Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

Abstract

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.

Revisiting the role of the Gcm transcription factor, from master regulator to Swiss army knife

“…In the immune system, Gcm is expressed at early embryonic stages and induces the expression of proteins involved in the defense against pathogen that will not be necessary before the larval stage. 30 Thus, the DamID data strongly suggest that the Gcm fate determinant directly contributes to late aspects of differentiation/function by inducing the expression of effector molecules, the expression of whose could be maintained/sustained by molecules such as the Repo transcription factor in glia. A first example is provided by Frazzled (Fra), the chemoattractant receptor for the Netrin ligand.…”

“…[28][29][30] This technique consists in building a fusion protein between Gcm and the Dam, expressing the fusion protein in the whole embryo and deducing the binding sites of Gcm from the adenine methylation profile of the genome. This approach allowed a comprehensive identification of the Gcm direct targets, a strong advantage over microarray assays that likely favor the identification of indirect targets.…”

“…For instance, Drosophila neuroblasts produce glia and neurons and Gcm induces the expression of the transcription factor Tramtrack (Ttk), which represses the neuronal fate, 51 as well as the expression of genes repressing neuroblast proliferation (mira, lola, pros and brat). 30 In the developing immune system, prohemocytes produce crystal cells and plasmatocytes and Gcm promotes the expression of the zinc finger transcription factor called U-shaped (Ush), which inhibits crystal cell formation. 30,52 Gcm also induces the transcription of genes that control the final differentiation of the cells.…”

“…30 In the developing immune system, prohemocytes produce crystal cells and plasmatocytes and Gcm promotes the expression of the zinc finger transcription factor called U-shaped (Ush), which inhibits crystal cell formation. 30,52 Gcm also induces the transcription of genes that control the final differentiation of the cells. The most described target of Gcm is Repo, a homeodomain transcription factor that remains expressed in glia until adulthood.…”

“…A) Tissues associated GO terms issued from the enrichment analysis carried out on the list of genes identified as Gcm direct target by the DamID screen, using DAVID. 30,69,70 The list displayed was arbitrarily limited to 3 GO terms per tissue, each GO term being enriched more than 2 folds with a P-value below 0.05. Dark gray indicates tissues/biological processes already known to require Gcm, intermediate intensity gray indicates a tissue analyzed in this report, pale gray indicates tissues/biological processes still not shown to require Gcm.…”