Joseph C. Pearson

Modulating Hox gene functions during animal body patterning

With their power to shape animal morphology, few genes have captured the imagination of biologists as the evolutionarily conserved members of the Hox clusters have done. Recent research has provided new insight into how Hox proteins cause morphological diversity at the organismal and evolutionary levels. Furthermore, an expanding collection of sequences that are directly regulated by Hox proteins provides information on the specificity of target-gene activation, which might allow the successful prediction of novel Hox-response genes. Finally, the recent discovery of microRNA genes within the Hox gene clusters indicates yet another level of control by Hox genes in development and evolution.

Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes—Ddc, ple, msn, and kkv—that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound–response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

Transcriptional autoregulation in development

The authors would like to thank Peter Harte and Mark Ptashne for helpful and stimulating comments on the manuscript. Our labs research is supported by grants from the NIH and NSF. The UNC Developmental Biology NIH training grant provided support to J.C.P.

Diverse modes of Drosophila tracheal fusion cell transcriptional regulation

Drosophila tracheal fusion cells play multiple important roles in guiding and facilitating tracheal branch fusion. Mechanistic understanding of how fusion cells function during development requires deciphering their transcriptional circuitry. In this paper, three genes with distinct patterns of fusion cell expression were dissected by transgenic analysis to identify the cis-regulatory modules that mediate their transcription. Bioinformatic analysis involving phylogenetic comparisons coupled with mutational experiments were employed. The dysfusion bHLH-PAS gene was shown to have two fusion cell cis-regulatory modules; one driving initial expression and another autoregulatory module to enhance later transcription. Mutational dissection of the early module identified at least four distinct inputs, and included putative binding sites for ETS and POU-homeodomain proteins. The ETS transcription factor Pointed mediates the transcriptional output of the branchless/breathless signaling pathway, suggesting that this pathway directly controls dysfusion expression. Fusion cell cis-regulatory modules of CG13196 and CG15252 require two Dysfusion:Tango binding sites, but additional sequences modulate the breadth of activation in different fusion cell classes. These results begin to decode the regulatory circuitry that guides transcriptional activation of genes required for fusion cell morphogenesis.

Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new ‘atlas-builder’ software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an ‘eNeuro’ atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes – cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters – and linking them to individual neurons.

Time-lapse imaging reveals stereotypical patterns of Drosophila midline glial migration.

The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. In this paper, we use time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. We identified 3 groups of MG that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function.

Enhancer Diversity and the Control of a Simple Pattern of Drosophila CNS Midline Cell Expression

Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, we compare the cis-regulation of multiple genes expressed in the Drosophila CNS midline primordium cells, and show that while the expression patterns may appear alike, the target genes are not equivalent in how these common expression patterns are achieved. Some genes utilize a single enhancer that promotes expression in all midline cells, while others utilize multiple enhancers with distinct spatial, temporal, and quantitative contributions. Two regulators, Single-minded and Notch, play key roles in controlling early midline gene expression. While Single-minded is expected to control expression of most, if not all, midline primordium-expressed genes, the role of Notch in directly controlling midline transcription is unknown. Midline primordium expression of the rhomboid gene is dependent on cell signaling by the Notch signaling pathway. Mutational analysis of a rhomboid enhancer reveals at least 5 distinct types of functional cis-control elements, including a binding site for the Notch effector, Suppressor of Hairless. The results suggest a model in which Notch/Suppressor of Hairless levels are insufficient to activate rhomboid expression by itself, but does so in conjunction with additional factors, some of which, including Single-minded, provide midline specificity to Notch activation. Similarly, a midline glial enhancer from the argos gene, which is dependent on EGF/Spitz signaling, is directly regulated by contributions from both Pointed, the EGF transcriptional effector, and Single-minded. In contrast, midline primordium expression of other genes shows a strong dependence on Single-minded and varying combinations of additional transcription factors. Thus, Single-minded directly regulates midline primordium-expressed genes, but in some cases plays a primary role in directing target gene midline expression, and in others provides midline specificity to cell signaling inputs.