Unknown,Transcriptomics,Genomics,Proteomics

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Differential Wnt and Hh signaling in head and growth zone of a short germ embryo

ABSTRACT: Purpose: Combination of systemic RNAi with RNAseq to identify target genes of important pathways in early development Drosophila early patterning occurs in the syncytial blastoderm where transcription factors diffuse between cells. However, in typical insect embryos, patterning occurs in a cellularized environment where signaling pathways are likely to play a more fundamental role. We use the short germ beetle Tribolium castaneum to investigate two putative Wnt and Hh signaling centers located in the anlagen of head and growth zone. These structures are known to develop in a different way in short germ insects. We find that Hh acts upstream of Wnt in the head, which is different from the Drosophila situation. In the growth zone Wnt signaling acts upstream. For the first time outside Drosophila, we comprehensively determine the Wnt and Hh target gene sets and distinguish the anterior from the posterior gene sets by genetically depleting head or growth zone. Surprisingly, there are significantly more targets in the growth zone than in the head for both pathways and we find that their growth zone gene sets are essentially non-overlapping. Furthermore, several pair rule genes, Tc-caudal, Tc-twist and hindgut patterning genes are regulated by Wnt signaling. In addition, Wnt controls growth zone metabolism and cell division. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. Unexpectedly, we find the Wnt target Tc-senseless to be required for hindgut development. 10-11 h old whole embyro mRNA profiles of the following treatments: reference: wild type 100bp single read triplicates reference: wild type 50bp single read triplicates treatment: Tc-arrow RNAi knockdown 50 bp single read triplicates treatment: Tc-frizzled1/2 RNAi double knockdown 50 bp single read triplicates treatment: Tc-hedgehog RNAi knockdown 100bp single reads quadruplicates treatment: Tc-orthodenticle RNAi knockdown 100bp single reads triplicates treatment: Tc-torso RNAi knockdown 100bp single reads triplicates treatment: Tc-wntless RNAi knockdown 50bp single reads triplicates Total: 2 references, 6 treatments, 25 sequencing runs

ORGANISM(S): Tribolium castaneum

SUBMITTER: Georg Oberhofer

PROVIDER: E-GEOD-54706 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Stomach and intestinal epithelial cells are maintained by the activity of stem cells located in the isthmus and crypt, respectively1,2. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in stomach and intestinal development and stem cells3,4. Although accumulating evidence suggests that intestinal stromal cells secrete Wnt ligands to promote stem cell renewal5-10, the source of stomach Wnt ligands is still unclear. Moreover, how these gastrointestinal stem cell niche signals are produced is currently unknown. By performing single cell analysis of gastrointestinal stromal cells, we identified cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to gastrointestinal epithelial cells, these cells highly expressed pericyte markers and Wnt ligands. They also were enriched for Hh signaling, which plays a key role in gut development11,12. A recent study has shown that intestinal pericryptal cells co-express Hh target and Wnt ligand genes8. To define their relationship, we analyzed mice with Hh gain of function in the pericyte-like stromal cells conserved between the stomach and intestine, and found increased levels of Wnt ligands, supporting Hh regulation of stromal Wnt ligand expression. Moreover, utilizing Sufu and Spop double knockout mice, which stabilized GLI2, a key Hh mediator in the gut, we were able to map GLI2 binding sites genome-wide and analyze super enhancers. This work demonstrates GLI2 activation of stromal Wnt ligands through enhancers that are conserved between the stomach and intestine. To determine the significance of Wnt secreting gastrointestinal stromal cells, we genetically inhibited Wnt secretion from the perictye-like or broad stromal cells, demonstrating their roles in gastrointestinal regeneration and development, respectively. Our work not only identifies the conserved gastrointestinal stromal niche cell populations but also reveals their underlying signaling and epigenetic mechanisms.

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Differential Wnt and Hh signaling in head and growth zone of a short germ embryo

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