1 2 3 Krüppel-like factor gene function in the ctenophore Mnemiopsis suggests an ancient role in 4 promoting cell proliferation in metazoan stem cell niches 5 6 Abstract 16All animals possess pluripotent stem cells that during an organism's lifespan give rise to 17 differentiated cell types and are capable of self-renewal. To date studies assessing the function of 18 explicit genes driving the regulation of stem cell renewal and pluripotency have been 19 characterized in bilaterians and a small number of cnidarians. Transcription factor genes critical 20 for regulation of stem cell self-renewal and pluripotency include Oct4, Sox2, Klf4, and c-myc 21 (OSKM) 1-3 . In particular, the role of Klfs in stem cell renewal and pluripotency has been 22 thoroughly investigated 1,2,4-9 . In mouse embryonic stem cells the three Krüppel-like factor (Klf) 23 genes Klf2, Klf4, and Klf5 participate in an auto-regulatory activation loop and maintain a 24 transcriptional circuit with Oct4, Sox2, c-myc, and Nanog regulating stem cell self-renewal 4 . 25While OSKM gene homologs have been found in the genomes of many metazoans 3,10,11 , their 26 function is unknown in non-bilaterians. Ctenophora, an ancient non-bilaterian lineage 12-14 , 27 possess putative stem cells 11,15,16 and have prodigious regenerative capacity 17,18 , making them 28 informative models for investigating the evolution and genetic underpinnings of stem cell 29 maintenance in Metazoa. Ctenophores possess homologs of the OSKM genes 11,19-21 , and while 30 the expression patterns of ctenophore Sox paralogs are associated with high rates of cell 31 proliferation in putative stem cell niches 11,19 , little is known about Pou, Klf, or c-myc expression 32 or function. Here we examine the expression of Klf homologs in the ctenophore Mnemiopsis 33 leidyi and disrupt zygotic Klf gene function during embryogenesis using both morpholino 34 oligonucleotides and CRISPR-Cas9. We find that zygotic MleKlf5a and MleKlf5b play a role in 35 patterning the gastrovascular cavity, pharynx, apical organ, and tentacle bulbs in M. leidyi. 36 Simultaneous zygotic knockdown of both MleKlf5a and MleKlf5b results in the diminution or 37 loss of cell proliferation in endodermal stem cell niches and aberrant endodermal patterning. Our 38 Running Title: Ctenophore Klf gene function 3 results highlight an ancestral functional role for Klf transcription factors in the regulation of rapid 39 cell proliferation in metazoan stem cell niches. 40 Main 41Ctenophores are marine, invertebrate predators characterized by rows of giant cilia fused 42 into 'ctenes' used for locomotion, an aboral and neuronal-rich gravity-sensing (apical) organ, and 43 muscular tentacles lined with sticky colloblast cells used for prey capture [22][23][24][25][26][27] . M. leidyi embryos 44 undergo a ctenophore-specific early cleavage program 28,29 , gastrulation takes place ~3-5 hours 45 post fertilization (hpf) followed by tissue organization and organogenesis over the course of the 46 next several hours before hatching as ...