Abstract
Abstract All animals possess pluripotent stem cells that during an organism’s 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 O ct4 , S ox2 , K lf4 , and c- m yc (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 Krüppel-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 , and 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. Significance All animals possess pluripotent stem cells that are capable of self-renewal and give rise to differentiated cell types. However studies assessing the function of genes regulating stem cells have been characterized only in bilaterians and cnidarians. Mnemiopsis leidyi , a member of the early diverging non-bilaterian phylum Ctenophora, has emerged as an important model system for understanding early animal evolution. M. leidyi possess putative stem cell niches in which the expression of ‘stemness’ genes have been described. Here we characterize the zygotic function of Krüppel-like factor ( Klf) genes during M. leidyi embryogenesis. Our results highlight an ancestral role for Klf genes in regulating cell proliferation in metazoan endodermal stem cell niches, suggesting very deep conservation of the core transcriptional circuitry critical for stem cell maintenance among extant metazoans.