Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling

ABSTRACT: Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia-Absent Radius (TAR) Syndrome is a rare congenital disease presenting with reduced blood platelets (megakaryocytic thrombocytopenia) and forelimb anomalies, concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay. How perturbing a general mRNA-processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here, we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity (PCP) signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function, we find a significant reduction of cd41-positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a-mutant zebrafish embryos already after gastrulation accumulate mRNAs with erroneously included introns, a hallmark of defective nonsense-mediated decay. Affected mRNAs include transcripts encoding components of the non-canonical Wnt pathway involved in PCP signaling, and rbm8a mutant-embryos show hallmarks of Wnt/PCP-dependent, early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b, wnt11f2, fzd7a, and vangl2. Following axis formation, rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm (LPM) that forms the hematopoietic, cardiovascular, kidney, and forelimb skeleton progenitors. Subsequently, rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a, kdrl, sox7, and the megakaryocyte regulator gfi1aa. Lastly, we document similar hematopoietic defects upon loss of vangl2. Together, our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Danio rerio

SUBMITTER: Mark Robinson

PROVIDER: E-MTAB-12503 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required for better treatment options for a variety of chronic intestinal diseases, however, current models of ISC lineage hierarchy and segregation are still under debate. Here we report the identification of Lgr5+ ISCs that express Flattop (Fltp), a Wnt/planar cell polarity (PCP) reporter and effector gene. Functional analysis and lineage tracing revealed that Wnt/PCP-activated Fltp+ ISCs are primed towards either the enteroendocrine or Paneth cell lineage in vivo, while retaining self-renewal and multi-lineage capacity in vitro. Surprisingly, canonical Wnt/beta-catenin- and non-canonical Wnt/PCP-activated Lgr5+ ISCs are indistinguishable by the expression of stem-cell signature or secretory lineage-specifying genes, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch of canonical to non-canonical Wnt signalling. Pseudotemporal ordering of targeted single-cell gene expression data allowed us to delineate the ISC differentiation path into enteroendocrine and Paneth cells. Strikingly, both lineages are directly recruited from ISCs via unipotent transition states, excluding the existence of formerly predicted bi- or multipotent secretory progenitors. Transitory cells that mature into Paneth cells are defined by label-retention and co-expression of stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells (LRCs). Taken together, we identified the Wnt/PCP pathway as a new niche signal and polarity cue regulating stem cell fate. Active Wnt/PCP signalling represents one of the earliest events in ISC lineage priming towards the Paneth and enteroendocrine cell fate, preceding lateral inhibition and expression of secretory lineage-specifying genes. Thus, our findings provide a better understanding of the niche signals and redefine the mechanisms underlying ISC lineage hierarchy and segregation. Here we establish the Wnt/Planar cell polarity (PCP) gene Flattop (Fltp) as a unique marker for intestinal LRCs and their distinct secretory fate. We show that Fltp+ cells are characterized by a combined stem cell and secretory gene signature. A subset of Fltp+ cells is classified by label-retention and predominantly locates at position +4, indicative of quiescent stem cells. Strikingly, Fltp+ LRCs are specified by Wnt/PCP signaling in contrast to actively cycling stem cells that rely on the canonical Wnt/β-catenin pathway. In mice with disturbed Wnt/PCP signaling, the differentiation of enteroendocrine cells from LRCs is impaired. These findings establish Fltp as a novel marker for intestinal LRCs and implicate Wnt/PCP signaling in cell-cycle exit and commitment of ISCs to the secretory lineage. Taken together, we not only provide a marker to study LRCs in homeostatic and diseased conditions, but also identify the Wnt/PCP signaling pathway as a therapeutic target for colorectal cancer and metabolic disease.

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RNA-seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling

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