DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

ABSTRACT Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.

Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

SummaryTissue homeostasis often requires a properly placed niche to support stem cells. The morphogenetic processes that position a niche are just being described. We recently showed that Drosophila testis pro niche cells, specified at disparate positions during early gonadogenesis, must assemble in one collective at the gonad anterior. Here, we identify Slit and FGF signals emanating from adjacent visceral mesoderm (Vm) that regulate assembly. In response to signaling, niche cells express islet, which we find is also required for positioning the niche. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of the dispersed niche is severely disrupted, with pro-niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as Alk target in the Drosophila visceral mesoderm

Development of the midgut visceral muscle of Drosophila crucially depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signalling, which is needed to specify founder cells (FCs) in the circular visceral mesoderm (VM). While activation of the Alk receptor by its ligand Jelly Belly (Jeb) is well characterized, only a small number of target molecules have been identified. Here, we assayed RNA polymerase II (Pol II) occupancy in VM cells by using the targeted DamID (TaDa) approach. To identify Alk targets we employed comparative analysis of embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differential expression of a number of genes, including the Snail/Scratch family transcription factor Kahuli (Kah). Upon further in vivo validation, we confirmed that Alk signalling regulates Kah mRNA expression in the VM. We show that Kah mutants display defects in the formation of midgut constrictions, similar to that of pointed (pnt) mutants. Analysis of publicly available ChIP data defined a Kah target-binding site similar to that of Snail. In addition, we compared genes that were differentially expressed in Kah mutants with publicly available Kah- and Pnt-ChIP datasets identifying a set of common target genes putatively regulated by Kah and Pnt in midgut constriction. Taken together, we (i) report a rich dataset of Alk responsive loci in the embryonic VM, (ii) provide the first functional characterization of the Kah transcription factor, identifying a role in embryonic midgut constriction, and (iii) suggest a model in which Kah and Pnt cooperate in embryonic midgut morphogenesis.

Collective Migrations of Drosophila Embryonic Trunk and Caudal Mesoderm-Derived Muscle Precursor Cells

Mesoderm migration in the Drosophila embryo is a highly conserved, complex process that is required for the formation of specialized tissues and organs, including the somatic and visceral musculature. In this FlyBook chapter, we will compare and contrast the specification and migration of cells originating from the trunk and caudal mesoderm. Both cell types engage in collective migrations that enable cells to achieve new positions within developing embryos and form distinct tissues. To start, we will discuss specification and early morphogenetic movements of the presumptive mesoderm, then focus on the coordinate movements of the two subtypes trunk mesoderm and caudal visceral mesoderm, ending with a comparison of these processes including general insights gained through study.

Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification

ABSTRACTPleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). We demonstrate here that for the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. Our results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lameduck and is likely a member of the ETS transcription factor family. Our findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a critical mechanism for determining unique responses to Ras signaling.SUMMARYA fundamentally different mechanism is shown for how Ras signaling governs cell fate specification in the Drosophila somatic versus visceral mesoderms, providing insight into how signaling specificity is achieved.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1

Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme.

The scaffolding protein Cnk Interacts with Alk to Promote Visceral Founder Cell Specification in Drosophila

ABSTRACTIn Drosophila, the receptor tyrosine kinase Alk and its ligand Jeb are required to drive founder cell (FC) specification in the visceral mesoderm (VM). Alk-signalling activates downstream MAPK/ERK- and PI3K-pathways in human and Drosophila but little is known about immediate downstream signalling events. Here we report that the scaffolding protein Cnk interacts directly with Alk via a novel c-terminal binding motif. Cnk is required for Alk-signalling as ectopic expression of the minimal interaction motif as well as loss of maternal and zygotic cnk blocks visceral FC-formation, resembling the phenotype of jeb and Alk mutants. We also show that the Cnk-interactor Aveugle/Hyphen (Ave/HYP) is critical, while the (pseudo-) kinase Ksr is not required for Alk-signalling in the developing VM. Taken together, Cnk and Ave represent the first molecules downstream of Alk whose loss genocopies the lack of visceral FC-specification of Alk and jeb mutants indicating an essential role in Alk-signalling.