A large reverse-genetic screen identifies numerous regulators of testis nascent myotube collective cell migration and collective organ sculpting

Abstract

Collective cell migration is critical for morphogenesis, homeostasis, and wound healing. During development migrating mesenchymal cells form tissues that shape some of the body's organs. We have developed a powerful model for examining this, exploring how Drosophila testis nascent myotubes migrate onto the testis during pupal development, forming the muscles that ensheath it and also creating its characteristic spiral shape. To define genes that regulate this process, we have carried out RNAseq to define the genes expressed in myotubes during migration. Using this dataset, we curated a list of 131 ligands, receptors and cytoskeletal regulators, including all Rho-family GTPase GAPs and GEFs, as candidates. We then used the GAL4/UAS system to express 279 shRNAs targeting these genes, using the muscle specific driver dMef2>GAL4, and examined the adult testis. We identified 29 genes with diverse roles in testis morphogenesis. Some have phenotypes consistent with defects in collective cell migration, while others alter testis shape in different ways, revealing some of the underlying logic of testis morphogenesis. We followed up one of these genes in more detail-that encoding the Rho-family GEF dPix. dPix knockdown leads to a drastic reduction in migration and a substantial loss of muscle coverage. Our data suggest different isoforms of dPix play distinct roles in this process, reveal a role for its protein partner Git. We also explore whether cdc42 activity regulation or cell adhesion are among the dPix mechanisms of action. Together, our RNAseq dataset and genetic analysis will provide an important resource for the community to explore cell migration and organ morphogenesis.

Fig. 1.

Diagram illustrating the steps used to generate our RNAseq datasets.

Fig. 2.

FACs sorting allowed us to isolate pure populations of living multinucleate myotubes. A. FACS profile along the propidium iodide versus the GFP-fluorescence [Alexa488] axis. Cells with high propidium iodide signals were likely dead cells and we discarded. B. FACS profile along the [v450] versus [Alexa488] axis. The Vybrant DyeCycleViolet [v450] signal assesses the DNA content of living cells and allowed us to separate the cells with high-GFP signal into three populations differing in DNA content. C-E. Examples of cells from the three populations revealing cells with one (C), two (D), or three (E) nuclei. Red=phalloidin (F-actin). Green=GFP Blue=DAPI (DNA). Insets in the upper right show the DNA channel of one cell from each image.

Fig. 3

Identifying genes up- and down-regulated at the early and late timepoints. A. Gene ontology (GO) terms of genes up-regulated (top, highlighted in gold) and down-regulated (bottom) at th 45 hr APF timepoint. At the right are examples of up-regulated GO terms that reflect muscle development or potential neuromuscular synapse assembly. B-D. Volcano plot of genes upregulated at the 30 hr APF (left, highlighted in teal) or the 45 hr APF timepoint (right, highlighted in magenta). B. Highlighted are selected examples of genes with known roles in muscles that are upregulated at the 45 hr APF timepoint. C. Highlighted are selected examples of genes that encode mesoderm transcription factors or proteins involved in myotube fusion that are upregulated at the 30 hr APF timepoint. D. Highlighted are 22 genes that are upregulated at the 30 hr APF timepoint which we chose to include in the RNAi screen.

Fig. 4.

Genes selected for our RNAi screen. Four lists derived from the Flybase-curated “Gene Group”-lists. In each genes are ranked by level of expression (reads per kilobase per million mapped reads), and genes included in our RNAi screen are boxed in red. A. Transmembrane receptors. B. Receptor tyrosine kinases. C. Cadherin family members. D. Integrin subunits.

Fig. 5.

RNAi lines that alter muscle coverage or distal testis shape. Adult testes from wildtype or adults expressing a UAS-driven RNAi line targeting the indicated gene under control of mef-GAL4. All are stained with fluorescently-labeled phalloidin to reveal F-actin, which highlights muscles. A. Wildtype. The wildtype testis is fully covered in circumferential muscle and has a spiral shape with a very gradual reduction in diameter toward the proximal end. B-T. Testis from knockdown lines, arranged according to phenotypic class. Detailed descriptions of each are in the text. B-D. Extreme loss of muscle coverage. The inset in D shows a close-up illustrating the “straited muscle phenotype”, E-I. Strong loss of distal muscle coverage (arrows). The more proximal arrow in G shows variation in testis diameter. J-M. Variable loss of distal muscles or gaps in muscle coverage (arrows). N-P. Strong (N) or weaker gaps that are not confined to the distal end (arrows). Q-T. Distal testis is enlarged (arrows).

Fig. 6.

RNAi lines that alter testis morphology in other ways. Adult testes from wildtype or adults expressing a UAS-driven RNAi line targeting the indicated gene under control of mef-GAL4. All are stained with fluorescently-labeled phalloidin to reveal F-actin, which highlights muscles. A. Wildtype, showing normal shape. Inset reveals normal aligned muscles. B-F. Variable testis diameter leading to a wavy margin (arrows). G-H. Broadened and shortened testis. Rlip knockdown also leads to variations in diameter (arrows). I-L. regional variations in testis diameter. M. Narrowed and elongated testis. O-R. Strong defects in muscle alignment (Inset in O, arrows) and loss of spiraling. S-U. Defects in muscle alignment (arrows) coupled with other defects in testis shape.

Fig. 7.

dPix knockdown dramatically slows myotube migration, and reducing N-cadherin partially suppresses the testis defects. A,B. Adult testis illustrating the range of phenotypes seen after dPix^RNAi. C. Wildtype myotube migration. D, E. Rpresentative examples of the delay in migration after dPix^RNAi. Some myotubes that are far apart remain interconnected by long processes that stretch over myotubes located between (arrows). F. Adult testis after Ncad^RNAi. G. Adult testis after dPix^RNAi; Ncad^RNAi. H. Quantification of phenotypic severity.

Fig 8.

Different dPix isoforms play different roles in testis morphogenesis and the dPix binding partner Git also plays a role. A. Diagram of the genomic structure of the dPix gene, scale at top, 5’ end left, exons are grey (non-coding) or tan (protein coding) boxes and introns lines. Multiple dPix isoforms are illustrated. Above at the locations of three shRNAs targeting different exons and the location of mobile element insertions in two mutant alleles. Below are some features of the dPix protein isoforms. B. RNAseq data from the 31 hrAPF and 45 hr APF timepoints. Colored arrows indicate exons discussed in the text. C-O. Adult testes from wildtype, adults expressing the noted UAS-driven RNAi line targeting dPix under control of mef-GAL4, or the dPix or Git mutant alleles indicated. All are stained with fluorescently-labeled phalloidin to reveal F-actin, which highlights muscles. Phenotypes are discussed in the text.

Fig. 9.

Knockdown of the AB isoforms of dPix elevates rather than reduces the signal froma cdc42 biosensor. A,B,D,G,H. Images of the cdc42 biosensor (the CRIB domain of Mbt fused to EGFP) expressed in the myotubes on the seminal vesicle at the base of the testis, using dMef2-GAL4. A. The biosensor signal is found at the cell cortex. B. cdc42 knockdown reduces cortical biosensor signal. C. Line scans perpendicular to the membrane reveal the reduction in the cortical signal. wt: N=7, RNAi: N=8. D, E. Slightly reduced biosensor signal after Rac2 knockdown. wt: N=6, RNAi: N=8. F. Quantification of normalized membrane signal in wildtype, two different cdc42 RNAi lines and a Rac2 RNAi line. G,H. dPix knockdown elevates the cortical biosensor signal. wt: N=17, RNAi: N=30. I. Line scans perpendicular to the membrane. J. Quantification of normalized membrane signal.

Fig 10:

Cartoon illustrating the sequential events of testis morphogenesis and the steps at which we hypothesize each gene may act. A. Diagrams of the entire testis and closeups of the myotubes at each step. B. Changes in actin and N-cadherin localization at each step. C. Stages of morphogenesis and proposed steps at which different gens act.