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Background: The three chick wing digits represent a classical example of a pattern specified by a morphogen gradient. Here we have investigated whether a mathematical model of a Shh gradient can describe the specification of the identities of the three chick wing digits and if it can be applied to limbs with more digits., Results: We have produced a mathematical model for specification of chick wing digit identities by a Shh gradient that can be extended to the four digits of the chick leg with Shh-producing cells forming a digit. This model cannot be extended to specify the five digits of the mouse limb., Conclusions: Our data suggest that the parameters of a classical-type morphogen gradient are sufficient to specify the identities of three different digits. However, to specify more digit identities, this core mechanism has to be coupled to alternative processes, one being that in the chick leg and mouse limb, Shh-producing cells give rise to digits; another that in the mouse limb, the cellular response to the Shh gradient adapts over time so that digit specification does not depend simply on Shh concentration., (Copyright © 2013 Wiley Periodicals, Inc.)

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N-myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh., (Copyright © 2011 Wiley-Liss, Inc.)

Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly., (Copyright © 2011 Wiley-Liss, Inc.)

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face., (Copyright 2001 Wiley-Liss, Inc.)

We tested a diffusion gradient model for setting up overlapping domains of Hoxa gene expression in the chick limb bud. The model is based on morphogen production at the limb bud tip where the apical ridge is located and assumes that cells respond to a series of concentration thresholds. Consistent with the model, Hoxa13 gene expression rapidly switches off when the ridge is removed from stage 21/22 buds, while Hoxa11 and Hoxa10 expression is stable; Hoxa13 expression can be initiated and maintained in absence of the ridge by FGF soaked beads; the Hoxa13 domain first expands quickly and then slows up and the size is related to the dose of FGF4. Contrary to the model, addition of FGF4 to early limb buds does not activate Hoxa13 prematurely nor extend the Hoxa13 expression domain proximally. Therefore FGF4 signalling is necessary but not sufficient for Hoxa gene expression in the limb bud., (Copyright 2001 Wiley-Liss, Inc.)

Pax1 expression in vertebrate limb buds is confined to cells in a discrete anterior proximal domain (Timmons et al. [1994] Development 120:2773-2785; Ebensperger et al. [1995] Anat. Embryol. 191:297-310). In dorsoventral patterning of Drosophila, expression of pox meso, an insect gene with high sequence similarity to Pax1, is repressed by decapentaplegic (dpp) in dorsal mesoderm and, thus, is restricted to a discrete ventral domain (Staehling-Hampton et al. [1994] Nature 372:783-786). In the chick wing, cells expressing a vertebrate homolog of dpp, bone morphogenetic protein 4 (Bmp4), abut the Pax1 domain, suggesting a similar relationship between homologous genes in both vertebrates and invertebrates. Here, we show that two BMPs (BMP4, and BMP2, also highly related to dpp) can repress Pax1 in the developing chick wing. Chick wing bud cells expressing Pax1 give rise to the shoulder girdle. Cells in an equivalent position in the mouse forelimb also express Pax1, and Pax1 mutant mice display shoulder girdle defects. Similarly in chick embryos, girdle defects are produced by treatments with signalling molecules that lead to expression of BMPs, which subsequently reduce Pax1 expression in the limb bud. Recently, BMP4 has been shown to inhibit Pax1 expression in the developing trunk (Monsoro-Burq et al. [1996] Development 122:3607-3616) and Pax9 expression in developing teeth (Neubüser et al. [1997] Cell 90:247-255). Thus, a property of BMPs appears to be to regulate pox meso homologs negatively and, thus, limit their expression domains.

Developing facial primordia change shape substantially in stages leading up to primary palate formation. We investigated expansion of cell populations within each of the four facial primordia of chick embryos between HH-stages 20 and 28, by using DiI labelling. Populations of cells centred around the nasal pits in the upper face, the midline of the paired mandibular primordia in the lower face, and at sites of fusion contribute most to overall expansion. Abundant Msx-1 transcripts are found in regions of high expansion, and Fgf-8 transcripts are seen in ectoderm associated with some of these regions. Many cell populations display preferential expansion along one axis. Maxillary and mandibular primordia cell populations expand along the proximodistal axis, whereas at the distal tip of the frontonasal mass, cell populations expand mediolaterally. Thus outgrowth occurs at the tips of mandibular and maxillary primordia, but at the base of the frontonasal mass. At regions where adjacent primordia abut each other, we found bidirectional movement of cells between primordia, unidirectional movement or could detect no movement at all. Regions of highest expansion in each primordium have the highest percentage of S phase labelled cells. Cell death occurs in some regions of low expansion but it seems likely that cell rearrangements and intercalations also contribute to shaping. These rearrangements could be associated with stretching of the primordia by neighbouring tissues. Treatment of chick embryos with retinoic acid causes clefts of the primary palate (Tamarin et al. [1984] J. Embryol. Exp. Morphol. 84:105-123). We found a decrease in expansion of cell populations that normally contribute to primary palate formation but surprisingly little ectopic cell death. Expansion of other cell populations in the treated upper face was more even rather than directed. This further supports the idea that tension exerted by neighbouring tissues plays a major role in global shaping of the upper face.

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.