During mammalian embryogenesis, cardiac progenitor cells constituting the second heart field (SHF) give rise to the right ventricle and primitive outflow tract (OFT). In zebrafish, previous lineage-tracing and mutant analyses suggested that SHF ventricular and OFT progenitors co-migrate to the arterial pole of the zebrafish heart tube soon after their specification in the field of anterior lateral plate mesoderm (ALPM). Using additional prospective lineage tracing, we demonstrate that while SHF ventricular progenitors migrate directly to the arterial pole, OFT progenitors become temporarily sequestered in the mesodermal cores of pharyngeal arch 2 (PA2), where they downregulate expression. While there, they intermingle with precursors for PA2-derived head muscles (HMs) and hypobranchial artery endothelium, which we demonstrate are co-specified with SHF progenitors in the ALPM. Soon after their sequestration in PA2, OFT progenitors migrate to the arterial pole of the heart and differentiate into OFT lineages. Lastly, we demonstrate that SHF ventricular and OFT progenitors exhibit unique sensitivities to a mutation in Our data highlight novel aspects of SHF, OFT and HM development in zebrafish that will inform mechanistic interpretations of cardiopharyngeal phenotypes in zebrafish models of human congenital disorders.

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.

SUMMARY The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that mature into critical segments of the aortic arch and its branches. Although defects in PAA development cause life-threating congenital cardiovascular defects, the molecular mechanisms that orchestrate PAA morphogenesis remain unclear. Through small-molecule screening in zebrafish, we identified TGF-β signaling as indispensable for PAA development. Specifically, chemical inhibition of the TGF-β type I receptor ALK5 impairs PAA development because nkx2.5+ PAA progenitor cells fail to differentiate into tie1+ angioblasts. Consistent with this observation, we documented a burst of ALK5-mediated Smad3 phosphorylation within PAA progenitors that foreshadows angioblast emergence. Remarkably, premature induction of TGF-β receptor activity stimulates precocious angioblast differentiation, thereby demonstrating the sufficiency of this pathway for initiating the PAA progenitor to angioblast transition. More broadly, these data uncover TGF-β as a rare signaling pathway that is necessary and sufficient for angioblast lineage commitment.

Background R-etomidate possesses unique desirable properties, but potently suppresses adrenocortical function. Consequently, efforts are being made to define structure-activity relationships with the goal of designing analogues with reduced adrenocortical toxicity. We explored the pharmacological impact of modifying etomidate’s chiral center utilizing R-etomidate, S-etomidate and two achiral etomidate analogues (cyclopropyl etomidate and dihydrogen etomidate). Methods The γ-aminobutyric acid type A (GABAA) receptor modulatory potencies of drugs were assessed in oocyte-expressed α1(L264T)β3γ2L and α1(L264T)β1γ2L GABAA receptors (for each drug, n=6 oocytes per subtype). In rats, hypnotic potencies and durations of action were measured using a righting reflex assay (n = 26 – 30 doses per drug) and adrenocortical potencies were quantified using an adrenocorticotropic hormone stimulation test (n = 20 experiments per drug). Results All four drugs activated both GABAA receptor subtypes in vitro and produced hypnosis and suppressed adrenocortical function in rats. However, drug potencies in each model ranged by 1–2 orders of magnitude. R-etomidate had the highest GABAA receptor moulatory, hypnotic, and adrenocortical inhibitory potencies. Respectively, R-etomidate, S-etomidate and cyclopropyl etomidate were 27.4-fold, 18.9-fold, and 23.5-fold more potent activators of receptors containing β3 subunits than β1 subunits; however, dihydrogen etomidate’s subunit selectivity was only 2.48-fold and similar to that of propofol (2.08-fold). S-etomidate was 1/23rd as potent an adrenocortical inhibitor as R-etomidate. Conclusion The linkage between the structure of etomidate’s chiral center and its pharmacology suggests that altering etomidate’s chiral center may be used as part of a strategy to design analogues with more desirable adrenocortical activities and/or subunit selectivities.

CPMM and propofol have similar potencies in GABAA receptors and tadpoles; however, CPMM provides more rapid and predictable recovery than propofol, particularly after prolonged infusion.