Unexpected mutual regulation underlies paralogue functional diversification and promotes epithelial tissue maturation in Tribolium

Abstract

Insect Hox3/zen genes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicated zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We dissect the paralogues’ expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression of Tc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose through multiple instances of zen subfunctionalization, leading to their complementary extant roles.

Persistent parental RNAi in the beetleTribolium castaneuminvolves maternal transmission of long double-stranded RNA

“…This corroborates the accuracy of the original, nested amplicon for quantification of Tc-zen1 transcript levels. Moreover, these findings with either amplicon are consistent with our previous work that documented a single early pulse of Tc-zen1 expression that peaks at 6-10 h before rapidly declining to undetectable levels for the rest of embryogenesis [20].…”

“…The homeodomain transcription factor Tc-Zen1 is a critical regulator in early development, specifying the identity of the extraembryonic serosal tissue that surrounds the embryo and confers mechanical, physiological, and immunological protection [18,20,26,27]. During routine verification of Tc-zen1 parental RNAi using RT-qPCR (as in [20]), we unexpectedly found that measured expression of Tc-zen1 mRNA was higher in RNAi samples than in wild type under certain assay conditions, despite strong phenotypic validation of systemic knockdown (see Methods).…”

“…However, due to the small size of the Tc-zen1 mRNA transcript, this amplicon was also nested within the region used as an established multi-purpose template for dsRNA and in situ hybridization (Fig. 1A: Fragment 2, compared to the long dsRNA, [20,25,30]). Using this amplicon, at young embryonic stages we observed strong reduction to 25% of wild type levels in the RNAi sample, consistent with our phenotypic validation (Fig.…”

“…For a gene of interest, primer design may be constrained such that an RT-qPCR amplicon is nested within the dsRNA region. To design intron-spanning primers for short, efficient amplicon sizes [28,29], while also avoiding conserved coding sequence regions that could cause off-target effects [15,20], both RT-qPCR and dsRNA primers may target the same region. Small genes with few introns are particularly constrained, such as Tc-zen1 (Fig 1A : Fragment 3 with respect to the short dsRNA that avoids the homeobox, as in [20]).…”

“…It is an effective RNAi screening platform [14][15][16]. pRNAi in Tribolium is regularly used for phenotypic investigation of development and to test genetic interactions singly or globally, such as by RNA-seq after RNAi [17][18][19][20]. Empirical work has shown that efficient RNAi is achieved through the introduction of long dsRNA into the organism, which persists longer in vivo and has more efficient cellular uptake than short interfering RNA (siRNA) [10,21].…”