Genetic and Comparative Mapping of Genes Dysregulated in Mouse Hearts Lacking the Hand2 Transcription Factor Gene Melissa P. Villanueva, 1, * Aparna R. Aiyer, 2 Shaine Muller, 2 Mathew T. Pletcher, 1 Xiao Liu, 1 Beverly Emanuel, 3 Deepak Srivastava, 2 and Roger H. Reeves 1,† 1 Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 2 University of Texas Southwestern Medical Center, Dallas, Texas 75235-9148 3 Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania *Current address: Department of Developmental Genetics, NYU Medical Center, Skirball Institute, 4th Floor, Room 2, 540 First Avenue, New York, NY 10016. † To whom correspondence and reprint requests should be addressed at The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2105. Fax: (410) 614-8731. E-mail: rreeves@jhmi.edu. The helix-loop-helix transcription factor HAND2 plays a vital role in the development of the heart, limb, facies, and other neural crest-derived structures. We used differential display analysis to identify 33 putative HAND2-regulated ESTs that are differentially expressed in Hand2 / vs wild-type mice. We determined the positions on mouse and human genetic maps of 29 of these by using the T31 mouse Radiation Hybrid panel, comparison to human genomic sequence, and comparative mapping. We examined the conserved chromosomal locations for phenotypes that involve development of heart, face, and limb structures that are affected by HAND2. One EST mapped to a region of conserved synteny between mouse chromosome 2 and human chromosome 10p. RACE analysis extended the sequence and identified this cDNA as the mouse ortholog of human nebulette, an actin-binding protein expressed in fetal heart. Nebulette was shown to be deleted in DiGeorge Syndrome 2 patients with the proximal deletion of human 10p13–p14 that is associated with cardiac and craniofacial abnormalities. INTRODUCTION Hand2, the gene encoding the basic helix-loop-helix tran- scription factor, HAND2, is expressed throughout prenatal development in the deciduum, heart, autonomic nervous system, and other neural crest-derived structures [1]. HAND2 is one of the earliest cardiac chamber-specific tran- scription factors identified to date [2]. By 9.5 days postco- itum (dpc) in the mouse embryo, HAND2 is expressed in the ventricular region, with abundant transcripts also in the outflow tract (conotruncus) and the first and second aortic arch arteries [1]. At 10.5 dpc, HAND2 is highly expressed in the first branchial arch (which gives rise to the craniofacial mesenchyme), the aortic sac, the third and fourth aortic arch arteries, and the truncus arteriosus [1]. HAND2 is also ex- pressed in the limb bud where it plays a role in anterior– posterior polarization [3]. The same studies showed that ectopic expression of HAND2 results in the shortening and broadening of the long bones of the forearms and digits and preaxial polydactyly. Mice homozygous for a null allele of Hand2 die from heart failure by 11 dpc [2]. Heart defects in the null embryos include lack of aortic arch arteries, a dilated aortic sac, and an abrupt connection between the outflow tract and the left ventricle, due to the absence of the right ventricle [2]. In these embryos, the first and second branchial arches become hypoplastic, most likely the result of extensive apoptosis, and the third and fourth branchial arches fail to form [4]. The lack of a critical amount of neural crest cells in the third and fourth branchial arches is thought to result in persistent truncus arteriosus (failure of septation of ascending aorta and pulmonary trunk) and interruption of the aorta, which are characteristic of DiGeorge syndrome (DGS)/velocardio- facial syndrome, also known as the 22q11 deletion syndrome [5]. In addition, Hand2 -/- mice demonstrated abnormal vas- cular development in the embryo and the yolk sac [6]. Recent investigations of the molecular basis for DGS have shown that deletions of both copies of the Tbx1 or the Crkol gene in mouse disrupts development of the heart, thyroid, parathyroids, and craniofacial structure in patterns reminis- cent of DGS [7–10]. Segmental monosomy for a 1.5-Mb re- doi:10.1006/geno.2002.7009 Article GENOMICS Vol. 80, Number 6, December 2002 593 Copyright © 2002 Elsevier Science (USA). All rights reserved. 0888-7543/02 $35.00