Role of dHAND in the anterior-posterior polarization of the limb bud: implications for the Sonic hedgehog pathway

Development ◽  
2000 ◽  
Vol 127 (10) ◽  
pp. 2133-2142 ◽  
Author(s):  
M. Fernandez-Teran ◽  
M.E. Piedra ◽  
I.S. Kathiriya ◽  
D. Srivastava ◽  
J.C. Rodriguez-Rey ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Limb Development ◽  
Mirror Image ◽  
Limb Bud ◽  
Bhlh Transcription Factor ◽  
Cardiovascular Development ◽  
Anterior Border ◽  
Lateral Plate ◽  
Shh Pathway ◽  
Anterior Posterior

dHAND is a basic helix-loop-helix (bHLH) transcription factor essential for cardiovascular development. Here we analyze its pattern of expression and functional role during chick limb development. dHAND expression was observed in the lateral plate mesoderm prior to emergence of the limb buds. Coincident with limb initiation, expression of dHAND became restricted to the posterior half of the limb bud. Experimental procedures that caused mirror-image duplications of the limb resulted in mirror-image duplications of the pattern of dHAND expression along the anterior-posterior axis. Retroviral overexpression of dHAND in the limb bud produced preaxial polydactyly, corresponding to mild polarizing activity at the anterior border. At the molecular level, misexpression of dHAND caused ectopic activation of members of the Sonic hedgehog (Shh) pathway, including Gli and Patched, in the anterior limb bud. A subset of infected embryos displayed ectopic anterior activation of Shh. Other factors implicated in anterior-posterior polarization of the bud such as the most 5′ Hoxd genes and Bmp2 were also ectopically activated at the anterior border. Our results indicate a role for dHAND in the establishment of anterior-posterior polarization of the limb bud.

The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2461-2470 ◽  
Author(s):  
J. Charite ◽  
D.G. McFadden ◽  
E.N. Olson
Keyword(s):  
Transcription Factor ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Target Genes ◽  
Ectopic Expression ◽  
Tissue Growth ◽  
Limb Bud ◽  
Bhlh Transcription Factor ◽  
Expression Domain ◽  
Zone Of Polarizing Activity

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development

Development ◽  
2000 ◽  
Vol 127 (23) ◽  
pp. 5133-5144
Author(s):  
A.T. Tavares ◽  
T. Tsukui ◽  
J.C. Izpisua Belmonte
Keyword(s):  
Limb Development ◽  
Limb Bud ◽  
Wing Margin ◽  
Lateral Plate ◽  
Posterior Limb ◽  
Downstream Target ◽  
Notch Pathways ◽  
Specialized Region ◽  
Dorsoventral Boundary ◽  
Cycle Regulation

In vertebrates, the apical ectodermal ridge (AER) is a specialized epithelium localized at the dorsoventral boundary of the limb bud that regulates limb outgrowth. In Drosophila, the wing margin is also a specialized region located at the dorsoventral frontier of the wing imaginal disc. The wingless and Notch pathways have been implicated in positioning both the wing margin and the AER. One of the nuclear effectors of the Notch signal in the wing margin is the transcription factor cut. Here we report the identification of two chick homologues of the Cut/Cux/CDP family that are expressed in the developing limb bud. Chick cux1 is expressed in the ectoderm outside the AER, as well as around ridge-like structures induced by (β)-catenin, a downstream target of the Wnt pathway. cux1 overexpression in the chick limb results in scalloping of the AER and limb truncations, suggesting that Cux1 may have a role in limiting the position of the AER by preventing the ectodermal cells around it from differentiating into AER cells. The second molecule of the Cut family identified in this study, cux2, is expressed in the pre-limb lateral plate mesoderm, posterior limb bud and flank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. The polarizing activity is determined by the ability of a certain region to induce digit duplications when grafted into the anterior margin of a host limb bud. Several manipulations of the chick limb bud show that cux2 expression is regulated by retinoic acid, Sonic hedgehog and the posterior AER. These results suggest that Cux2 may have a role in generating or mediating polarizing activity. Taking into account the probable involvement of Cut/Cux/CDP molecules in cell cycle regulation and differentiation, our results raise the hypothesis that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively.

Autoregulation of Shh expression and Shh induction of cell death suggest a mechanism for modulating polarising activity during chick limb development

Development ◽  
2000 ◽  
Vol 127 (22) ◽  
pp. 4811-4823 ◽  
Author(s):  
J.J. Sanz-Ezquerro ◽  
C. Tickle
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Retroviral Vector ◽  
Cellular Mechanism ◽  
Limb Bud ◽  
Drug Induced ◽  
Signalling Molecule ◽  
Vertebrate Limb

The polarising region expresses the signalling molecule sonic hedgehog (Shh), and is an embryonic signalling centre essential for outgrowth and patterning of the vertebrate limb. Previous work has suggested that there is a buffering mechanism that regulates polarising activity. Little is known about how the number of Shh-expressing cells is controlled but, paradoxically, the polarising region appears to overlap with the posterior necrotic zone, a region of programmed cell death. We have investigated how Shh expression and cell death respond when levels of polarising activity are altered, and show an autoregulatory effect of Shh on Shh expression and that Shh affects cell death in the posterior necrotic zone. When we increased Shh signalling, by grafting polarising region cells or applying Shh protein beads, this led to a reduction in the endogenous Shh domain and an increase in posterior cell death. In contrast, cells in other necrotic regions of the limb bud, including the interdigital areas, were rescued from death by Shh protein. Application of Shh protein to late limb buds also caused alterations in digit morphogenesis. When we reduced the number of Shh-expressing cells in the polarising region by surgery or drug-induced killing, this led to an expansion of the Shh domain and a decrease in the number of dead cells. Furthermore, direct prevention of cell death using a retroviral vector expressing Bcl2 led to an increase in Shh expression. Finally, we provide evidence that the fate of some of the Shh-expressing cells in the polarising region is to undergo apoptosis and contribute to the posterior necrotic zone during normal limb development. Taken together, these results show that there is a buffering system that regulates the number of Shh-expressing cells and thus polarising activity during limb development. They also suggest that cell death induced by Shh could be the cellular mechanism involved. Such an autoregulatory process based on cell death could represent a general way for regulating patterning signals in embryos.

scholarly journals Intercalation and the cellular origin of supernumerary limbs in Xenopus

Development ◽  
1987 ◽  
Vol 99 (4) ◽  
pp. 521-526 ◽  
Author(s):  
K. Muneoka ◽  
E.H. Murad
Keyword(s):  
Limb Development ◽  
Limb Bud ◽  
Posterior Limb ◽  
Cellular Origin ◽  
Supernumerary Limbs ◽  
Anterior Posterior ◽  
Anterior Posterior Axis

The hypothesis that a specialized polarizing zone controls the pattern of the anterior-posterior axis during limb development in Xenopus has been tested by analysing the cellular contribution to supernumerary limbs. Supernumerary limbs were generated by grafting hindlimb buds contralaterally between X. borealis and X. laevis to appose anterior and posterior limb tissues. Cells derived from these two species of Xenopus are readily identified by staining with quinacrine. The analysis of cellular contribution showed that supernumerary limbs consist of approximately half anterior-derived (57%) and half posterior-derived (43%) cells. These data are not consistent with the polarizing zone theory but are consistent with the hypothesis that both supernumerary limbs and normally developing limbs arise from intercalary interactions between limb bud cells with different positional values.

The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor

Development ◽  
1997 ◽  
Vol 124 (11) ◽  
pp. 2235-2244 ◽  
Author(s):  
H. Ohuchi ◽  
T. Nakagawa ◽  
A. Yamamoto ◽  
A. Araga ◽  
T. Ohata ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Limb Bud ◽  
Fibroblast Growth ◽  
Lateral Plate Mesoderm ◽  
Lateral Plate ◽  
Bud Formation ◽  
Limb Mesenchyme ◽  
Vertebrate Limb ◽  
Fgf10 Expression ◽  
Zone Of Polarizing Activity

Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. In this report, we provide evidence that a member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs.

Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2537-2547 ◽  
Author(s):  
E. Marti ◽  
R. Takada ◽  
D.A. Bumcrot ◽  
H. Sasaki ◽  
A.P. McMahon
Keyword(s):  
Long Range ◽  
Sonic Hedgehog ◽  
Motor Neurons ◽  
Close Association ◽  
Small Region ◽  
Floor Plate ◽  
Limb Bud ◽  
Intercellular Signaling ◽  
Anterior Posterior

Sonic hedgehog (Shh) encodes a signal that is implicated in both short- and long-range interactions that pattern the vertebrate central nervous system (CNS), somite and limb. Studies in vitro indicate that Shh protein undergoes an internal cleavage to generate two secreted peptides. We have investigated the distribution of Shh peptides with respect to these patterning events using peptide-specific antibodies. Immunostaining of chick and mouse embryos indicates that Shh peptides are expressed in the notochord, floor plate and posterior mesenchyme of the limb at the appropriate times for their postulated patterning functions. The amino peptide that is implicated in intercellular signaling is secreted but remains tightly associated with expressing cells. The distribution of peptides in the ventral CNS is polarized with the highest levels of protein accumulating towards the luminal surface. Interestingly, Shh expression extends beyond the floor plate, into ventrolateral regions from which some motor neuron precursors are emerging. In the limb bud, peptides are restricted to a small region of posterior-distal mesenchyme in close association with the apical ectodermal ridge; a region that extends 50–75 microns along the anterior-posterior axis. Temporal expression of Shh peptides is consistent with induction of sclerotome in somites and floor plate and motor neurons in the CNS, as well as the regulation of anterior-posterior polarity in the limb. However, we can find no direct evidence for long-range diffusion of the 19 × 10(3) Mr peptide which is thought to mediate both short- and long-range cell interactions. Thus, either long-range signaling is mediated indirectly by the activation of other signals, or alternatively the low levels of diffusing peptide are undetectable using available techniques.

Why we have (only) five fingers per hand: hox genes and the evolution of paired limbs

Development ◽  
1992 ◽  
Vol 116 (2) ◽  
pp. 289-296 ◽  
Author(s):  
C.J. Tabin
Keyword(s):  
Limb Development ◽  
Hox Genes ◽  
Expression Patterns ◽  
Limb Bud ◽  
Developmental Constraint ◽  
Limb Morphogenesis ◽  
Different Types ◽  
Embryonic Limb ◽  
Limb Pattern ◽  
Anterior Posterior

Limb development has long been a model system for studying vertebrate pattern formation. The advent of molecular biology has allowed the identification of some of the key genes that regulate limb morphogenesis. One important class of such genes are the homeobox-containing, or Hox genes. Understanding of the roles these genes play in development additionally provides insights into the evolution of limb pattern. Hox gene expression patterns divide the embryonic limb bud into five sectors along the anterior/posterior axis. The expression of specific Hox genes in each domain specifies the developmental fate of that region. Because there are only five distinct Hox-encoded domains across the limb bud there is a developmental constraint prohibiting the evolution of more than five different types of digits. The expression patterns of Hox genes in modern embryonic limb buds also gives clues to the shape of the ancestral fin field from which the limb evolved, hence elucidating the evolution of the tetrapod limb.

Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4817-4826 ◽  
Author(s):  
C.J. Neumann ◽  
H. Grandel ◽  
W. Gaffield ◽  
S. Schulte-Merker ◽  
C. Nusslein-Volhard
Keyword(s):  
Retinoic Acid ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Normal Development ◽  
Limb Bud ◽  
Pectoral Fin ◽  
Anteroposterior Patterning ◽  
Vertebrate Limb ◽  
Anteroposterior Polarity

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Here we report an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene. We show that Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independently of Shh, and only later come to depend on Shh for their maintenance. We also demonstrate that Shh is required for the activation of posterior HoxD genes by retinoic acid. Finally, we show that Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton.

Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 537-542 ◽  
Author(s):  
T. Ogura ◽  
I.S. Alvarez ◽  
A. Vogel ◽  
C. Rodriguez ◽  
R.M. Evans ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Sonic Hedgehog ◽  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Retinoic Acid Receptor ◽  
Mirror Image ◽  
Limb Bud ◽  
Embryonal Carcinoma Cell ◽  
Embryonal Carcinoma Cell Line ◽  
Vertebrate Limb

Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554–566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R. L., Laufer, E. and Tabin, C. J. (1993) Cell 25, 1401–1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994 Development 120, 3339–3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARalpha). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process.

Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1821-1829
Author(s):  
M.A. Ros ◽  
M. Sefton ◽  
M.A. Nieto
Keyword(s):  
Zinc Finger ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Limb Bud ◽  
Mesenchymal Phenotype ◽  
Signalling Molecules ◽  
Slug Expression ◽  
Snail Family ◽  
Zone Of Polarizing Activity ◽  
Signalling Systems

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.

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