Terri A. Williams

The evolution of patterning of serially homologous appendages in insects.

Arthropod bodies are formed by a series of appendage-bearing segments, and appendages have diversified both along the body axis within species and between species. Understanding the developmental basis of this variation is essential for addressing questions about the evolutionary diversification of limbs. We examined the development of serially homologous appendages of two insect species, the beetle Tribolium castaneum and the grasshopper Schistocerca americana. Both species retain aspects of ancestral appendage morphology and development that have been lost in Drosophila, including branched mouthparts and direct development of appendages during embryogenesis. We characterized the expression of four genes important in proximodistal axis development of Drosophila appendages: the secreted signaling factors wingless and decapentaplegic, and the homeodomain transcription factors extradenticle and Distal-less. Our comparisons focus on two aspects of appendage morphology: differentiation of the main axis of serial homologues and the appearance of proximal branches (endites) in the mouthparts. Although Distal-less expression is similar in endites and palps of the mouthparts, the expression of other genes in the endites does not conform to their known roles in axial patterning, leading us to reject the hypothesis that branched insect mouthparts develop by reiteration of the limb patterning network. With the exception of decapentaplegic, patterning of the main appendage axis is generally more similar in direct homologues than in serial homologues. Interestingly, however, phylogenetic comparisons suggest that patterning of serial homologues was more similar in ancestral insects, and thus that the observed developmental differences did not cause the evolutionary divergence in morphology among serial homologues.

Overexpression of transcription factor AP-2α suppresses mammary gland growth and morphogenesis

AP-2 transcription factors are key regulators of mouse embryonic development. Aberrant expression of these genes has also been linked to the progression of human breast cancer. Here, we have investigated the role of the AP-2 gene family in the postnatal maturation of the mouse mammary gland. Analysis of AP-2 RNA and protein levels demonstrates that these genes are expressed in the mammary glands of virgin and pregnant mice. Subsequently, AP-2 expression declines during lactation and then is reactivated during involution. The AP-2alpha and AP-2gamma proteins are localized in the ductal epithelium, as well as in the terminal end buds, suggesting that they may influence growth of the ductal network. We have tested this hypothesis by targeting AP-2alpha expression to the mouse mammary gland using the MMTV promoter. Our studies indicate that overexpression of AP-2alpha inhibits mammary gland growth and morphogenesis, and this coincides with a rise in PTHrP expression. Alveolar budding is severely curtailed in transgenic virgin mice, while lobuloalveolar development and functional differentiation are inhibited during pregnancy and lactation, respectively. Our studies strongly support a role for the AP-2 proteins in regulating the proliferation and differentiation of mammary gland epithelial cells in both mouse and human.

Decoupling elongation and segmentation: Notch involvement in anostracan crustacean segmentation

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ‐secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss‐of‐function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.

Evidence for a novel role for dachshund in patterning the proximal arthropod leg

The branchiopod crustacean Triops longicaudatus has paddlelike thoracic appendages with few joints and multiple marginal lobes. Here, we explore the degree to which the Triops limb is patterned by the same network of genes known to pattern the uniramous, multi-jointed insect appendage. Insect leg patterning proceeds through a process of subdividing the leg into proximal, intermediate, and distal regions by the activity of the transcription factors hth/exd, dac, and Dll. The immature Triops limb is subdivided into large, discrete regional domains (proximal and distal) as defined by nuclear-EXD and DLL. We show that HTH expression in Triops overlaps cell-to-cell with n-EXD expression. In addition, dac is expressed in two domains: (1) adjacent to and partially overlapping the distal Dll domain and (2) along the medial margin of the developing leg. The DAC domain adjacent to the distal Dll domain supports the early establishment of the expected intermediate domain of DAC expression. The medial expression domain resolves over time into a series of reiterated stripes located on the lower side of each medial lobe. Later, this expression pattern correlates with the sclerotized regions associated with limb flexion. We propose that these stripes of DAC expression play a role in forming reiterated medial lobes. Unlike Drosophila, where the proximal distal patterning of the leg is coincident with patterning of reiterated structures (segments), we hypothesize that the patterning in Triops may reflect an ancestral state where the patterning of reiterated medial structures was not coincident with proximodistal limb patterning.

Linking gene regulation to cell behaviors in the posterior growth zone of sequentially segmenting arthropods.

Virtually all arthropods all arthropods add their body segments sequentially, one by one in an anterior to posterior progression. That process requires not only segment specification but typically growth and elongation. Here we review the functions of some of the key genes that regulate segmentation: Wnt, caudal, Notch pathway, and pair-rule genes, and discuss what can be inferred about their evolution. We focus on how these regulatory factors are integrated with growth and elongation and discuss the importance and challenges of baseline measures of growth and elongation. We emphasize a perspective that integrates the genetic regulation of segment patterning with the cellular mechanisms of growth and elongation.

Early Distal-less expression in a developing crustacean limb bud becomes restricted to setal-forming cells

SUMMARY Distal‐less (Dll) plays a well‐known role in patterning the distal limb in arthropods. However, in some taxa, its expression even during early limb development is not always limited to the distal limb. Here, I trace the expression of Distal‐less in a crustacean (Thamnocephalus platyurus) from the early limb bud to later stages of limb development, a period that includes differentiation of juvenile and adult morphology. During early development, I find two distinct types of DLL expression: one correlated with proximal distal leg patterning and the other restricted to setal‐forming cells. Later in development, all the DLL expression is restricted to setal‐forming cells. Based on the particular cells expressing DLL, I hypothesize an ancestral role for Dll function in the formation accessory cells of sensilla.

Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus. We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited. Summary: A detailed analysis of posterior segment addition in an insect reveals that the growth zone is divided into two functional domains responsible for growth and differentiation.

Wnt repertoire and developmental expression patterns in the crustacean Thamnocephalus platyurus.

Wnt genes are a family of conserved glycoprotein ligands that play a role in a wide variety of cell and developmental processes, from cell proliferation to axis elongation. There are 13 Wnt subfamilies found among metazoans. Eleven of these appear conserved in arthropods with a pattern of loss during evolution of as many as six subfamilies among hexapods. Here we report on Wnt genes in the branchiopod crustacean, Thamnocephalus platyurus, including the first documentation of the expression of the complete Wnt gene family in a crustacean. Our results suggest fewer Wnt genes were retained in Thamnocephalus than in the related crustacean, Daphnia, although the Thamnocephalus Wnt repertoire is larger than that found in insects. We also find an intriguing pattern of staggered expression of Wnts—an anterior‐posterior stagger within the posterior growth zone and a dorsal‐ventral stagger in the developing segments—suggesting a potential for subfunctionalization of Wnts in these regions.

Evodevo and the Promise of Understanding Morphological Transitions in Evolution1

Abstract The field of Evolutionary Developmental biology arose with the promise of new approaches to answering longstanding questions of comparative biology. Here we review the fruits of that promise some decades later. We chose three areas of arthropod EvoDevo—evolution of body plans, segment number, and appendage morphology—to provide an overview for the nonspecialist of how these issues have been clarified by the comparative analysis of regulatory gene networks. In all cases, we identify substantial progress and novel insights provided by the tools and perspective of EvoDevo. We also recognize that some core questions remain unanswered, and we reflect on how discoveries in EvoDevo fit in the landscape of other progress in phylogenetics, population biology, and genomics, facilitated by a new and ever-expanding set of molecular tools for comparative studies in evolution.