Susan M. Parkhurst

Parallels between tissue repair and embryo morphogenesis

Wound healing involves a coordinated series of tissue movements that bears a striking resemblance to various embryonic morphogenetic episodes. There are several ways in which repair recapitulates morphogenesis. We describe how almost identical cytoskeletal machinery is used to repair an embryonic epithelial wound as is involved during the morphogenetic episodes of dorsal closure in Drosophila and eyelid fusion in the mouse foetus. For both naturally occurring and wound-activated tissue movements, JNK signalling appears to be crucial, as does the tight regulation of associated cell divisions and adhesions. In the embryo, both morphogenesis and repair are achieved with a perfect end result, whereas repair of adult tissues leads to scarring. We discuss whether this may be due to the adult inflammatory response, which is absent in the embryo.

Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration

Aa robust inflammatory response to tissue damage and infection is conserved across almost all animal phyla. Neutrophils and macrophages, or their equivalents, are drawn to the wound site where they engulf cell and matrix debris and release signals that direct components of the repair process. This orchestrated cell migration is clinically important, and yet, to date, leukocyte chemotaxis has largely been studied in vitro. Here, we describe a genetically tractable in vivo wound model of inflammation in the Drosophila melanogaster embryo that is amenable to cinemicroscopy. For the first time, we are able to examine the roles of Rho-family small GTPases during inflammation in vivo and show that Rac-mediated lamellae are essential for hemocyte motility and Rho signaling is necessary for cells to retract from sites of matrix– and cell–cell contacts. Cdc42 is necessary for maintaining cellular polarity and yet, despite in vitro evidence, is dispensable for sensing and crawling toward wound cues.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and Hairy-mediated transcriptional repression

hairy is a Drosophila pair‐rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy‐mediated repression, we used a yeast interaction screen and identified a Hairy‐interacting protein, the Drosophila homolog of the human C‐terminal‐binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C‐terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two‐hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C‐terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix–loop–helix protein class. We identified a P‐element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose‐sensitive genetic interactions between dCtBP and hairy mutations.

Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairys structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

Myc and Max homologs in Drosophila.

The proteins encoded by the myc proto-oncogene family are involved in cell proliferation, apoptosis, differentiation, and neoplasia. Myc acts through dimerization with Max to bind DNA and activate transcription. Homologs of the myc and max genes were cloned from the fruit fly Drosophila melanogaster and their protein products (dMyc and dMax) were shown to heterodimerize, recognize the same DNA sequence as their vertebrate homologs, and activate transcription. The dMyc protein is likely encoded by the Drosophila gene diminutive (dm), a mutation in which results in small body size and female sterility caused by degeneration of the ovaries. These findings indicate a potential role for Myc in germ cell development and set the stage for genetic analysis of Myc and Max.

Human Subtelomeric WASH Genes Encode a New Subclass of the WASP Family

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASHs location in these dynamic regions could have advantageous as well as pathologic consequences.

Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.

Drosophila Sir2 Is Required for Heterochromatic Silencing and by Euchromatic Hairy/E(Spl) bHLH Repressors in Segmentation and Sex Determination

Yeast SIR2 is a NAD+-dependent histone deacetylase required for heterochromatic silencing at telomeres, rDNA, and mating-type loci. We find that the Drosophila homolog of Sir2 (dSir2) also encodes deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres. We show that dSir2 interacts genetically and physically with members of the Hairy/Deadpan/E(Spl) family of bHLH euchromatic repressors, key regulators of Drosophila development. dSir2 is an essential gene whose loss of function results in both segmentation defects and skewed sex ratios, associated with reduced activities of the Hairy and Deadpan bHLH repressors. These results indicate that Sir2 in higher organisms plays an essential role in both euchromatic repression and heterochromatic silencing.

Hairy transcriptional repression targets and cofactor recruitment in Drosophila.

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2–6 h) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box–containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairys cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for Hairy in segmentation, we find groups of targets suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairys three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find here that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development.

Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling.

Single-cell wound repair in Drosophila involves mechanistically distinct expansion, contraction, and closure phases.