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Developmental Expression of "Germline"- And "Sex Determination"-Related Genes in the Ctenophore Mnemiopsis Leidyi

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Developmental Expression of "Germline"- And "Sex Determination"-Related Genes in the Ctenophore Mnemiopsis Leidyi

Adam M Reitzel et al. Evodevo.

Abstract

Background: An essential developmental pathway in sexually reproducing animals is the specification of germ cells and the differentiation of mature gametes, sperm and oocytes. The "germline" genes vasa, nanos and piwi are commonly identified in primordial germ cells, suggesting a molecular signature for the germline throughout animals. However, these genes are also expressed in a diverse set of somatic stem cells throughout the animal kingdom leaving open significant questions for whether they are required for germline specification. Similarly, members of the Dmrt gene family are essential components regulating sex determination and differentiation in bilaterian animals, but the functions of these transcription factors, including potential roles in sex determination, in early diverging animals remain unknown. The phylogenetic position of ctenophores and the genome sequence of the lobate Mnemiopsis leidyi motivated us to determine the compliment of these gene families in this species and determine expression patterns during development.

Results: Our phylogenetic analyses of the vasa, piwi and nanos gene families show that Mnemiopsis has multiple genes in each family with multiple lineage-specific paralogs. Expression domains of Mnemiopsis nanos, vasa and piwi, during embryogenesis from fertilization to the cydippid stage, were diverse, with little overlapping expression and no or little expression in what we think are the germ cells or gametogenic regions. piwi paralogs in Mnemiopsis had distinct expression domains in the ectoderm during development. We observed overlapping expression domains in the apical organ and tentacle apparatus of the cydippid for a subset of "germline genes," which are areas of high cell proliferation, suggesting that these genes are involved with "stem cell" specification and maintenance. Similarly, the five Dmrt genes show diverse non-overlapping expression domains, with no clear evidence for expression in future gametogenic regions of the adult. We also report on splice variants for two Mnemiopsis Dmrt genes that impact the presence and composition of the DM DNA binding domain for these transcription factors.

Conclusion: Our results are consistent with a potential role for vasa, piwi and nanos genes in the specification or maintenance of somatic stem cell populations during development in Mnemiopsis. These results are similar to previous results in the tentaculate ctenophore Pleurobrachia, with the exception that these genes were also expressed in gonads and developing gametes of adult Pleurobrachia. These differences suggest that the Mnemiopsis germline is either specified later in development than hypothesized, the germline undergoes extensive migration, or the germline does not express these classic molecular markers. Our results highlight the utility of comparing expression of orthologous genes across multiple species. We provide the first description of Dmrt expression in a ctenophore, which indicates that Dmrt genes are expressed in distinct structures and regions during development but not in future gametogenic regions, the only sex-specific structure for this hermaphroditic species.

Keywords: Cell proliferation; Dmrt; Germline; Mnemiopsisleidyi; Stem cell.

Figures

Fig. 1
Fig. 1
Splice variants for MlDmrtA and MlDmrtB. (Top) MlDmrtA is a three-exon gene with the start site located in the first exon, the DM DNA binding domain spanning exons 1 and 2 and the stop codon located in the third exon. The DM domain is indicated in the alignment with a box and the double zinc binding modules indicated by circles (Zn-binding module 1) and squares (Zn-binding module 2) below the alignment [regions defined as in 81]. We recovered a splice variant sharing the same start and stop positions but lacking exon 2 and thus the complete DM domain. The splice variant lacking exon 2 also had a shorter 3′ UTR indicated by a different polyadenylation site. (Bottom) MlDmrtB is a ten-exon gene locus. However, both our RACE sequencing and the assembled gene models (Trinity and Cufflinks) available at the Mnemiopsis genome database indicated two nine-exon genes, none with all ten exons. Both isoforms shared the same start and stop codons. IsoformA lacks exon 3, and IsoformB lacks exon 4. Contrary to the splice variant for MlDmrtA, both splice variants of MlDmrtB appear to have complete, but different, DM domains (indicated by white and black symbols), with a portion of each Zn-finger domain located in exon 2 and shared by both splice variants. The DM domain for IsoformB is indicated with a box. Like MlDmrtA, the two splice variants have different polyadenylation sites
Fig. 2
Fig. 2
Expression of nanos, vasa and piwi homologs during Mnemiopsis development. All views are lateral, unless otherwise specified, with the asterisk marking the position of the blastopore or mouth. (Top row) MlNanos1 was expressed uniformly in the egg and through cleavage stages and blastulae (0–2 hpf). From gastrulation (4 hpf) onward, expression was confined to the developing comb plates and pharynx. In later stages (10–12 hpf), there was also additional expression in the tentacle bulbs. (Middle row) MlVasa1 was not detected until after gastrulation, at 7–9 hpf. It was expressed in the blastopore and eventually the pharynx. MlVasa1 was also expressed in the ectoderm along the sagittal axis. Additionally, it was expressed in mesodermal derivatives. In later stages (10–12 hpf), expression was detected in the pharynx, tentacle bulbs and in the ectoderm between the comb rows. (Bottom row) MlPiwi1 is expressed uniformly in the egg. At gastrulation, it was downregulated except for the invaginating pharynx, mesodermal derivatives and four other groups of cells. In later stages, it remained expressed in the pharynx, tentacle bulb and the muscle that connects the two tentacle bulbs
Fig. 3
Fig. 3
Expression of four vasa paralogs in Mnemiopsis embryos at 10–12 hpf. Views for each gene are shown in aboral (left column) and lateral (right column) orientation. MlVasa1 was expressed in the ectoderm between the comb plates and the sagittal axis as well as in the tentacle bubs and pharynx. MlVasa3 was expressed in the developing comb plates. MlVasa4 also showed expression in the ectoderm between the comb rows and the tentacle bulbs. MlVasa5 expression was detected aboral ectoderm in a region corresponding to the apical organ floor and the polar fields. Expression for MlVasa2 was not determined
Fig. 4
Fig. 4
Expression of nanos, vasa and piwi genes in cydippid stages, approximately 18–24 hpf. (Top row) MlNanos1 remained expressed in the comb rows (cr), as well as in two parts of the tentacle bulb, an interior portion (black arrowheads), as well as a region surrounding the exterior (black arrows). (Middle row) MlVasa1 was expressed in cells adjacent to the comb rows (cr), different from that of MlNanos1. There was also expression detected in the interior part of the tentacle bulb (black arrowhead) similar to that of MlNanos1 and an additional domain in the aboral part of the tentacle bulb (white arrowhead). (Bottom row) MlPiwi1 was expressed in similar tentacular domains as MlVasa1 (white arrowhead), as well as in the pharynx, and muscle connecting the tentacle bulbs (black arrowhead)
Fig. 5
Fig. 5
Confocal projections of EdU labeling of Mnemiopsis cydippids (18–24 hpf) to assay for cell proliferation. ad Confocal projection of aboral portion of cydippid stage labeled with anti-tyrosine tubulin in red (a), which stains the nervous system as well as the cilia of the ciliated groove (cg) and comb rows (cr), Hoechst-stained nuclei in blue (b), EdU-labeled nuclei in green (c) and the merged (d). The apical organ (ao) was at the center, along with the two tentacle bulbs (tent) adjacent. White arrows show EdU labeling in the apical organ floor, and white arrowheads show labeling in the tentacle bulb apparati. eh Same embryo as in ad zoomed in close to the tentacle bulb
Fig. 6
Fig. 6
Expression of all 5 Dmrt genes during development of Mnemiopsis. (1st row) MlDmrtA was broadly expressed in the ectoderm outside of the developing comb rows, mouth and aboral organ. (2nd row) MlDmrtB was specifically expressed in the four developing comb rows. (3rd row) MlDmrtC was initially detected at the base of the tentacle bulbs with later expression in diffuse cells lining the pharynx in the cydippid larvae. (4th row) MlDmrtD was expressed in the oral and aboral regions of the gastrulating embryo, where expression remained in the cydippid stage. Expression was also observed in the tentacle buds, apical organ and an ectodermal band extending from the mouth. (5th row) MlDmrtE expression was initially observed in four regions of the gastrulating embryo, which continued into the cydippid stage, and later in the mouth
Fig. 7
Fig. 7
Double in situ of MlDmrtA and MlDmrtD in embryos 10–12 hpf showing adjacent expression in the oral ectoderm for these two Dmrt genes

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