A bHLH code for cardiac morphogenesis

Abstract

Cell specification and differentiation of cardiomyocytes from mesodermal precursors is orchestrated by epigenetic and transcriptional inputs throughout heart formation. Of the many transcription factor super families that play a role in this process, the basic Helix-loop Helix (bHLH) family of proteins is well represented. The bHLH protein by design allows for dimerization-both as homodimers and heterodimers with other proteins within the family. Although DNA binding is mediated via a short variable cis-element termed an E-box, it is clear that DNA-affinity for these elements as well as the transcriptional input conveyed is dictated largely by the transcriptional partners within the dimer complex. Dimer partner choice has a number of inputs requiring co-expression within a given cell nucleus and dimerization modulation by the level of protein present, and post-translational modifications that can both enhance or reduce protein-protein interactions. Due to these complex interrelationships, it has been difficult to identity bona-fide downstream transcriptional targets and define the molecular pathways regulated of bHLH factors within cardiogenesis, despite the clear roles suggested via loss-of-function animals models. This review focuses on the Hand bHLH proteins-key members of the Twist-family of bHLH factors. Despite over a decade of investigation, questions regarding functional redundancy, downstream targets, and biological role during heart specification and differentiation have still not been fully addressed. Our goal is to review what is currently known and address strategies for gaining further understanding of Hand/Twist gene dosage and functional redundancy relationships within the developing heart that may underlie congenital heart defect pathogenesis.

Keywords: Basic helix-loop-helix transcription factors; Congenital heart diseases; Gene dosage.

Fig. 1

a Schematic of Hand gene expression during early cardiac development. Published findings describe early expression within the anterior later plate mesoderm, which in the chick persists for both Hand genes. In mouse, Hand2 persists until E8.0 and upon rightward looping, restricts to second field lineage cells on the right ventricle and OFT. Hand1 prior to heart tube fusion already displays chamber-specific expression within the primary heart field derived left ventricle and OFT such that by E9.5 coexpression with the myocardial cuff and OFT is observed along with unique expression domains that define the left and right ventricles. b Schematic of Hand1 and Hand2 coding regions showing amino acid identities within the amino terminal, bHLH, and carboxy terminal domains. The positions of the basic DNA-binding domain are shown in black and poly-histidine and polyalanine tracks in the amino terminal portion of each respective protein are indicated. The evolutionarily conserved threonine and serine within Helix I of the bHLH are phosphoregulated via PKA and B56d-containing PP2A. Regulation alters dimer partner choice and effects DNA-binding affinities to various cis-elements. Amino acid alignment of Hand1 and 2 over the basic domain and Helix I showing high level of conservation within the DNA binding and dimerization motifs

Fig. 2

In situ hybridization of Twist1, Hand1, and Hand2 in sagitally sectioned E10.5 day mouse embryos. Expression is observed within a broad domain of mesodermally and neural crest-derived cells that make up the craniofacial, cardiac, sympathetic, and enteric nervous system as well as lateral mesoderm derived organs. Given the broad overlap of expression of Twist-family genes during development and the ability of the coded proteins to form transcriptional complexes modulation of where these genes are expressed as well as levels of overall expression are critical to normal development

Fig. 3

Schematic of Hand1 tethered dimers expressed via the αMHC-promoter. Three F0 E9.5 day embryos expressing Hand1-Hand1 (top row) within the heart display pericardial bleeding cardiac morphological defects. Hand1-E12 tethered expression (middle row) results in loss of right ventricular structures phenocopying the Hand2 systemic knockout (lower row). Wild-type mice included for comparison