Kathleen Gajewski

The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.

D-mef2 is a target for Tinman activation during Drosophila heart development

The NK‐type homeobox gene tinman and the MADS box gene D‐mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D‐mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D‐mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D‐mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila.

The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in Drosophila.

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.

Genetically distinct cardial cells within the Drosophila heart

Summary: Although often viewed as a simple pulsating tube, the Drosophila dorsal vessel is intricate in terms of its structure, cell types, and patterns of gene expression. Two nonidentical groups of cardial cells are observed in segments of the heart based on the differential expression of transcriptional regulators. These include sets of four cell pairs that express the homeodomain protein Tinman (Tin), alternating with groups of two cell pairs that express the orphan steroid hormone receptor Seven Up (Svp). Here we show that these myocardial cell populations are distinct in terms of their formation and gene expression profiles. The Svp‐expressing cells are generated by asymmetric cell divisions of precursor cells based on decreases or increases in their numbers in numb or sanpodo mutant embryos. In contrast, the numbers of Tin‐expressing cardial cells are unchanged in these genetic backgrounds, suggesting they arise from symmetric cell divisions. One function for Svp in the two pairs of cardial cells is to repress the expression of the tin gene and at least one of its targets, the β3 tubulin gene. Further differences in the cells are substantiated by the identification of separable enhancers for D‐mef2 gene transcription in the distinct cardioblast sets. Taken together, these results demonstrate a greater cellular and genetic complexity of the Drosophila heart than previously appreciated. genesis 28:36–43, 2000.

Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis

The GATA factor Serpent (Srp) is required for hemocyte precursor formation during Drosophila hematopoiesis. These blood cell progenitors give rise to two distinct lineages within the developing embryo. Lozenge, a Runx protein homologue, and Glial cells missing-1 and -2 are essential for crystal cell and plasmatocyte production, respectively. In contrast U-shaped, a Friend of GATA class factor, antagonizes crystal cell formation. Here we show that Srp, Lozenge, and U-shaped interact in different combinations to regulate crystal cell lineage commitment. Coexpression of Srp and Lozenge synergistically activated the crystal cell program in both embryonic and larval stages. Furthermore, expression of Lozenge and SrpNC, a Srp isoform with N- and C-terminal zinc fingers, inhibited u-shaped expression, indicating that crystal cell activation coincided with the down-regulation of this repressor-encoding gene. In contrast, whereas U-shaped and SrpNC together blocked crystal cell production, coexpression of U-shaped with noninteracting Srp proteins failed to prevent overproduction of this hemocyte population. Such results indicated that U-shaped and SrpNC must interact to block crystal cell production. Taken together, these studies show that the specialized SrpNC isoform plays a pivotal role during crystal cell lineage commitment, acting as an activator or repressor depending on the availability of specific transcriptional coregulators. These findings provide definitive proof of the combinatorial regulation of hematopoiesis in Drosophila and an in vivo demonstration of GATA and Runx functional interaction in a blood cell commitment program.

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth.

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.

Phosphorylation by the DHIPK2 Protein Kinase Modulates the Corepressor Activity of Groucho

Groucho function is essential for Drosophila development, acting as a corepressor for specific transcription factors that are downstream targets of various signaling pathways. Here we provide evidence that Groucho is phosphorylated by the DHIPK2 protein kinase. Phosphorylation modulates Groucho corepressor activity by attenuating its protein-protein interaction with a DNA-bound transcription factor. During eye development, DHIPK2 modifies Groucho activity, and eye phenotypes generated by overexpression of Groucho differ depending on its phosphorylation state. Moreover, analysis of nuclear extracts fractionated by column chromatography further shows that phospho-Groucho associates poorly with the corepressor complex, whereas the unphosphorylated form binds tightly. We propose that Groucho phosphorylation by DHIPK2 and its subsequent dissociation from the corepressor complex play a key role in relieving the transcriptional repression of target genes regulated by Groucho, thereby controlling cell fate determination during development.

Expression, regulation, and requirement of the toll transmembrane protein during dorsal vessel formation in Drosophila melanogaster.

ABSTRACT Early heart development in Drosophila and vertebrates involves the specification of cardiac precursor cells within paired progenitor fields, followed by their movement into a linear heart tube structure. The latter process requires coordinated cell interactions, migration, and differentiation as the primitive heart develops toward status as a functional organ. In the Drosophila embryo, cardioblasts emerge from bilateral dorsal mesoderm primordia, followed by alignment as rows of cells that meet at the midline and morph into a dorsal vessel. Genes that function in coordinating cardioblast organization, migration, and assembly are integral to heart development, and their encoded proteins need to be understood as to their roles in this vital morphogenetic process. Here we prove the Toll transmembrane protein is expressed in a secondary phase of heart formation, at lateral cardioblast surfaces as they align, migrate to the midline, and form the linear tube. The Toll dorsal vessel enhancer has been characterized, with its activity controlled by Dorsocross and Tinman transcription factors. Consistent with the observed protein expression pattern, phenotype analyses demonstrate Toll function is essential for normal dorsal vessel formation. Such findings implicate Toll as a critical cell adhesion molecule in the alignment and migration of cardioblasts during dorsal vessel morphogenesis.

Requirement of the LIM Homeodomain Transcription Factor Tailup for Normal Heart and Hematopoietic Organ Formation in Drosophila melanogaster

ABSTRACT Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. We used a cardioblast-expressed Toll-GFP transgene to screen for additional genes involved in heart development and identified tailup as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loop-helix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes.

Combinatorial control of Drosophila mef2 gene expression in cardiac and somatic muscle cell lineages

Abstract The Drosophila mef2 gene encodes a MADS domain transcription factor required for the differentiation of cardiac, somatic, and visceral muscles during embryogenesis and the patterning of adult indirect flight muscles assembled during metamorphosis. A prerequisite for D-MEF2 function in myogenesis is its precise expression in multiple cell types during development. Novel enhancers for D-mef2 transcription in cardiac and adult muscle precursor cells have been identified and their regulation by the Tinman and Twist myogenic factors have been demonstrated. However, these results suggested the existence of additional regulators and provided limited information on the specification of progenitor cells for different muscle lineages. We have further characterized the heart enhancer and show it is part of a complex regulatory region controlling the activation and repression of D-mef2 transcription in several cell types. The mutation of a GATA sequence in the enhancer changes its specificity from cardial to pericardial cells. Also, the addition of flanking sequences to the heart enhancer results in expression in a new cell type, that being the founder cells of a subset of body wall muscles. As tinman function is required for D-mef2 expression in both the cardial and founder cells, these results define a shared regulatory DNA that functions in distinct lineages due to the combinatorial activity of Tinman and other factors that work through adjacent sequences. The analysis of D-mef2-lacZ fusion genes in mutant embryos revealed that the specification of the muscle precursor cells involved the wingless gene and the activation of a receptor tyrosine kinase signaling pathway.