A collagenous extracellular matrix regulates germline gene expression in the sea star embryo
The sea star Patiria miniata uses cell-cell signaling mechanisms in the inductive process of germline formation and here we test if these cells require the extracellular matrix (ECM) in this process. The ECM is a network of proteins and long-chain sugar polymers necessary for structural support, cell signaling, and migration. Collagen is a fundamental component of the ECM network that provides tensile strength and structural integrity, in addition to interacting with other proteins such as laminin and integrins.  Here we used b-aminopropionitrile (BAPN), a well-known inhibitor of lysyl oxidase (LOX), an enzyme important for cross-linking mature collagen, a morpholino antisense oligonucleotide to LOX2A, and Col003, an inhibitor of collagen processing to test for a role of the ECM with cells that form the germ line. A DEG-RNA-seq approach was used to broadly test for genes responsive to the ECM at different times in development. A qPCR-based analysis of these resultant candidates show that some ECM responsive genes markedly increase gene expression, including eIF5A1, LvN1.2, ATF3, and the newly described BAPN-Associated Target with Malformed Archenteron (BATMAN), identified here. Furthermore, genes involved in germline formation in this embryo differentially respond to the ECM, including Nanos3, Vasa, PIWI, and the signaling factors BMP2/4, Nodal, and Wnt8. Our results show that disruption of the ECM dysregulates germline gene expression and normal gut morphogenesis during specific development stages. This paradigm adds to a growing list of mechanisms in how primordial germ cells use the ECM in an inductive mode Overall design: Sea star embryos were treated with BAPN at different time points and at different concentrations. Total RNA was extracted using at least 200 embryos for each sample and sent to Novogene for next-generation bulk RNA sequencing following the manufacturer's instructions. Libraries were sequenced by Novogene in a paired-end stranded fashion.