Failed Progenitor Specification Underlies the Cardiopharyngeal Phenotypes in a Zebrafish Model of 22q11.2 Deletion Syndrome

Abstract

SUMMARYMicrodeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5+ progenitors in pharyngeal arches 2–6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5+ pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5+ pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and cranio-facial defects observed in the 22q11.2DS population.

Pbx4 limits heart size and fosters arch artery formation by partitioning second heart field progenitors and restricting proliferation

“…The majority of the sequenced cells expressed nkx2.5 (Figs 10 and 11), validating our isolation of nkx2.5 + cells. Smaller clusters that had lower levels or below detectable levels of nkx2.5 expression were indicated to be ECs (C1) and skeletal muscle progenitors (C14) ( Figs 9A,B, 10 and 11), populations that transiently express endogenous nkx2.5 (Guner-Ataman et al, 2018;Nagelberg et al, 2015;Paffett-Lugassy et al, 2013). Hence, some discordance between expression from the nkx2.5:ZsYellow transgene used for sorting and endogenous nkx2.5 was not unexpected due to the persistence of ZsYellow (Zhou et al, 2011).…”

“…To test this hypothesis, we investigated posterior pharyngeal arch artery ( pPAA) development in lzr mutants because Nkx2.5 + progenitors contribute to both the heart and pPAAs (3-6) (Abrial et al, 2017;Guner-Ataman et al, 2018;Paffett-Lugassy et al, 2013. Furthermore, similar to Nkx2.5 in zebrafish, recent lineage tracing has demonstrated that Isl1 + SHFPs in mice give rise to ECs within the posterior 3-6 PAAs in addition to the OFT (Wang et al, 2017), implying that a conserved aspect of vertebrate SHFPs is that they may contribute to the OFT of the heart and ECs of the pPAAs.…”

Cardiopharyngeal Progenitor Specification: Multiple Roads to the Heart and Head Muscles

“…As discussed above, the CPF is able to differentiate into both cardiac tissue and skeletal muscles of the head and neck. Several studies in mice, chick and fish have shown that there is a conserved regulatory network (including Tbx1, Pitx2, Nkx2-5 and Isl1) that governs cardiac and skeletal muscle progenitor specification (Tirosh-Finkel et al 2006;Nathan et al 2008;Sambasivan et al 2009;Nevis et al 2013;Paffett-Lugassy et al 2017;Guner-Ataman et al 2018). Interestingly, fundamental insights on how the CPF is able to differentiate into cardiac or skeletal muscle lineages arise from studies in Ciona robusta.…”

“…Tbx1 mutant embryos show defects in both craniofacial skeletal muscles and heart (Lindsay et al 2001;Kelly et al 2004;Lescroart et al 2015). Through RNA-sequencing of wild-type versus tbx1 mutant zebrafish embryos, gdf3-ALK4, a TGFµ superfamily ligand has been identified as a downstream effector of tbx1 involved in CPC specification (Guner-Ataman et al 2018). In fish, tbx1 is also required to specify the nkx2.5+ CPF.…”

Cell – ECM interactions play distinct and essential roles at multiple stages during the development of the aortic arch

“…PAAs arise by vasculogenesis from endothelial precursors originating in the lateral plate mesoderm, also known as the second heart field (SHF) [7][8][9][10][11][12][13] . Experiments in zebrafish and mice have demonstrated that PAA formation is a multistage process that entails endothelial specification in the SHF, migration of SHF-derived endothelial progenitors into the pharyngeal region, differentiation into ECs, and the assembly of SHF-derived ECs into a plexus of small blood vessels 9,[13][14][15][16] . Thereafter, the pharyngeal endothelial plexus becomes connected with the ventral and dorsal aortae.…”