Getting a handle on embryo limb development: Molecular interactions driving limb outgrowth and patterning

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.

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An intrinsic cell cycle timer terminates limb bud outgrowth

eLife ◽

10.7554/elife.37429 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 4

Author(s):

Joseph Pickering ◽

Constance A Rich ◽

Holly Stainton ◽

Cristina Aceituno ◽

Kavitha Chinnaiya ◽

...

Keyword(s):

Cell Cycle ◽

Rna Sequencing ◽

Late Stage ◽

Limb Development ◽

Limb Bud ◽

Limb Outgrowth ◽

Bmp Signalling ◽

Mesenchyme Cells

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

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The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development

Development ◽

10.1242/dev.125.22.4417 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4417-4425 ◽

Cited By ~ 6

Author(s):

M. Takahashi ◽

K. Tamura ◽

D. Buscher ◽

H. Masuya ◽

S. Yonei-Tamura ◽

...

Keyword(s):

Negative Feedback ◽

Limb Development ◽

Feedback Loop ◽

Limb Bud ◽

Negative Regulator ◽

Negative Feedback Loop ◽

Vertebrate Limb ◽

Limb Outgrowth ◽

Vertebrate Limb Development

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

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Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421949112 ◽

2015 ◽

Vol 112 (16) ◽

pp. 5075-5080 ◽

Cited By ~ 33

Author(s):

Ryutaro Akiyama ◽

Hiroko Kawakami ◽

Julia Wong ◽

Isao Oishi ◽

Ryuichi Nishinakamura ◽

...

Keyword(s):

Progenitor Cells ◽

Sonic Hedgehog ◽

Limb Development ◽

Critical Role ◽

Dependent Manner ◽

Skeletal Element ◽

Proximal Development ◽

Limb Outgrowth ◽

Common Regulator

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

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Novel Cellular and Molecular Interactions During Limb Development, Revealed from Studies on the Split Hand Foot Congenital Malformation

New Discoveries in Embryology ◽

10.5772/60402 ◽

2015 ◽

Cited By ~ 2

Author(s):

Daniele Conte ◽

Luisa Guerrini ◽

Giorgio R. Merlo

Keyword(s):

Congenital Malformation ◽

Molecular Interactions ◽

Limb Development

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Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds

Development ◽

10.1242/dev.116.3.811 ◽

1992 ◽

Vol 116 (3) ◽

pp. 811-818 ◽

Cited By ~ 11

Author(s):

M.A. Ros ◽

G. Lyons ◽

R.A. Kosher ◽

W.B. Upholt ◽

C.N. Coelho ◽

...

Keyword(s):

Pattern Formation ◽

Limb Development ◽

Apical Ectodermal Ridge ◽

Limb Bud ◽

Rapid Decline ◽

Limb Buds ◽

Limb Outgrowth ◽

Removal Experiments ◽

The Relationship

The homeobox-containing genes GHox-7 and GHox-8 have been proposed to play fundamental roles in limb development. The expression of GHox-8, by the apical ridge cells, and GHox-7, in the subridge mesoderm, suggests the involvement of these two genes in limb outgrowth and proximo-distal pattern formation. A straightforward way to test this is to remove the apical ridge. Here we report the relationship between the mesodermal expression of GHox-7 and GHox-8 and the apical ectodermal ridge in the chick limb bud. The data from ridge removal experiments indicate that there are at least two domains of GHox-7 expression in the apical limb bud mesoderm. The posterior subridge GHox-7 domain in the progress zone requires the influence of the apical ridge for continued expression, while the anterior GHox-7 domain continues expression after ridge removal. Posterior subridge mesoderm is exquisitely sensitive to the loss of the ridge in that GHox-7 expression by these cells is reduced in only two hours and undetectable by three hours after ridge removal. It would appear that one of the ways progress zone cells respond to the apical ridge signal is by expressing GHox-7. The loss of ridge influence whether by growth at the apex or by ridge removal is followed by an unusually rapid decline in detectable GHox-7 transcripts. Maintenance of GHox-8 expression by the anterior mesoderm appears to be independent of the presence of the apical ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

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The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10

Development ◽

10.1242/dev.129.22.5161 ◽

2002 ◽

Vol 129 (22) ◽

pp. 5161-5170 ◽

Cited By ~ 1

Author(s):

Jennifer K. Ng ◽

Yasuhiko Kawakami ◽

Dirk Büscher ◽

Ángel Raya ◽

Tohru Itoh ◽

...

Keyword(s):

Limb Development ◽

Gene Families ◽

Chick Embryos ◽

Loss Of Function ◽

T Box ◽

Vertebrate Limb ◽

Limb Outgrowth ◽

Tbx5 Expression ◽

Necessary And Sufficient ◽

Limb Initiation

A major gap in our knowledge of development is how the growth and identity of tissues and organs are linked during embryogenesis. The vertebrate limb is one of the best models to study these processes. Combining mutant analyses with gain- and loss-of-function approaches in zebrafish and chick embryos, we show that Tbx5, in addition to its role governing forelimb identity,is both necessary and sufficient for limb outgrowth. We find thatTbx5 functions downstream of WNT signaling to regulateFgf10, which, in turn, maintains Tbx5 expression during limb outgrowth. Furthermore, our results indicate that Tbx5 andWnt2b function together to initiate and specify forelimb outgrowth and identity. The molecular interactions governed by members of the T-box,Wnt and Fgf gene families uncovered in this study provide a framework for understanding not only limb development, but how outgrowth and identity of other tissues and organs of the embryo may be regulated.

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Normal limb development in conditional mutants of Fgf4

Development ◽

10.1242/dev.127.5.989 ◽

2000 ◽

Vol 127 (5) ◽

pp. 989-996 ◽

Cited By ~ 5

Author(s):

A.M. Moon ◽

A.M. Boulet ◽

M.R. Capecchi

Keyword(s):

Limb Development ◽

Expression Patterns ◽

Large Body ◽

Embryonic Lethality ◽

Limb Bud ◽

Recombination Reaction ◽

Site Specific ◽

Early Embryonic Lethality ◽

Limb Outgrowth

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.

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An intrinsic cell cycle timer terminates limb bud outgrowth

10.1101/296350 ◽

2018 ◽

Author(s):

Joseph Pickering ◽

Kavitha Chinnaiya ◽

Constance A. Rich ◽

Patricia Saiz-Lopez ◽

Maria A. Ros ◽

...

Keyword(s):

Cell Cycle ◽

Rna Sequencing ◽

Late Stage ◽

Limb Development ◽

Limb Bud ◽

Limb Outgrowth ◽

Bmp Signalling ◽

Mesenchyme Cells

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the mesenchyme and the overlying epithelium (e-m signalling). However, by grafting mesenchyme cells from late stage chick wing buds to an early epithelial environment we show that this mechanism is not required. RNA sequencing reveals that mesenchyme cells terminate growth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

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The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb

Development ◽

10.1242/dev.126.23.5515 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5515-5522 ◽

Cited By ~ 5

Author(s):

R. Merino ◽

J. Rodriguez-Leon ◽

D. Macias ◽

Y. Ganan ◽

A.N. Economides ◽

...

Keyword(s):

Cell Death ◽

Soft Tissue ◽

Programmed Cell Death ◽

Limb Development ◽

Limb Bud ◽

Central Core ◽

Limb Outgrowth ◽

Bmp Antagonists ◽

And Function ◽

Bmp Antagonist

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.

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