Coordinated expression of Hoxa-11 and Hoxa-13 during limb muscle patterning

The limb muscle precursor cells migrate from the somites and congregate into the dorsal and ventral muscle masses in the limb bud. Complex muscle patterns are formed by successive splitting of the muscle masses and subsequent growth and differentiation in a region-specific manner. Hox genes, known as key regulator genes of cartilage pattern formation in the limb bud, were found to be expressed in the limb muscle precursor cells. We found that HOXA-11 protein was expressed in the premyoblasts in the limb bud, but not in the somitic cells or migrating premyogenic cells in the trunk at stage 18. By stage 24, HOXA-11 expression began to decrease from the posterior halves of the muscle masses. HOXA-13 was expressed strongly in the myoblasts of the posterior part in the dorsal/ventral muscle masses and weakly in a few myoblasts of the anterior part of the dorsal muscle mass. Transplantation of the lateral plate of the presumptive wing bud to the flank induced migration of premyoblasts from somites to the graft. Under these conditions, HOXA-11 expression was induced in the migrating premyoblasts in the ectopic limb buds. Application of retinoic acid at the anterior margin of the limb bud causes duplication of the autopodal cartilage and transformation of the radius to the ulna, and at the same time induces duplication of the muscle pattern along the anteroposterior axis. Under these conditions, HOXA-13 was also induced in the anterior region of the ventral muscles in the zeugopod. These results suggest that Hoxa-11 and Hoxa-13 expression in the migrating premyoblasts is under the control of the limb mesenchyme and the polarizing signal(s). In addition, these results indicate that these Hox genes are involved in muscle patterning in the limb buds.     

Visual demonstration of the limb-forming zone in the chick embryo lateral plate

The present study was undertaken to determine whether a visible Wolffian ridge, distinct from the lateral fold, can be identified in chick embryos. Ectoderm thickness was measured in stage 11-17 chick embryos. There was a general trend, from thin ectoderm in the midline, to an ectodermal thickening over the somites, intermediate mesoderm, and lateral plate. Other embryos were cut from the yolk, pinned out, and photographed. The lateral fold was then eliminated, and the embryo was rephotographed. The photographs reveal a definite opaic zone, distinct from the lateral fold, in stage 11-18 chick embryos. Furthermore, there is a direct correlation between the opacity of this cellular band and the limb-forming potential of grafted wing, flank, and leg regions (see Stephens et al., '89). At stages 11-14, the wing, flank, and leg exhibit a uniform opacity, and a uniform capacity for limb formation when grafted to a host celom. From stage 15 to stage 18, the opacity in the flank diminishes, and its limb-forming capability disappears. This study demonstrates the presence of an opaic zone, which we have called the limb-forming zone (LFZ) along the lateral side of early chick embryos, which is independent of the lateral fold, is not as extensive as the lateral plate, and is not simply associated with ectodermal thickening, but which is directly correlated with limb-forming potential in the lateral plate.     

The role of lin-22, a hairy/enhancer of split homolog, in patterning the peripheral nervous system of C. elegans

In C. elegans, six lateral epidermal stem cells, the seam cells V1-V6, are located in a row along the anterior-posterior (A/P) body axis. Anterior seam cells (V1-V4) undergo a fairly simple sequence of stem cell divisions and generate only epidermal cells. Posterior seam cells (V5 and V6) undergo a more complicated sequence of cell divisions that include additional rounds of stem cell proliferation and the production of neural as well as epidermal cells. In the wild type, activity of the gene lin-22 allows V1-V4 to generate their normal epidermal lineages rather than V5-like lineages. lin-22 activity is also required to prevent additional neurons from being produced by one branch of the V5 lineage. We find that the lin-22 gene exhibits homology to the Drosophila gene hairy, and that lin-22 activity represses neural development within the V5 lineage by blocking expression of the posterior-specific Hox gene mab-5 in specific cells. In addition, in order to prevent anterior V cells from generating V5-like lineages, wild-type lin-22 gene activity must inhibit (directly or indirectly) at least five downstream regulatory gene activities. In anterior body regions, lin-22(+) inhibits expression of the Hox gene mab-5. It also inhibits the activity of the achaete-scute homolog lin-32 and an unidentified gene that we postulate regulates stem cell division. Each of these three genes is required for the expression of a different piece of the ectopic V5-like lineages generated in lin-22 mutants. In addition, lin-22 activity prevents two other Hox genes, lin-39 and egl-5, from acquiring new activities within their normal domains of function along the A/P body axis. Some, but not all, of the patterning activities of lin-22 in C. elegans resemble those of hairy in Drosophila.     

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.     

Epithelial-mesenchymal signaling during the regionalization of the chick gut

The development of the vertebrate gut requires signaling between the endoderm and mesoderm for establishing its normal anteroposterior (AP) axis and for tissue-specific differentiation. Factors implicated in positional specification of the AP regions of the gut include endodermally expressed Sonic hedgehog (Shh), mesodermally expressed Bmp4 and members of the Hox gene family. We have investigated the roles of these factors during AP regional specification of the chick embryonic gut. Early in gut development, the endoderm sends inductive signals to the mesoderm. Shh has been implicated as one of these signals. We find a differential response to exposure of the inductive influence of Shh along the AP axis of the gut. Virally mediated misexpression of Shh results in ectopic upregulation of its receptor Ptc and a cellular proliferation throughout the gut mesoderm. Although ectopic Shh can induce Bmp4 in the mesoderm of the midgut and hindgut, Bmp4 is not induced in the stomach region of the foregut. The stomach region has a thicker layer of mesoderm than the rest of the gut suggesting that the normal function of Bmp4 could be to limit mesodermal growth in the non-stomach regions of the gut. Ectopic Bmp4 expression in the stomach results in a reduction of the mesodermal component consistent with this hypothesis. In addition to the regional restriction on Bmp4 induction, Shh can only induce Hoxd-13 in the mesoderm of the hindgut. These findings suggest that a prepattern exists in the primitive gut mesoderm prior to expression of Shh in the endoderm. The gut mesoderm is subsequently responsible for inducing region-specific differentiation of its overlying endoderm. We tested the role of Hoxd-13, normally restricted in its mesodermal expression to the most posterior region of the hindgut (cloaca), in controlling adjacent endodermal differentiation. When virally mediated Hoxd-13 is misexpressed in the primitive midgut mesoderm, there is a transformation of the endoderm to the morphology and mucin content of the hindgut. Thus, the positionally restricted expression of a Hox gene in the gut mesoderm influences the inductive signaling that leads to regionally specific differentiation of gut endoderm.     

Control of antennal versus leg development in Drosophila

During the evolution of insects from a millipede-like ancestor, the Hox genes are thought to have promoted the diversification of originally identical body structures. In Drosophila melanogaster, antennae and legs are homologous structures that differ from each other as a result of the Hox gene Antennapedia (Antp), which promotes leg identities by repressing unknown antennal-determining genes. Here we present four lines of evidence that identify extradenticle (exd) and homothorax (hth) as antennal-determining genes. First, removing the function of exd or hth, which is required for the nuclear localization of Exd protein, transforms the antenna into leg; such transformations occur without activation of Antp. Second, hth is expressed and Exd is nuclear in most antennal cells, whereas both are restricted to proximal cells of the leg. Third, Antp is a repressor of hth. Fourth, ectopic expression of Meis1, a murine hth homologue, can trigger antennal development elsewhere in the fly. Taken together, these data indicate that hth is an antennal selector gene, and that Antp promotes leg development by repressing hth and consequently nuclear Exd.     

Analysis of K-ras gene mutations in lung carcinomas: correlation with gender, histological subtypes, and clinical outcome

The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord's role in imparting anteroposterior information to adjacent structures is less well understood, especially as the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Here we report that four zebrafish hox genes: hoxb1, hoxb5, hoxc6 and hoxc8 are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3' in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date.     

Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut

Reciprocal inductive signals between the endoderm and mesoderm are critical to vertebrate gut development. Sonic hedgehog encodes a secreted protein known to act as an inductive signal in several regions of the developing embryo. In this report, we provide evidence to support the role of Sonic hedgehog and its target genes Bmp-4 and the Abd-B-related Hox genes in the induction and patterning the chick hindgut. Sonic is expressed in the definitive endoderm at the earliest stage of chick gut formation. Immediately subjacent to Sonic expression in the caudal endoderm is undifferentiated mesoderm, later to become the visceral mesoderm of the hindgut. Genes expressed within this tissue include Bmp-4 (a TGF-beta relative implicated in proper growth of visceral mesoderm) and members of the Abd-B class of Hox genes (known regulators of pattern in many aspects of development). Using virally mediated misexpression, we show that Sonic hedgehog is sufficient to induce ectopic expression of Bmp-4 and specific Hoxd genes within the mesoderm. Sonic therefore appears to act as a signal in an epithelial-mesenchymal interaction in the earliest stages of chick hindgut formation. Gut pattern is evidenced later in gut morphogenesis with the presence of anatomic boundaries reflecting phenotypically and physiologically distinct regions. The expression pattern of the Abd-b-like Hox genes remains restricted in the hindgut and these Hox expression domains reflect gut morphologic boundaries. This finding strongly supports a role for these genes in determining the adult gut phenotype. Our results provide the basis for a model to describe molecular controls of early vertebrate hindgut development and patterning. Expression of homologous genes in Drosophila suggest that aspects of gut morphogenesis may be regulated by similar inductive networks in the two organisms.     

Archetypal organization of the amphioxus Hox gene cluster

Organization into gene clusters is an essential and diagnostic feature of Hox genes. Insect and nematode genomes possess single Hox gene clusters (split in Drosophila); in mammals, there are 38 Hox genes in four clusters on different chromosomes. A collinear relationship between chromosomal position, activation time and anterior expression limit of vertebrate Hox genes suggests that clustering may be important for precise spatiotemporal gene regulation and hence embryonic patterning. Hox genes have a wide phylogenetic distribution within the metazoa, and are implicated in the control of regionalization along the anteroposterior body axis. It has been suggested that changes in Hox gene number and genomic organization played a role in metazoan body-plan evolution, but identifying significant changes is difficult because Hox gene organization is known from only very few and widely divergent taxa (principally insects, nematodes and vertebrates). Here we analyse the complexity and organization of Hox genes in a cephalochordate, amphioxus, the taxon thought to be the sister group of the vertebrates. We find that the amphioxus genome has only one Hox gene cluster. It has similar genomic organization to the four mammalian Hox clusters, and contains homologues of at least the first ten paralogous groups of vertebrate Hox genes in a collinear array. Remarkably, this organization is compatible with that inferred for a direct ancestor of the vertebrates; we conclude that amphioxus is a living representative of a critical intermediate stage in Hox cluster evolution.     

Identification of a novel dual angiotensin II/vasopressin receptor

The development and patterning of the Drosophila wing relies on interactions between cell populations that have the anteroposterior (AP) axis and dorsoventral (DV) axis of the wing imaginal disc as frames of reference [1-3]. Each of these cell populations gives rise to a compartment - a group of cells that have their fates restricted by cell lineage - within which cells acquire specific identities through the expression of 'selector' genes [1,2,4]. The genes engrailed (en) and invected (inv), for example, label cells in the posterior compartment and mediate a set of cell interactions that direct the patterning and growth of the wing along the AP axis [1,2,4]. A similar situation has been proposed to exist across the DV axis, along with apterous (ap) as a dorsal selector gene [5], mediating cell interactions by regulating the expression of Serrate (Ser) [6] [7] and fringe (fng) [8]. In ap mutants, the wing is lost [5] [9], and here we report that this phenotype can be rescued by ectopic expression of either Ser or fng and that, surprisingly, the resulting wings have both dorsal and ventral cell fates.     

A Hox class 3 orthologue from the spider Cupiennius salei is expressed in a Hox-gene-like fashion

The class 3 Hox gene orthologue in insects, zerknüllt (zen), is not expressed along the anterior-posterior axis, but only in extra-embryonic tissues, suggesting that it has lost its function as a normal Hox gene. To analyse whether this loss of Hox gene function has already occurred in a basal arthropod lineage, we have isolated a Hox3 orthologue from the spider Cupiennius salei. In contrast to the insect zen sequences, which have a highly diverged homeobox, the spider Hox3 gene orthologue, Cs-Hox3, shows a high sequence similarity to the class 3 Hox genes of other phyla, including chordates. In situ hybridization in early embryos shows that it is expressed in a continuous region covering the pedipalp segment and the four leg-bearing segments. This expression pattern suggests a Hox-gene-like function for Cs-Hox3. On the other hand, the expression pattern does not strictly follow the colinearity rule, as it overlaps fully with the expression domain of the class 1 orthologue of the spider, Cs-lab. Still, our data suggest that the ancestor of the arthropods must have had a class 3 Hox gene with a function in anterior-posterior axis specification and that this function has been lost in the lineage leading to the insects.     

decapentaplegic overexpression affects Drosophila wing and leg imaginal disc development and wingless expression

We have used the GAL4-UAS expression system to increase the level of expression of the Drosophila gene decapentaplegic (dpp) in a pattern approximating its normal pattern in leg and wing imaginal discs. Intermediate increases of dpp expression have little effect in wing discs but high levels of dpp overexpression lead to reduction of the scutellum and duplication of posterior wing structures. In leg discs intermediate increases cause supernumerary outgrowths of ventral leg structures in the anterior-ventral region. Greater increases of dpp expression cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. The defects observed in both legs and wings appear to arise through dose-dependent effects of dpp on wingless (wg) expression. A high level of dpp overexpression in the wing disc causes reduction of wg expression in the presumptive scutellar region, consistent with the subsequent reduction of the scutellum. An intermediate increase of dpp expression in leg discs induces the expansion of wg expression into the ventral outgrowths. At higher dpp expression levels, ventral wg expression in leg discs is eliminated, consistent with the loss of ventral leg cuticle. In the leg disc end knob and in the wing margin primordium, where wg and dpp cooperate in producing distal outgrowth, dpp overexpression has no detectable effect either on patterning or on wg expression. We propose that a critical role for dpp in other regions of the leg and wing discs is to reduce or block the expression of wg. This role of dpp is supported by the observation that ectopic wg expression is detected in imaginal discs where dpp signaling is compromised by lowering the activity of one of its receptors, tkv. This antagonism between dpp and wg expression may be critical to assigning only one disc region as the distal organizer.     

Functional differences among Xenopus nodal-related genes in left-right axis determination

An association has been noted previously in chick, mouse and frog embryos between asymmetric nodal-related gene expression and embryonic situs, implying an evolutionarily conserved role in left-right specification. Of the four Xenopus nodal-related genes expressed during gastrulation, only Xnr-1 is re-expressed unilaterally in the left lateral plate mesoderm at neurula/tailbud stages. Here, we show that the asymmetric expression of Xnr-1 can be made bilaterally symmetric by right-sided microinjection of RNA encoding active Xenopus hedgehog proteins. Moreover, we provide the first evidence that Xnr-1 expression per se is a causal factor in left-right axis determination. When plasmids expressing Xnr-1 were delivered unilaterally to the right side of Xenopus embryos, a reversed laterality of both the heart and gut (homotaxic reversal) was induced in 40% of surviving embryos, while an additional 10-20% showed reversal of the heart or gut alone (heterotaxia). This effect on laterality was specific to Xnr-1, since neither Xnr-2 nor Xnr-3 plasmids had this activity. In addition, we find that Xnr-1 and Xnr-2, which have both been defined as mesoderm inducers from overexpression studies, show quantitative differences in their ability to induce dorsal mesoderm. Together, these findings suggest that the various Xnrs perform substantially different functions during Xenopus embryogenesis. Moreover, they strongly support the hypothesis that left lateral plate expression of nodal-related genes is a causative factor in the determination of asymmetry in vertebrate embryos.     

Ethanol impairs migration of the prechordal plate in the zebrafish embryo

Exposure of vertebrate embryos to ethanol causes cyclopia, but little is known about the underlying mechanisms of this effect. Here we show that cyclopia can be induced in the zebrafish by a short ethanol treatment during early gastrula stages and is accompanied by loss of gene expression characteristic of the ventral aspects of the fore- and midbrain. Interestingly, defects in the expression of ventral brain markers are linked to impaired migration of the prechordal plate mesoderm indicating that the correct position of the prechordal plate mesoderm under the anterior neural plate in the zebrafish embryo is required for specification of the anterior neural midline. Ethanol-induced cyclopia does not, however, impair the induction of anterior neuroectodermal structures in general. Finally, as genes like goosecoid and islet-1 are expressed in prechordal plate cells in a temporal pattern similar to control embryos despite the ectopic position of expressing cells, it appears that regulation of prechordal plate-specific gene expression is largely independent of the final position of the prechordal plate.     

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-beta (TGF-beta) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-beta superfamily that is related to mouse nodal. cyc encodes another nodal-related proteins, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.