Expression patterns of Slit and Robo family members during vertebrate limb development.

We report the cloning and expression during limb development of the chicken Slit1, Slit2, and Slit3 ligands, and Robo1 and Robo2 receptor genes. We also compare the expression patterns of Robo1 and Robo2 in developing chick and mouse hindlimbs. These genes are expressed in regions of muscle development, chrondrogenesis, and axon guidance.

Characterisation of hoxa gene expression in the chick limb bud in response to FGF.

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.

Smad7 misexpression during embryonic angiogenesis causes vascular dilation and malformations independently of vascular smooth muscle cell function.

Numerous in vitro and in vivo studies implicate transforming growth factor-beta (TGFbeta) superfamily signaling in vascular development and maintenance. Mice and humans with mutations in TGFbeta superfamily signaling pathway genes exhibit a range of vascular defects that include dilated, fragile and hemorrhagic vessels, defective angiogenic remodeling, severe vascular malformations including arterio-venous malformations, and disrupted vascular smooth muscle cell recruitment and maintenance. Despite a wealth of data, the functions of TGFbeta superfamily signals during angiogenesis are poorly defined, since early embryonic lethality and difficulty distinguishing between primary and secondary defects frequently confound phenotypic interpretation. To perturb TGFbeta superfamily signaling during angiogenesis, we have misexpressed Smad7, an intracellular antagonist of TGFbeta superfamily signaling, in the developing chick limb and head. We find that the great vessels are strikingly dilated and frequently develop intra and intervascular shunts. Neither noggin nor dominant negative BMP receptor misexpression causes similar vascular phenotypes. However, simultaneous misexpression of constitutively active BMP receptors with Smad7 suppresses the Smad7-induced phenotype, suggesting that a BMP-like intracellular pathway is the target of Smad7 action. Despite the gross morphological defects, further analyses find no evidence of hemorrhage and vessel structure is normal. Furthermore, enlarged vessels and vascular malformations form in either the presence or absence of vascular smooth muscle, and vascular smooth muscle cell recruitment is unperturbed. Our data define the TGFbeta superfamily pathway as an integral regulator of vessel caliber that is also essential for appropriate vessel connectivity. They demonstrate that dilation need not result in vessel rupture or hemorrhage, and dissociate vessel maintenance from the presence of a vascular smooth muscle cell coat. Furthermore they uncouple vascular smooth muscle cell recruitment and differentiation from TGFbeta superfamily signaling.

Expression patterns of Notch1, Serrate1, Serrate2 and Delta1 in tissues of the developing chick limb.

Signalling via the receptor Notch, delivered by the ligands Delta and Serrate, plays a key role in many cell fate decisions in both Drosophila and vertebrate development (for review seeArtavanis-Tsakonas, S., Matsuno, K. and Fortini, M.E., 1995. Notch signalling. Science 268, 225-232; Lewis, J., 1996. Neurogenic genes and vertebrate neurogenesis. Curr. Opin. Neurobiol. 6, 3-10; Blair, S.S., 1997. Limb development: marginal fringe benefits. Curr Biol. 7, 686-690; Irvine, K.D. and Vogt, T.F., 1997. Dorsal-ventral signaling in limb development. Curr. Opin. Cell Biol. 9, 867-876). Recently vertebrate homologues of Notch (Notch1; Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J., 1996. A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogeneis. Dev. Biol. 174, 233-247) and Serrate (Serrate1 and 2; Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J., 1996. A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogeneis. Dev. Biol. 174, 233-247; Hayashi, H., Mochii, M., Kodama, R., Hamada, Y., Mizuno, N., Eguchi, G. and Tachi, C., 1996. Isolation of a novel chick homolog of Serrate and its coexpression with Notch-1 in chick development. Int. J. Dev. Biol. 40, 1089-96; Laufer, E., Dahn, R., Orozco, O.E., Yeo, C.Y., Pisenti, J., Henrique, D., Abbott, U., Fallon, J.F. and Tabin, C., 1996. Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366-373; Rodriguez-Esteban, C., Schwabe, J.W., De La Pena, J., Foys, B., Eshelman, B. and Izpisua-Belmonte, J.C., 1997. Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. Nature 386, 360-366) were shown to be expressed in early chick limb mesenchyme and apical ridge. However, later expression patterns of these genes and of Delta 1 (Henrique, D. , Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D., 1995. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787-790) in vertebrate limbs have not been documented. We have used whole mount in-situ hybridization to document expression patterns of Notch1, Serrate1, Serrate2 and Delta1 within the mesenchyme of the developing chick limb up to stage 31 of development. We show these genes are expressed, in different combinations, in the vasculature, the musculature and the tissues of the handplate.

Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb.

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.

Cell fate in the chick limb bud and relationship to gene expression.

We have produced detailed fate maps for mesenchyme and apical ridge of a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme of the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fatemaps of the apical ridge show that there is also a marked anterior expansion and cells that were in anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert--the apical ridge extends more anteriorly. We used the fatemaps to investigate the relationship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. We found that at the proximal edge of the Hoxd-13 domain, cell populations stopped expressing the gene as development proceeded and found no evidence that the changes in extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to expansion and fanning out of cell populations initially expressing the genes.

Local origin of cells in FGF-4 - induced outgrowth of amputated chick wing bud stumps.

Urodele amphibians are the only vertebrates that can regenerate amputated limbs, even as adults. However, we have previously shown that amputated chick wing bud stumps can be induced to ((regenerate)) and to form a complete set of correctly-patterned skeletal elements, following implantation of beads soaked in fibroblast growth factor-4 (FGF-4). We have now performed Dil injection experiments to determine which cells contribute to FGF-4-induced chick wing bud ((regenerates)). We show that the FGF-4-induced outgrowth of the regenerating wing bud stump is comprised of mesenchyme cells that originate from a region within 200 microm of the FGF-4 bead, and that cells proximal to the bead move distally.

The ectodermal control in chick limb development: Wnt-7a, Shh, Bmp-2 and Bmp-4 expression and the effect of FGF-4 on gene expression.

We have manipulated the chick limb bud by dorsoventrally inverting the ectoderm, by grafting the AER to the dorsal or ventral ectoderm and by insertion of an FGF-4 soaked heparin bead into the mesoderm. After dorso-ventral reversal of the ectoderm, Wnt-7a expression is autonomous from an early stage of limb development in the original dorsal ectoderm. Exogenous FGF-4 causes ectopic Wnt-7a expression and induces ectopic Shh. In addition, exogenous FGF-4 increases the thickness of cartilages and also shortens them, and both Bmp-2 and Bmp-4 may mediate this effect. The ectoderm outside the AER can regulate not only the dorso-ventral polarity of the underlying mesenchyme cells but also the cartilage formation, and both Bmp-2 and Bmp-4 may mediate this control.

Exogenous retinoic acid causes specific alterations in the development of the midbrain and hindbrain of the zebrafish embryo including positional respecification of the Mauthner neuron.

Exogenously applied retinoic acid given at the early stages of gastrulation causes abnormal development of the caudal midbrain and anterior hindbrain in vertebrate embryos. We describe the limits of the brain regions that are affected using neuroanatomical criteria in the zebrafish embryo. Analysis of the reticulospinal complex shows that the Mauthner cell, which normally differentiates in rhombomere 4, is duplicated either in this rhombomere or in rhombomere 2. Using probes for zebrafish krx20 and pax2, it is demonstrated that retinoic acid affects the expression domains of these regulatory genes in a manner that is consistent with the neuroanatomical data. Expression of the goosecoid gene, which expressed in the prospective anterior mesoderm from the onset of gastrulation, is unaffected by the doses of retinoic acid used in this study, reflecting the normal development of the anterior end of the embryo.