Krüppel-like factor gene function in the ctenophore Mnemiopsis suggests an ancient role in promoting cell proliferation in metazoan stem cell niches

All animals possess pluripotent stem cells that during an organism9s lifespan give rise to differentiated cell types and are capable of self-renewal. To date studies assessing the function of explicit genes driving the regulation of stem cell renewal and pluripotency have been characterized in bilaterians and a small number of cnidarians. Transcription factor genes critical for regulation of stem cell self-renewal and pluripotency include Oct4, Sox2, Klf4, and c-myc (OSKM) 1-3 . In particular, the role of Klfs in stem cell renewal and pluripotency has been thoroughly investigated 1,2,4-9 . In mouse embryonic stem cells the three Kruppel-like factor ( Klf ) genes Klf2, Klf4, and Klf5 participate in an auto-regulatory activation loop and maintain a transcriptional circuit with Oct4, Sox2, c-myc, and Nanog regulating stem cell self-renewal 4 . While OSKM gene homologs have been found in the genomes of many metazoans 3,10,11 , their function is unknown in non-bilaterians. Ctenophora, an ancient non-bilaterian lineage 12-14 , possess putative stem cells 11,15,16 and have prodigious regenerative capacity 17,18 , making them informative models for investigating the evolution and genetic underpinnings of stem cell maintenance in Metazoa. Ctenophores possess homologs of the OSKM genes 11,19-21 , and while the expression patterns of ctenophore Sox paralogs are associated with high rates of cell proliferation in putative stem cell niches 11,19 , little is known about Pou, Klf, or c-myc expression or function. Here we examine the expression of Klf homologs in the ctenophore Mnemiopsis leidyi and disrupt zygotic Klf gene function during embryogenesis using both morpholino oligonucleotides and CRISPR-Cas9. We find that zygotic MleKlf5a and MleKlf5b play a role in patterning the gastrovascular cavity, pharynx, apical organ, and tentacle bulbs in M. leidyi . Simultaneous zygotic knockdown of both MleKlf5a and MleKlf5b results in the diminution or loss of cell proliferation in endodermal stem cell niches and aberrant endodermal patterning. Our results highlight an ancestral functional role for Klf transcription factors in the regulation of rapid cell proliferation in metazoan stem cell niches.