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The Genetic Control of Morphogenesis
Summary: Thomas Kaufman is interested in understanding the role of genes in the developmental process, particularly how the essentially two-dimensional information encoded in the DNA is translated into a three-dimensional organism.
Our laboratory has focused over the past several years on trying to understand the role of the homeotic Antennapedia complex (ANT-C) in Drosophila melanogaster. This group of tightly linked Hox genes was defined in this lab and shown to be responsible for the specification of segmental identity in the anterior thorax and head of the embryo and adult. We have recovered and characterized mutations in all of the genes of the complex and cloned and sequenced the coding portions of those genes.
The genes proboscipedia (pb) and Sex combs reduced (Scr), members of the ANT-C, are required for the proper specification of the adult mouthparts. The proboscis of the fly is one of the most derived appendages, and its segmental composition and development are not well understood. However, recent studies in Drosophila have revealed a system for the patterning of legs and antennae and several genes have been shown to be required for this process. Among these are extradenticle (exd), homothorax (hth), dachshund (dac), Distal-less (Dll), spalt (sal), and wingless (wg). We have demonstrated that the gene products of pb and Scr regulate the appendage genes during proboscis development. Pb inhibits exd, hth, and dac expression and down-regulates Dll. This explains the ability of Pb to inhibit the effects of ectopically expressed trunk genes in the proboscis or to suppress leg identity in the trunk. Scr suppresses wg and Dll expression and, together with Pb, negatively regulates sal. We conclude that the proboscis constitutes a genetically distinct type of appendage that does not require leg or antennal patterning systems for its morphogenesis.
Evolution of the Homeotic genes
The discovery of the homeobox and more particularly the homeotic complex (HOM-C) has had at least two major impacts on evolutionary thought and research. First, the presence of the clusters and their colinear linkage and expression patterns in all of the bilaterian phyla indicate that many of the basic molecular mechanisms for the regulation of development evolved very early. Thus the evolutionary biologist must be concerned with explaining how an essentially common set of underlying molecular paradigms have been used to produce the rich array of morphologies found in extant and fossil animal forms. Fortunately the second virtue of the Hox genes—the sequence conservation of the homeobox itself—has allowed the ready cloning of this motif from a large number of organisms that would normally not be investigated at the molecular level. These cloned fragments have been used to determine the expression domains of these genes over broad phylogenetic distances.
In Drosophila the Hox genes have stereotypical spatiotemporal patterns of expression, and we have investigated the conservation or divergence of these patterns within the Arthropoda. Using recovered cDNAs and polymerase chain reactions, we have cloned homeobox-containing fragments from members of several different orders of insects. Probes derived from these clones have shown that the expression domains of the Hox genes are generally conserved among the insects. We have extended this analysis by recovering homologous fragments from higher crustaceans. Representatives of the Insecta and the higher crustaceans have highly derived body plans subdivided into several tagma, groups of segments united by a common function and/or morphology. The tagmatization of segments in the trunk, the part of the body between head and telson, in both lineages is thought to have evolved independently from ancestors with a distinct head but a homonymous, undifferentiated trunk. In the lower crustacean Artemia franciscana, the trunk Hox genes are expressed in broad overlapping domains suggesting a conserved ancestral state. In comparison, in insects, the Antennapedia-class genes of the homeotic clusters are more regionally deployed into distinct domains where they serve to control the morphology of the different trunk segments. Thus an originally Artemia-like pattern of homeotic gene expression has apparently been modified in the insect lineage associated with and perhaps facilitating the observed pattern of tagmatization.
We examined the expression patterns of the Hox genes Antp, Ultrabithorax, and abdominal-A in the higher crustacean Porcellio scaber. Unlike the pattern seen in Artemia, these genes are expressed in well-defined discrete domains coinciding with tagmatic boundaries that are distinct from those of the insects. Our observations suggest that during the independent tagmatization in insects and higher crustaceans, the homologous "trunk" genes evolved to perform different developmental functions and that in each lineage the changes in Hox gene expression pattern may have been important in trunk tagmatization.
It has been proposed that during the evolution of several crustacean lineages, changes in the expression patterns of the homeotic genes Ubx andabd-A have played a role in transformation of the anterior thoracic appendages into mouthparts termed maxillipeds. This homeotic-like transformation is recapitulated at the late stages of embryonic development of Porcellio. Interestingly, this morphological change is associated with apparent novelties both in the transcriptional and post-transcriptional regulation of the Porcellio ortholog of Drosophila Scr. Specifically, we find that Scr mRNA is present in the second maxillary segment and the first pair of thoracic legs (T1) in early embryos, whereas protein accumulates only in the second maxillae. In later stages, however, high levels of Scr appear in the T1 legs, which correlates temporally with the transformation of these appendages into maxillipeds. Our observations provide further insight into the process of the homeotic leg-to-maxilliped transformation in the evolution of crustaceans and suggest a novel regulatory mechanism for this process in this group of arthropods.
What remains to be determined, of course, is whether the function of the Hox genes and their products have diverged in the same way that their deployment might indicate. To approach this problem, we have begun to use interference RNA (RNAi) to ask questions about function. We have successfully produced phenocopies of several homeotic mutations by the injection of double-stranded RNA into cleavage-stage embryos of the milkweed bug Oncopeltus faciatus, which has specialized suctorial mouthparts. The Hox genes Dfd, pb, and Scr have previously been shown to be expressed in the gnathal appendages of this species. By analyzing single and combination RNAi depletions, we find that Dfd, pb, and Scr are used in the milkweed bug to specify the identity of the mouthparts. The exact roles of the genes, however, are different from what is known in Drosophila and Tribolium. The maxillary appendages in the bug are determined by the activities of the genes Dfd and Scr, rather than Dfd and pb as in the fly and beetle. The mandibular appendages are specified by Dfd, but their unique morphology in Oncopeltus suggests that Dfd's targets are different. As in flies and beetles, the labium is specified by the combined activities of pb and Scr, but again, the function of pb appears to be different. Additionally, the regulatory control of pb by the other two genes seems to be different in the bug than in either of the other species. These novelties in Hox function, expression pattern, and regulatory relationships may have been important for the evolution of the unique hemipteran head. We are now attempting to broaden this functional analysis to other insects and crustaceans.
Last updated June 20, 2001
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