Arhat Abzhanov

The calmodulin pathway and evolution of elongated beak morphology in Darwin's finches

A classic textbook example of adaptive radiation under natural selection is the evolution of 14 closely related species of Darwins finches (Fringillidae, Passeriformes), whose primary diversity lies in the size and shape of their beaks. Thus, ground finches have deep and wide beaks, cactus finches have long and pointed beaks (low depth and narrower width), and warbler finches have slender and pointed beaks, reflecting differences in their respective diets. Previous work has shown that even small differences in any of the three major dimensions (depth, width and length) of the beak have major consequences for the overall fitness of the birds. Recently we used a candidate gene approach to explain one pathway involved in Darwins finch beak morphogenesis. However, this type of analysis is limited to molecules with a known association with craniofacial and/or skeletogenic development. Here we use a less constrained, complementary DNA microarray analysis of the transcripts expressed in the beak primordia to find previously unknown genes and pathways whose expression correlates with specific beak morphologies. We show that calmodulin (CaM), a molecule involved in mediating Ca2+ signalling, is expressed at higher levels in the long and pointed beaks of cactus finches than in more robust beak types of other species. We validated this observation with in situ hybridizations. When this upregulation of the CaM-dependent pathway is artificially replicated in the chick frontonasal prominence, it causes an elongation of the upper beak, recapitulating the beak morphology of the cactus finches. Our results indicate that local upregulation of the CaM-dependent pathway is likely to have been a component of the evolution of Darwins finch species with elongated beak morphology and provide a mechanistic explanation for the independence of beak evolution along different axes. More generally, our results implicate the CaM-dependent pathway in the developmental regulation of craniofacial skeletal structures.

Regulation of skeletogenic differentiation in cranial dermal bone

Although endochondral ossification of the limb and axial skeleton is relatively well-understood, the development of dermal (intramembranous) bone featured by many craniofacial skeletal elements is not nearly as well-characterized. We analyzed the expression domains of a number of markers that have previously been associated with endochondral skeleton development to define the cellular transitions involved in the dermal ossification process in both chick and mouse. This led to the recognition of a series of distinct steps in the dermal differentiation pathways, including a unique cell type characterized by the expression of both osteogenic and chondrogenic markers. Several signaling molecules previously implicated in endochondrial development were found to be expressed during specific stages of dermal bone formation. Three of these were studied functionally using retroviral misexpression. We found that activity of bone morphogenic proteins (BMPs) is required for neural crest-derived mesenchyme to commit to the osteogenic pathway and that both Indian hedgehog (IHH) and parathyroid hormone-related protein (PTHrP, PTHLH) negatively regulate the transition from preosteoblastic progenitors to osteoblasts. These results provide a framework for understanding dermal bone development with an aim of bringing it closer to the molecular and cellular resolution available for the endochondral bone development.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro

During development neural crest cells give rise to a wide variety of specialized cell types in response to cytokines from surrounding tissues. Depending on the cranial-caudal level of their origin, different populations of neural crest cells exhibit differential competence to respond to these signals as exemplified by the unique ability of cranial neural crest to form skeletal cell types. We show that in addition to differences in whether they respond to particular signals, cranial neural crest cells differ dramatically from the trunk neural crest cells in how they respond to specific extracellular signals, such that under identical conditions the same signal induces dissimilar cell fate decisions in the two populations in vitro. Conversely, the same differentiated cell types are induced by different signals in the two populations. These in vitro differences in neural crest response are consistent with in vivo manipulations. We also provide evidence that these differences in responsiveness are modulated, at least in part, by differential expression of Hox genes within the neural crest.

Chelicerate Hox genes and the homology of arthropod segments

Genes of the homeotic complex (HOM‐C) in insects and vertebrates are required for the specification of segments along the antero‐posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM‐C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia (pb) and Deformed (Dfd  ). In addition, we have cloned larger cDNA fragments of pb, zerknullt (zen/Hox3) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co‐linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD‐A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.

Peripheral arterial occlusive disease: Global gene expression analyses suggest a major role for immune and inflammatory responses

BackgroundPeripheral arterial disease (PAD), a major manifestation of atherosclerosis, is associated with significant cardiovascular morbidity, limb loss and death. However, mechanisms underlying the genesis and progression of the disease are far from clear. Genome-wide gene expression profiling of clinical samples may represent an effective approach to gain relevant information.ResultsAfter histological classification, a total of 30 femoral artery samples, including 11 intermediate lesions, 14 advanced lesions and 5 normal femoral arteries, were profiled using Affymetrix microarray platform. Following real-time RT-PCR validation, different algorithms of gene selection and clustering were applied to identify differentially expressed genes. Under a stringent cutoff, i.e., a false discovery rate (FDR) <0.5%, we found 366 genes were differentially regulated in intermediate lesions and 447 in advanced lesions. Of these, 116 genes were overlapped between intermediate and advanced lesions, including 68 up-regulated genes and 48 down-regulated ones. In these differentially regulated genes, immune/inflammatory genes were significantly up-regulated in different stages of PAD, (85/230 in intermediate lesions, 37/172 in advanced lesions). Through literature mining and pathway analysis using different databases such as Gene Ontology (GO), and the Kyoto Encyclopedia of Gene and Genomics (KEGG), genes involved in immune/inflammatory responses were significantly enriched in up-regulated genes at different stages of PAD(p < 0.05), revealing a significant correlation between immune/inflammatory responses and disease progression. Moreover, immune-related pathways such as Toll-like receptor signaling and natural killer cell mediated cytotoxicity were particularly enriched in intermediate and advanced lesions (P < 0.05), highlighting their pathogenic significance during disease progression.ConclusionLines of evidence revealed in this study not only support previous hypotheses, primarily based on studies of animal models and other types of arterial disease, that inflammatory responses may influence the development of PAD, but also permit the recognition of a wide spectrum of immune/inflammatory genes that can serve as signatures for disease progression in PAD. Further studies of these signature molecules may eventually allow us to develop more sophisticated protocols for pharmaceutical interventions.

ROLES FOR MODULARITY AND CONSTRAINT IN THE EVOLUTION OF CRANIAL DIVERSITY AMONG ANOLIS LIZARDS

Complex organismal structures are organized into modules, suites of traits that develop, function, and vary in a coordinated fashion. By limiting or directing covariation among component traits, modules are expected to represent evolutionary building blocks and to play an important role in morphological diversification. But how stable are patterns of modularity over macroevolutionary timescales? Comparative analyses are needed to address the macroevolutionary effect of modularity, but to date few have been conducted. We describe patterns of skull diversity and modularity in Caribbean Anolis lizards. We first diagnose the primary axes of variation in skull shape and then examine whether diversification of skull shape is concentrated to changes within modules or whether changes arose across the structure as a whole. We find no support for the hypothesis that cranial modules are conserved as species diversify in overall skull shape. Instead we find that anole skull shape and modularity patterns independently converge. In anoles, skull modularity is evolutionarily labile and may reflect the functional demands of unique skull shapes. Our results suggest that constraints have played little role in limiting or directing the diversification of head shape in Anolis lizards.

Closely related bird species demonstrate flexibility between beak morphology and underlying developmental programs

The astonishing variation in the shape and size of bird beaks reflects a wide range of dietary specializations that played an important role in avian diversification. Among Darwin’s finches, ground finches (Geospiza spp.) have beaks that represent scaling variations of the same shape, which are generated by alterations in the signaling pathways that regulate growth of the two skeletal components of the beak: the prenasal cartilage (pnc) and the premaxillary bone (pmx). Whether this developmental mechanism is responsible for variation within groups of other closely related bird species, however, has remained unknown. Here, we report that the Caribbean bullfinches (Loxigilla spp.), which are closely related to Darwin’s finches, have independently evolved beaks of a novel shape, different from Geospiza, but also varying from each other only in scaling. However, despite sharing the same beak shape, the signaling pathways and tissues patterning Loxigilla beaks differ among the three species. In Loxigilla noctis, as in Geospiza, the pnc develops first, shaped by Bmp4 and CaM signaling, followed by the development of the pmx, regulated by TGFβIIr, β-catenin, and Dkk3 signaling. In contrast, beak morphogenesis in Loxigilla violacea and Loxigilla portoricensis is generated almost exclusively by the pmx through a mechanism in which Ihh and Bmp4 synergize to promote expansion of bone tissue. Together, our results demonstrate high flexibility in the relationship between morphology and underlying developmental causes, where different developmental programs can generate identical shapes, and similar developmental programs can pattern different shapes.

Embryonic expression patterns of the Hox genes of the crayfish Procambarus clarkii (Crustacea, Decapoda)

SUMMARY Higher crustaceans (class Malacostraca) represent the most species‐rich and morphologically diverse group of non‐insect arthropods. The superorders Eucarida and Peracarida, two large groups that separated over 350 million years ago, encompass most malacostracan diversity. Recently, the Hox genes of the peracarid woodlouse Porcellio scaber (Isopoda) were shown to be expressed in domains that coincide with morphological boundaries of body tagmata, which differ from those in insects ( Abzhanov and Kaufman 1999a,b ). Moreover, observed changes in Hox expression domains during ontogeny correlate with morphological remodeling, such as a transformation of the first thoracic leg into mouthpart maxillipeds, which occurs in the trunk of the embryo. Decapods have a different modification of the malacostracan bodyplan, with up to three pairs of maxillipeds and extensive fusion and cephalization of the thorax. Here we describe expression patterns of the trunk Hox genes Scr, Antp, Ubx, abd‐A and cad in the eucarid crayfish Procambarus clarkii (Decapoda). We find that the crayfish expression patterns, for the most part, resemble those of the woodlouse Porcellio scaber (Isopoda), but are more modulated and complex. Nevertheless, as in Porcellio the boundaries of the Hox expression domains do correlate with morphological features and their modulations to transformations in the embryo. Thus we propose that the trunk Hox genes were likely important in the evolution of and currently play an essential role in the development of the complex decapod bodyplan.

CONVERGENT EVOLUTION OF SEXUAL DIMORPHISM IN SKULL SHAPE USING DISTINCT DEVELOPMENTAL STRATEGIES

Studies integrating evolutionary and developmental analyses of morphological variation are of growing interest to biologists as they promise to shed fresh light on the mechanisms of morphological diversification. Sexually dimorphic traits tend to be incredibly divergent across taxa. Such diversification must arise through evolutionary modifications to sex differences during development. Nevertheless, few studies of dimorphism have attempted to synthesize evolutionary and developmental perspectives. Using geometric morphometric analysis of head shape for 50 Anolis species, we show that two clades have converged on extreme levels of sexual dimorphism through similar, male‐specific changes in facial morphology. In both clades, males have evolved highly elongate faces whereas females retain faces of more moderate proportion. This convergence is accomplished using distinct developmental mechanisms; one clade evolved extreme dimorphism through the exaggeration of a widely shared, potentially ancestral, developmental strategy whereas the other clade evolved a novel developmental strategy not observed elsewhere in the genus. Together, our analyses indicate that both shared and derived features of development contribute to macroevolutionary patterns of morphological diversity among Anolis lizards.