Cliff Tabin

Sonic hedgehog mediates the polarizing activity of the ZPA

The zone of polarizing activity (ZPA) is a region at the posterior margin of the limb bud that induces mirror-image duplications when grafted to the anterior of a second limb. We have isolated a vertebrate gene, Sonic hedgehog, related to the Drosophila segment polarity gene hedgehog, which is expressed specifically in the ZPA and in other regions of the embryo, that is capable of polarizing limbs in grafting experiments. Retinoic acid, which can convert anterior limb bud tissue into tissue with polarizing activity, concomitantly induces Sonic hedgehog expression in the anterior limb bud. Implanting cells that express Sonic hedgehog into anterior limb buds is sufficient to cause ZPA-like limb duplications. Like the ZPA, Sonic hedgehog expression leads to the activation of Hox genes. Sonic hedgehog thus appears to function as the signal for antero-posterior patterning in the limb.

p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.

The p63 gene, a homologue of the tumour-suppressor p53 (refs 1–5), is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63 −/− mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.

Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud

Proper limb growth and patterning requires signals from the zone of polarizing activity in the posterior mesoderm and from the overlying apical ectodermal ridge (AER). Sonic hedgehog and Fgf-4, respectively, have recently been identified as candidates for these signals. We have dissected the roles of these secreted proteins in early limb development by ectopically regulating their activities in a number of surgical contexts. Our results indicate that Sonic hedgehog initiates expression of secondary signaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in the ectoderm. The mesoderm requires ectodermally derived competence factors, which include Fgf-4, to activate target gene expression in response to Sonic hedgehog. The expression of Sonic hedgehog and Fgf-4 is coordinately regulated by a positive feedback loop operating between the posterior mesoderm and the overlying AER. Taken together, these data provide a basis for understanding the integration of growth and patterning in the developing limb.

A molecular pathway determining left-right asymmetry in chick embryogenesis

While significant progress has been made in understanding the molecular events underlying the early specification of the antero-posterior and dorso-ventral axes, little information is available regarding the cellular or molecular basis for left-right (LR) differences in animal morphogenesis. We describe the expression patterns of three genes involved in LR determination in chick embryos: activin receptor IIa, Sonic hedgehog (Shh), and cNR-1 (related to the mouse gene nodal). These genes are expressed asymmetrically during and after gastrulation and regulate the expression of one another in a sequential pathway. Moreover, manipulation of the sidedness of either activin protein or Shh expression alters heart situs. Together, these observations identify a cascade of molecular asymmetry in that determines morphological LR asymmetry in the chick embryo.

Fossils, genes and the evolution of animal limbs

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.

Deep homology and the origins of evolutionary novelty

Do new anatomical structures arise de novo, or do they evolve from pre-existing structures? Advances in developmental genetics, palaeontology and evolutionary developmental biology have recently shed light on the origins of some of the structures that most intrigued Charles Darwin, including animal eyes, tetrapod limbs and giant beetle horns. In each case, structures arose by the modification of pre-existing genetic regulatory circuits established in early metazoans. The deep homology of generative processes and cell-type specification mechanisms in animal development has provided the foundation for the independent evolution of a great variety of structures.

Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites

Differentiation of somites into sclerotome, dermatome, and myotome is controlled by a complex set of inductive interactions. The ability of axial midline tissues, the notochord and floor plate, to induce sclerotome has been well documented and has led to models in which ventral somite identity is specified by signals derived from the notochord and floor plate. Herein, we provide evidence that Sonic hedgehog, a vertebrate homolog of the Drosophila segment polarity gene hedgehog, is a signal produced by the notochord and floor plate that directs ventral somite differentiation. Sonic hedgehog is expressed in ventral midline tissues at critical times during somite specification and has the ability, when ectopically expressed, to enhance the formation of sclerotome and antagonize the development of dermatome.

Induction of the LIM homeobox gene Lmx1 by WNT6a establishes dorsoventral pattern in the vertebrate limb

During vertebrate limb development, the ectoderm directs the dorsoventral patterning of the underlying mesoderm. To define the molecular events involved in this process, we have analyzed the function of WNT7a, a secreted factor expressed in the dorsal ectoderm, and LMX1, a LIM homeodomain transcription factor expressed in the dorsal mesenchyme. Ectopic expression of Wnt7a is sufficient to induce and maintain Lmx1 expression in limb mesenchyme, both in vivo and in vitro. Ectopic expression of Lmx1 in the ventral mesenchyme is sufficient to generate double-dorsal limbs. Thus, the dorsalization of limb mesoderm appears to involve the WNT7a-mediated induction of Lmx1 in limb mesenchymal cells.

The initiation of the limb bud: Growth factors, Hox genes, and retinoids

Department of Genetics Harvard Medical School Boston, Massachusetts 02115 The undifferentiated vertebrate limb bud is a self- organizing system. If transplanted to a favorable ectopic location, the limb bud is capable of developing into a mor- phologically normal limb (Harrison, 1918), and the ante- rior-posterior polarity of the transplanted limb is deter- mined by the graft rather than the host environment. Great progress has recently been made in understanding the molecular steps by which pattern emerges within an undif- ferentiated limb bud (for recent reviews see Johnson et al., 1994; Tickle and Eichele, 1994). But what first initiates the formation of the limb bud? And what molecular steps provide the information necessary for polarized self- organization? Two recent papers (Charit~ et al., 1994; Cohn et al., 1995 [this issue of

The long and short of hedgehog signaling

The past decade has seen a revolution in our understanding of the molecular basis of embryonic development in higher organisms. As our understanding of vertebrate development has grown, a number of completely unanticipated and truly remarkable parallels between mechanisms of patterning in vertebrates and Drosophila have been revealed (Scott, 1994). These findings suggest that the wealth of genetic and molecular information available concerning fly development will continue to provide an enormous resource for gaining further insight into vertebrate development. Indeed, many significant genes known to control various aspects of fly development have vertebrate homologs. Although their developmental roles may not be specifically conserved, analysis of their function will provide clues to the general processes they control and mechanisms by which they act. The role of hedgehog (hh) genes as intercellular signals in establishing embryonic pattern provides a dramatic example of this transfer of developmental insight from Drosophila to vertebrates and shows how studies in both organisms can synergistically lead to rapid elucidation of the molecular mechanisms underlying embryological processes. One mechanism by which developing embryos attain proper position-specific cell differentiation is to organize cell fates relative to a discrete inducing tissue. In principle, such induction could be achieved by a single long-range secreted signal instructing cell fate in a concentrationdependent manner (Figure la). Molecules acting via this mechanism have been termed morphogens. Alternatively, the primary inductive response could be quite local, initiating a cascade of short-range signals that are then propagated through responding tissues (Figure 1 b). Finally, the inductive trigger could act locally to initiate long-range and graded secondary signals (Figure 1 c). The identification of hh genes as key signals in a variety of embryonic inductive processes provides an opportunity to determine which of these theoretical mechanisms are actually used in regulating pattern. Shortand Long.Range Signaling by hh hh was identified by NLisslein-Volhard and Wieschaus (1980) in a saturation screen for mutants that affect larval cuticular patterning in Drosophila. Subsequent studies have shown that hh encodes a secreted protein that plays multiple inductive roles during fly development (reviewed by Perrimon, 1995). Via short-range action, over 1 or 2-cell diameters, hh regulates aspects of embryonic segmentation and patterning of adult appendages. In establishing early segmental borders, the inductive targets of hh signaling cells are directly adjacent cells. A cascade of shortrange interactions is thereby initiated that programs cell fate at different positions within the segment, corresponding to the model diagrammed in Figure lb. In the case of appendages, hh again acts locally to pattern cells within the larval appendage anlage, the imaginal discs. In this instance, however, cells respond locally by secreting decapentaplegic (dpp), which then may serve to pattern the disc in a graded manner over considerable distances, as shown in Figure lc. Besides these short-range activities, hh also is responsible for long-range specification of cell types in the dorsal epidermis. While at times cited as evidence of long-range hh induction, this latter process could result either from a direct action of hh on both adjacent and distant cells, as shown in Figure la, or it could depend upon the secretion of a second (as yet unidentified) longrange factor, as shown in Figure lc. Vertebrate homologs of hh have been isolated in screens utilizing the cloned Drosophila gene. One homolog in particular, Sonic hedgehog (Shh), displays a surprisingly wide range of activities in vertebrate embryos (Smith, 1994). SH H regulates dorsal-ventral patterning of the neural tube, the somites, and the anterior-posterior axis of the limb bud. As with its Drosophila homolog, the Shh gene product appears to act locally in some circumstances (floor