limbs and returned to the limbless condition. Examples include eels, caecilians, snakes, slow worms and several marine mammals. Analyses of fossil and extant vertebrates show that evolution of limblessness frequently occurred together with elongation of the trunk and loss of clear morphological boundaries in the vertebral column. This may be suggestive of a common developmental mechanism linking these two processes. We have addressed this question by analysing python embryonic development at tissue, cellular and molecular levels, and we have identified a developmental mechanism which may account for evolution of limb loss in these animals.

PMID: 11277086

Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws

Evol Dev. 2001 Sep-Oct;3(5):355-63.

Hamrick MW.

Department of Anthropology & School of Biomedical Sciences, Kent State University, OH 44242, USA. Abstract Paleontological evidence indicates that the evolutionary diversification of mammals early in the Cenozoic era was characterized by an adaptive radiation of distal limb structures. Likewise, neontological data show that morphological variation in distal limb integumentary appendages (e.g., nails, hooves, and claws) can be observed not only among distantly related mammalian taxa but also among closely related species within the same clade. Comparative analysis of nail, claw, and hoof morphogenesis reveals relatively subtle differences in mesenchymal and epithelial patterning underlying these adult differences in distal limb appendage morphology. Furthermore, studies of regulatory gene expression during vertebrate claw development demonstrate that many of the signaling molecules involved in patterning ectodermal derivatives such as teeth, hair, and feathers are also involved in organizing mammalian distal limb appendages. For example, Bmp4 signaling plays an important role during the recruitment of mesenchymal cells into the condensations forming the terminal phalanges, whereas Msx2 affects the length of nails and claws by suppressing proliferation of germinal epidermal cells. Evolutionary changes in the form of distal integumentary appendages may therefore result from changes in gene expression during formation of mesenchymal condensations (Bmp4, posterior Hox genes), induction of the claw fold and germinal matrix (shh), and/or proliferation of epidermal cells in the claw matrix (Msx1, Msx2). The prevalence of convergences and parallelisms in nail and claw structure among mammals underscores the existence of multiple morphogenetic pathways for evolutionary change in distal limb appendages.

PMID: 11710767


Fossils, genes and the evolution of animal limbs

Nature. 1997 Aug 14;388(6643):639-48.

Shubin N, Tabin C, Carroll S.

Department of Biology, University of Pennsylvania, Philadelphia 19104, USA. Abstract The morphological and functional evolution of appendages has played a crucial role in the adaptive radiation of tetrapods, arthropods and winged insects. The origin and diversification of fins, wings and other structures, long a focus of palaeontology, can now be approached through developmental genetics. Modifications of appendage number and architecture in each phylum are correlated with regulatory changes in specific patterning genes. Although their respective evolutionary histories are unique, vertebrate, insect and other animal appendages are organized by a similar genetic regulatory system that may have been established in a common ancestor.

PMID: 9262397


imaging -

scapula -


Harrison transplant Experiments

Proc Natl Acad Sci U S A. 1924 Feb;10(2):69-74. Some Unexpected Results of the Heteroplastic Transplantation of Limbs. Harrison RG1. Author information PMID: 16576784 PMCID: PMC1085516

Proc Natl Acad Sci U S A. 1917 Apr;3(4):245-51. Transplantation of Limbs. Harrison RG1. Author information PMID: 16586709 PMCID: PMC1091226

Experiments on the Development of the Limbs in Amphibia. Harrison RG. Proc Natl Acad Sci U S A. 1915 Nov;1(11):539-44. No abstract available. PMID: 16576066