Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Abstract

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

Species-specific deployment of Runx2 isoforms and differential regulation of target genes during avian jaw development and evolution

“…To overexpress Runx2 isoforms in chick and duck cells we utilized a dox-inducible promoter system that we characterized previously (Chu et al, 2020). We confirmed Runx2 isoform overexpression by mScarlet (RFP) fluorescence and by measuring expression of total Runx2 mRNA.…”

“…Full length Runx2 was amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (M0493L, NEB, Ipswich, MA, USA) and cloned using CloneJET PCR Cloning Kit (K1231, Thermo Fisher Scientific, Waltham, MA, USA). Following confirmation of obtaining full length Runx2 by Sanger sequencing, Runx2 was cloned into the pPIDNB inducible-promoter system (Chu et al, 2020), which was digested with AflII (R0520S, NEB, Ipswich, MA, USA) and PstI (R3140S, NEB, Ipswich, MA, USA), using NEBuilder HiFi DNA Assembly Master Mix. pPIDNB contains 1) a piggyBac transposon (Lacoste et al, 2009;Yusa et al, 2011;Yusa, 2015), which allows for stable integration of the construct into a host genome when combined with a pNano-hyPBase plasmid; 2) a constitutively active mNeongreen (GFP) (Shaner et al, 2013), which serves as a reporter for transfection or electroporation efficiency; and 3) a doxycycline (dox)-inducible (Gossen et al, 1995;Loew et al, 2010;Heinz et al, 2011) mScarlet-I (RFP) (Bindels et al, 2017), which serves as a reporter for overexpression of the gene of interest (Chu et al, 2020).…”

“…We focused on these two isoforms because they are the most distinct from one another with each defined by either an alternative promoter (P1 or P2), the presence or absence of the domain encoded by exon 5 (AS+ or AS-), or an alternative C-terminus (C1 or C2) (Figure 4A). To restrict overexpression to late stages of jaw development, we utilized a tool we previously developed that combines a piggyBac transposon system, which integrates into the host genome and expresses GFP, and a dox-inducible promoter, which drives overexpression of RFP and a gene of interest (Chu et al, 2020). We unilaterally electroporated presumptive NCM at HH8.5 so that the non-electroporated contralateral side could serve as an internal control (Figure 7A), treated embryos with dox at HH34 (Figure 7B), and then examined the extent of isoform overexpression at HH37 by screening embryos for RFP using epifluorescent or confocal microscopy (Figure 7C).…”

“…One gene in particular, Runt related transcription factor 2 (Runx2), shows intriguing species-specific patterns with quail expressing higher endogenous levels than duck, coincident with their smaller jaw size. Runx2 is considered a master regulator of osteogenesis as evidenced by its effects on osteoblasts and the loss of bone in Runx2 mutant mice (Ducy et al, 1997;Komori et al, 1997;Otto et al, 1997;Karsenty et al, 1999;Ducy, 2000;Teplyuk et al, 2008;Schneider, 2009;Ainsworth et al, 2010;Mitgutsch et al, 2011;Fish and Schneider, 2014a;Gammill et al, 2019;Chu et al, 2020;Smith et al, 2020). As Galliformes, chick and quail are separated by approximately 50 million years, and they diverged from a common ancestor with Anseriformes, which include duck, around 100 million years ago (Pereira and Baker, 2006;Hackett et al, 2008;Kan et al, 2010).…”

“…Then we perform a series of in vitro and in vivo functional analyses of Runx2 primarily using a three-taxon comparison in which two taxa are more closely related than either are to a third, since this is considered a robust strategy for making parsimonious inferences about evolution (Nelson and Platnick, 1991;Mavrodiev et al, 2019;Rineau et al, 2020). We exploit duck, chick, and quail in particular, because their jaw skeletons differ in size (from relatively large to small), their genomes are sequenced and annotated, and their embryos can be easily matched using the Hamburger and Hamilton (HH) staging system (Hamburger and Hamilton, 1951;Koecke, 1958;Padgett and Ivey, 1960;Zacchei, 1961;Hamilton, 1965;Yamashita and Sohal, 1987;Le Douarin et al, 1996;Ricklefs and Starck, 1998;Starck and Ricklefs, 1998;Nakane and Tsudzuki, 1999;Schneider and Helms, 2003;Stern, 2005;Lwigale and Schneider, 2008;Sauka-Spengler and Barembaum, 2008;Jheon and Schneider, 2009;Ainsworth et al, 2010;Mitgutsch et al, 2011;Fish and Schneider, 2014a;Gammill et al, 2019;Chu et al, 2020;Smith et al, 2020). As Galliformes, chick and quail are separated by approximately 50 million years, and they diverged from a common ancestor with Anseriformes, which include duck, around 100 million years ago (Pereira and Baker, 2006;Hackett et al, 2008;Kan et al, 2010).…”