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[Preprint]. 2024 Jan 12:2024.01.12.575210.
doi: 10.1101/2024.01.12.575210.

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans

Karin Kiontke  1 , R Antonio Herrera  2 , D Adam Mason  3 , Alyssa Woronik  4 , Stephanie Vernooy  3 , Yash Patel  1 , David H A Fitch  1
Affiliations

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans

Karin Kiontke et al. bioRxiv. .

Update in

Abstract

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the transcription factor DMD-3. To find genes regulated by DMD-3, We performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males vs. dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the UPR (unfolded protein response) pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are coregulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

Keywords: DMD-3; GRN; chondroitin proteoglycan; differential expression; gene network.

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Figures

Figure 1
Figure 1
Tail tips in C. elegans. The larvae of both sexes have a long, pointed tail consisting of 4 epithelial cells, hyp8–11. Hermaphrodites retain this shape as adults. In males, the morphogenetic process TTM creates a short and round tail. Adherens junctions between the tail tip cells disassemble. One cell of each phasmid socket transdifferentiates into the male-specific PHD neuron.
Figure 2
Figure 2
(A) Principal component analysis using all samples, (left) and only samples for WT males (wtM) WT hermaphrodites (wtH) and dmd-3(−) males (lfM) excluding dmd-3(gf) hermaphrodites (gfH). (B) log2 fold changes for each gene in the comparisons of WT males vs. WT hermaphrodites plotted against the log2 fold change in the comparison of WT males with dmd-3(−) males. (C) Venn diagram showing the overlap of DE genes found in the comparison of WT males with hermaphrodites and WT males with dmd-3(−) males.
Figure 3
Figure 3
Tail tip expression of transcriptional reporters for DE genes activated by DMD-3 and for dmd-6, which is repressed by DMD-3.
Figure 4
Figure 4
Expression of three collagens that are activated by DMD-3. (A, B) GFP::BLI-1 expression in an L4.4 male (A) and L4.5 hermaphrodite (B). In males, BLI-1 is expressed in the tail tip cells and in hyp13 and weakly in puncta at the apical surface of hypodermal cells, where it is also observed in hermaphrodites. (C, D) GFP::COL-20 begins in late L4 in the seam and hyp7 in both sexes but not in the hermaphrodite tail tip. In young adult males (C, C*), COL-20 is expressed in the ventral tail hyp and the rays. (E, F) GFP::COL-89 is expressed in the proctodeum of both sexes and occasionally in R9.p in males.
Figure 5
Figure 5
The chondroitin synthesis pathway plays a role in TTM. (A) Expression of transcriptional reporters sqv-4, sqv-5 and sqv-7 during L4 in males and hermaphrodites. (B) Schematic of the pathway (modified after Hwang et al. 2003) with genes found to be involved in TTM in red. (C) Examples of the mutant or RNAi tail tip phenotype for 4 sqv-genes and mig-22. (D) Examples for the protein expression pattern in the L4 male tail tip of 5 components of the sqv-pathway and the putative core protein FBN-1.
Figure 6
Figure 6
Network models for TTM. (A) DMD-3 controls only few targets, all of them GEFs, GAPs, kinases and phosphatases (effectors). (B) Between DMD-3 and the effectors or conditional modules are chains of transcription factors. (C) Composite model with a combination of transcription factors and effectors controlling molecular machines and conditional modules.
Figure 7
Figure 7
Hypothetical role of chondroitin proteoglycans and components of the HTSR in TTTM. The tail tip secretes CPGs into the extracellular space between tail tip tissue and cuticle (pink). This hygroscopic matrix attracts water, which passes through aquaporin-8 water channels that are located in the membrane of the tail tip or in vesicles similar to those constituting the canaliculi of the excretory duct. The thus created pressure aids/stabilizes tail tip cell shortening and retraction. The osmolarity in the tail tip tissue is balanced by glycerol production through upregulation of gpdh-1 activity via the kinases DRL-1 and WNK-1.
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