Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum

Abstract

In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a smallscale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

Hexadecameric structure of an invertebrate gap junction channel

“…This led to some extrapolation in the literature that innexin channels may also contain hexameric hemichannels and dodecameric junction channels like connexin [29][30][31][32]. On the other hand, it has also been reported that invertebrate gap junction channels are larger than vertebrate junction channels [12][13][14][15]20].…”

Transcriptomic and functional analysis of the oosome, a unique form of germ plasm in the waspNasonia vitripennis

“…Nv-coronin ’s functional annotation is consistent with a role in cellularization, as it is predicted to interact with both the microtubule and actin cytoskeletons, both of which are crucial for mitosis and cell formation. While Innexin1 is most well known as a crucial component of gap junctions and a role in the nervous system [57], a role for at least one Innexin in cellurization has been demonstrated in the beetle Tribolium [58], suggesting that the full potential of these proteins in regulating cellular processes has not been fully explored.…”

Fog signaling has diverse roles in epithelial morphogenesis in insects

“…The different requirement for Fog signaling during cellularisation in Drosophila and three insects we have studied may be linked to variations in the modes of cellularisation. The formation of a high-columnar blastoderm, which has been well studied in Drosophila, is an exception among insects and even among flies (Bullock et al, 2004;van der Zee et al, 2015). In many insects, cellularisation generates a blastoderm of cuboidal cells, while some insects initially form individual cells that then migrate to form a continuous epithelium (Ho et al, 1997;Nakamura et al, 2010).…”

“…However, the way in which this may occur at a mechanistic level is a completely open question. The discovery of special Innexin-based cell junctions that are essential for cellularisation in Tribolium, but do not exist in Drosophila, highlights the potential diversity in mechanisms underlying cellularisation in different insects (van der Zee et al, 2015). Thus, as with the spreading of the serosa, future work is required to uncover the mechanism of Fog signaling during blastoderm formation in Tribolium and other insects.…”

“…To test whether Fog signaling does have a more widely conserved role in early development, we have analyzed Fog pathway components in the beetle Tribolium castaneum. In contrast to Drosophila and other dipteran species like Chironomus, many features of Tribolium embryogenesis are more typical of insects in general, including the mechanism and timing of blastoderm cellularisation (van der Zee et al, 2015), the mode of germ cell formation (Schroder, 2006), germband formation (Benton, 2018), extraembryonic tissue development (Hilbrant et al, 2016;Horn and Panfilio, 2016;van der Zee et al, 2005) and segmentation (Clark and Peel, 2018;Sommer and Tautz, 1993). Therefore, analyzing Fog signaling in Tribolium will reveal the role of the pathway within a developmental context that is more representative of insects.…”