Germ cell specification and differentiation in planarians

Morgridge Institute For Research, Inc., Madison WI

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Abstract

Project Summary/Abstract Germ cells give rise to gametes–oocytes and sperm–that fuse to create a totipotent zygote, which divides and generates all the cells of an organism. During development, a new germ cell lineage must be established and differentiate properly to ensure propagation of the species. Defects in germ cell development can lead to infertility or germ cell tumors; furthermore, many cancers are associated with mis-expression of germ cell genes. Much knowledge of germ cell development comes from model organisms in which maternal determinants specify germ cells early in embryogenesis. By contrast, many animals (including mammals) specify their germ cells in response to inductive cues from surrounding cells later in development. Despite these differences, proper development of all germ cells relies on extrinsic factors provided by somatic support cells, which act as a niche. The free-living planarian flatworm, Schmidtea mediterranea, is well known for its extraordinary regenerative prowess based upon a population of pluripotent somatic stem cells. Strikingly, planarians can regenerate new germ cells de novo from their somatic stem cells. Recent work has identified b- alanyl-tryptamine (BATT) as a critical, niche-derived signal that triggers planarian germ cell development. This dipeptide is the product of a nonribosomal peptide synthetase that conjugates b-alanine to tryptamine (produced by decarboxylating tryptophan). Tryptophan, an essential amino acid, is taken up by the intestine; b- alanine synthetic enzymes are also expressed in the gut. However, the nonribosomal peptide synthetase that produces BATT is expressed in somatic gonadal cells. Thus, BATT may serve to integrate nutritional status and reproductive maturation. To test this hypothesis, the following two aims are proposed: (i) to characterize BATT production in somatic gonadal cells; and (ii) to identify the BATT receptor(s). In Aim 1, mass spectrometry-based metabolic profiling will measure changes in b-alanine and tryptophan metabolism during reproductive system development. Spatial transcriptomics will map the expression patterns of candidate genes involved in the synthesis, metabolism, or transport of b-alanine and tryptophan. Genes required for the production or transport of BATT will be characterized by RNA interference (RNAi) combined with mass spectrometry and chemical rescue experiments. In Aim 2, candidate G protein-coupled receptors (GPCRs) expressed in somatic gonadal cells and germline stem cells will be identified by a combination of single-cell RNA sequencing and spatial transcriptomics. Potential BATT receptors will be identified by RNAi screening for knockdowns that phenocopy loss of BATT. Candidate BATT receptor(s) will be validated by cell-based GPCR assays. The proposed experiments capitalize upon the planarian’s inductive germ cell specification and the functional genomic tools available for studying these animals to characterize a new signaling pathway. In addition, this work could provide new approaches for disrupting reproduction in parasitic flatworms, whose prolific reproductive output enables them to propagate and afflict hundreds of millions of people worldwide.