Hox genes limit germ cell formation in the short germ insectGryllus bimaculatus

Abstract

20Hox genes are conserved transcription factor-encoding genes that specify the identity of body regions in 21 bilaterally symmetrical animals. In the cricket Gryllus bimaculatus, a member of the hemimetabolous 22 insect group Orthoptera, the induction of a subset of mesodermal cells to form the primordial germ cells 23 (PGCs) is restricted to the second through the fourth abdominal segments (A2-A4). In numerous insect 24 species, the Hox genes Sex-combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and 25 abdominal-A (abd-A) jointly regulate the identities of middle and posterior body segments, suggesting that 26 these genes may restrict PGC formation to specific abdominal segments in G. bimaculatus. Here we 27show that all of these Hox genes, either individually or in segment-specific combinations, restrict PGC 28 formation. Our data provides evidence for a segmental Hox code used to regulate the placement of PGC 29 formation, reminiscent of the segmental Hox codes used in other arthropod groups to establish other 30 aspects of segmental identity. These data also provide, to our knowledge, the first evidence for this 31 ancient group of genes in determining PGC placement within the context of axial patterning in any animal 32 studied thus far. specifying the body regions of bilaterally symmetrical animals during development (reviewed in 1). In 36 arthropods, Hox genes act to specify the distinct identities of different body segments (reviewed in 2), with 37 mutations in Hox genes usually resulting in switches of segmental type called homeotic transformations 38 (reviewed in 3). We previously showed that in the cricket Gryllus bimaculatus, which belongs to the 39 hemimetabolous insect order Orthoptera, the primordial germ cells (PGCs) form from the mesoderm of 40 the second to the fourth abdominal segments (A2-A4) (4) via a bone morphogenetic protein (BMP)-41 dependent mechanism (5). Given that BMP activity is not limited to the segments where PGCs form, but 42 rather, is present in the dorsal regions of all body segments (5), these data suggested that some 43 unidentified factor or factors must ensure that PGCs form specifically in A2-A4. As Hox genes play a 44 conserved role in establishing segmental identity, here we test the hypothesis that Hox genes contribute 45 to regulating the placement of the PGCs in A2-A4. 46Data implicating Hox genes in embryonic germ line placement in other animal taxa are scarce. In 48 transcription factor Blimp-1 (5-7). Mouse embryonic cells that take on PGC fate repress the Hox genes 49 Hoxa1 and Hoxb1 via activity of the BMP-activated transcription factor Blimp-1 (7-9). This has been 50 interpreted as reflecting the loss of somatic differentiation programs that is associated with adopting PGC 51 fate. Repression of Hox genes during differentiation of human induced pluripotent stem cells (hIPS) into in 52 vitro derived PGCs (iPGCs) (10) is consistent with the hypothesis that Hox gene expression and PGC fate 53 are mutually exclusive. In a system where germ cells are spec...

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricketGryllus bimaculatus

“…4.7% of G0 embryos showed mosaic GFP expression in the T3 legs (Table 3). In G1 stage X embryos, the GFP expression pattern was identical to that of the previously reported expression pattern of Gb-Ubx (Barnett et al, 2019;Matsuoka et al, 2015;Zhang et al, 2005). (C) GFP expression in heterozygous and homozygous G2 mutants.…”

“…Four G0 adults were individually backcrossed with wild type counterparts of the opposite sex to obtain multiple G1 crickets. We obtained one stable transgenic line, in which GFP expression in G2 embryos was similar to the previously documented expression pattern of Gb-abd-A transcript (Barnett et al, 2019;Matsuoka et al, 2015;Zhang et al, 2005) (compare Fig. 4B with Supplementary Fig.…”

“…CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made previously reported to cross-react in multiple Gryllus species including G. bimaculatus (Barnett et al, 2019;Mahfooz et al, 2004). In wild type embryos, the UbdA antibody revealed the expected combination of the Gb-Ubx and Gb-Abd-A expression patterns (Barnett et al, 2019;Mahfooz et al, 2004) (Fig.…”

“…It is made perform functional analyses of the G. bimaculatus ortholog of the Hox gene Ultrabithorax (Gb-Ubx) (Table 1). RNAi-induced phenotypes for Gb-Ubx have been previously examined in developing abdominal segments (Barnett et al, 2019;Matsuoka et al, 2015), providing a basis for comparison with CRISPR-induced mutants. We designed sgRNA for a sequence within an exon upstream of the homeodomain (Fig.…”

“…CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A (Barnett et al, 2019;Matsuoka et al, 2015;Zhang et al, 2005). (C) Assessment of knock-in event by using PCR and Sanger sequencing.…”