Chapter Three - The Hand2 Gene Dosage Effect in Developmental Defects and Human Congenital Disorders

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Abstract

Heart- and neural crest derivatives-expressed (Hand) proteins belong to the Twist family of the basic helix–loop–helix (bHLH) transcription factors, and play crucial roles in the development of several organs. They form heterodimers with Twist1 via their HLH domain. Disruption of the expression balance between Hand2 and Twist1 causes limb malformation, indicating that the expression level of Hand2 relative to Twist1 is essential for limb development. Mutations of the TWIST1 and TWIST2 genes are involved in human diseases. Although, the functions of the Hand proteins are indispensable for limb, heart, and craniofacial development, mutations of the Hand genes that are causative of human diseases remain elusive. Recently, comparative analyses of a human chromosomal disorder, partial trisomy distal 4q, and its mouse model, which is a spontaneously occurring mutant, clearly demonstrated that over dosage of Hand2 results in developmental defects of limbs, craniofacial, and lumbar vertebrae, and that trisomy of the Hand2 gene directly causes a human congenital disorder. In this review, we focus on gene dosage effect of Hand2 in limb, heart, and craniofacial development, and discuss its implication in human diseases.

Introduction

Basic helix–loop–helix (bHLH) transcription factors have an evolutionarily conserved domain that comprises a short stretch of basic amino acids and two alpha helices separated by a loop motif (Murre et al., 1989). Phenotype analyses of mutant flies and mice revealed that the bHLH transcription factors play essential roles to regulate cell-fate determination and the development of several organs (Massari & Murre, 2000). To date, extensive searches for homologs of bHLH genes in higher vertebrate species, including mouse and human, have been carried out, and many homologs and novel bHLH transcription factors have been identified. From a functional aspect, they are divided into several groups, such as neurogenic differentiation (NeuroD), Hairy/enhancer-of-split (Hes), and myogenic differentiation antigen (MyoD) families, which act on neural differentiation, neural inhibition, and myogenic differentiation, respectively. On the other hand, based on their expression patterns, the bHLH transcription factors are categorized into two classes. One is the ubiquitously expressed Class A, which includes E12, E47, and HEB; and the other is tissue-specifically expressed Class B, which includes MyoD, Neurog1, and Mash1. The Class B proteins form homo- and/or hetero dimers with Class A or other Class B proteins through their HLH domains, and bind to genomic DNA through their basic amino acid stretch, which recognizes a consensus sequence, CANNTG, termed an E-box, which resides in cis-regulatory elements of downstream target genes (Murre et al., 1989, Wilson-Rawls et al., 2004).

Heart- and neural crest derivatives-expressed 2 (Hand2), also known as dHand, Hed, and Thing2, is a typical bHLH transcription factor. It belongs to the Twist family together with Hand1, which is also knows as eHand, Hxt, and Thing1 (Fig. 3.1). Hand2 forms a heterodimer with Twist1, which also belongs to the Twist family (Firulli et al., 2005). Haploinsufficiency of human TWIST1 resulting from mutation of the TWIST1 gene causes a congenital disorder, Saethre–Chotzen Syndrome (OMIM 101400), which manifests as craniofacial anomaly, facial asymmetry, premature closing of suture, polydactyly, and clinodactyly (el Ghouzzi et al., 1997, Howard et al., 1997, Jabs, 2001). Indeed, approximately 80% of Saethre–Chotzen Syndrome patients have a point mutation or deletion in TWIST1 (Gripp et al., 2000, Jabs, 2001, Johnson et al., 1998). Moreover, it was reported that mutations of the class A bHLH transcription factor, TCF12, which forms a dimer with Twist1, as does Hand2, is also responsible for Saethre–Chotzen Syndrome (Sharma et al., 2013).

Although, the Hand proteins form heterodimers with the Twist1 protein and play pivotal roles during the heart and limb development, human diseases caused by mutations in the Hand genes are largely unknown. Recently, a genetic study of a mouse mutant revealed that over dosage of Hand2 caused numerous developmental defects, and is responsible for several symptoms of a human chromosomal disorder named partial trisomy distal 4q (Tamura et al., 2013). In this review, we focus on the gene dosage effect of Hand2 on the cardiovascular, craniofacial, and limb development and its implication in human diseases.

Section snippets

Cloning of Hand Genes and Their Expression Patterns

Several research groups independently cloned the mouse Hand1 and Hand2 genes using the Yeast two-hybrid system with the E-protein as the bait, or by screening of homologous sequences in cDNA libraries using known sequences encoding the bHLH domain as probes (Cross et al., 1995, Cserjesi et al., 1995, Hollenberg et al., 1995, Srivastava et al., 1995). These studies revealed that the Hand1 and Hand2 proteins are members of the Hand subfamily, and belong to the Twist family, which include

Developmental Functions of Hand2

Developmental functions of the vertebrate Hand genes have been investigated by phenotype analyses of mice mutated in these genes. The first conventional KO mouse of Hand2 was generated soon after cloning of the gene (Srivastava et al., 1997). Hand2 comprises two exons, and this structure is evolutionally conserved from teleosts to mammals, including humans (Firulli, 2003). In the first KO mouse, a neomycin resistance cassette replaced a segment containing the two exons. This Hand2 KO mouse

Gene Dosage Effect of Hand2 in Mouse Embryogenesis

Hand2 forms a heterodimer with Twist1 (Firulli et al., 2005). Heterozygotes of the Twist1 KO mutant mouse exhibited an anterior ectopic expression of Shh, leading to excess digits in the anterior limb bud (Bourgeois et al., 1998, el Ghouzzi et al., 1997, Zhang et al., 2010). This suggests that Twist1 has an inhibitory effect on the Shh expression in the normal limb bud. The opposing actions of Hand2 and Twist1 in A–P patterning are controlled in a dose dependent manner. Polydactyly in

Disruption of Hand2 Dosage Causes Human Diseases

The human HAND2 gene is located on human chromosome 4, at 4q34.1 (UCSC Human Genome Browser; GRCh37/hg19). Duplication of the distal end of the long arm of human chromosome 4, which includes 4q34, leads to the congenital disorder, “Partial trisomy distal 4q” (denoted 4q +), whose typical phenotypes include growth retardation, psychomotor retardation, microcephaly, epicantic folds, high nasal bridge, short philtrum, micrognathia, low set/malformed ear, preaxial polydactyly, epilepsy, renal

Future Perspectives

It is intriguing that only expression level changes of Hand2, but not its ectopic expression, elicits several developmental defects, including preaxial polydactyly in mice and humans. Accumulating data have provided evidence that balanced expression of Hand2 and Twist1 is essential for proper limb development, and disruption of the expression balance between these two genes causes developmental defects.

Human 4q syndrome (4q −), which might be caused by lower levels of Hand2, shows several

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (12013551 to M. T.), and by the Ministry of Education, Culture, Sports, Science and Technology in Japan (17018033 to T. S.).

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