Neuropeptides are evolutionarily ancient peptide hormones of the nervous and neuroendocrine systems, and are thought to have regulated metamorphosis in early animal ancestors. In particular, the deeply conserved Wamide family of neuropeptides—shared across Bilateria (e.g. insects and worms) and its sister group Cnidaria (e.g. jellyfishes and corals)—has been implicated in mediating life-cycle transitions, yet their endogenous roles remain poorly understood. By using CRISPR-Cas9-mediated reverse genetics, we show that cnidarian Wamide—referred to as GLWamide—regulates the timing of life cycle transition in the sea anemone cnidarian Nematostella vectensis. We find that mutant planula larvae lacking GLWamides transform into morphologically normal polyps at a rate slower than that of the wildtype control larvae. Treatment of GLWamide null mutant larvae with synthetic GLWamide peptides is sufficient to restore a normal rate of metamorphosis. These results demonstrate that GLWamide plays a dispensable, modulatory role in accelerating metamorphosis in a sea anemone.
All data generated or analyzed during this study are included in the manuscript and supporting files. A source data file has been provided for Figures 1, 2 and 5.
- Nagayasu Nakanishi
- Mark Q Martindale
- Nagayasu Nakanishi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Alejandro Sánchez Alvarado, Stowers Institute for Medical Research, United States
© 2018, Nakanishi & Martindale
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies.
Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function and the transcriptome profiles will provide a valuable resource for future studies.