Distinct enhancers regulate neural expression of Pax7 Deborah Lang, Christopher B. Brown, Rita Milewski, Yue Qin Jiang, Min Min Lu, and Jonathan A. Epstein* Cardiovascular Division, Department of Medicine, University of Pennsylvania, 954 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104, USA Received 16 March 2003; accepted 30 May 2003 Abstract The murine Pax7 gene has emerged as an important regulator of neural and somite development. It is expressed in discrete domains of the central nervous system, including cranial neural crest, dorsal neural tube, and mesencephalic tectum, pretectum, and base, and at the midbrain– hindbrain boundary. It is also expressed by nasal epithelia and neural crest-derived facial structures. Here, we define the 5' end of the cDNA for murine Pax7 and identify the transcriptional start site. We clarify gene structure and the murine coding sequence, and we define regions of noncoding sequence that are conserved between mice and humans. Using transgenic approaches, we identify upstream and intronic regulatory elements that confer distinct domains of neural expression in the cranial neural crest, facial mesenchyme, mesencephalon, and pontine reticular nucleus. These enhancer regions will be useful for gene expression studies and for the identification of upstream regulators of Pax7 expression. © 2003 Elsevier Inc. All rights reserved. Keywords: Brain; Transcription factors; Homeodomain proteins; Gene expression regulation; Molecular sequence data; Mice, transgenic; Cell lineage Pax7 is a paired-domain-containing transcription factor that is expressed in distinct populations within the central nervous system during embryonic development [1]. Pax7 is thought to play an early role in the specification of prolif- erating neuronal precursors and it is expressed in the ven- tricular zone of the dorsal neural tube where rapidly divid- ing neuronal precursors reside. It is also expressed at the midbrain– hindbrain boundary and in distinct regions of the pons and mesencephalon [2]. Inactivation of Pax7 in the mouse leads to neonatal lethality and significant craniofacial abnormalities related to defects of cranial neural crest. Mice deficient in Pax7 also show myogenic abnormalities during embryogenesis and a failure of satellite cell maintenance in mature muscle [3,4]. Pax7 is one of nine mammalian Pax genes. Each gene encodes a similar DNA binding domain (the paired domain) and some Pax genes also encode a second DNA binding domain, a homeodomain [5]. Pax2–8 are expressed in the central nervous system in a complex and overlapping pat- tern. For instance, Pax2 and 5, like Pax7, are expressed at the midbrain– hindbrain boundary, while Pax6 expression overlaps with that of Pax7 in the pons [2]. Functional redundancy between Pax genes has been described [6,7]. Inactivation of both Pax2 and Pax5 leads to loss of neural tissues derived from the midbrain– hindbrain junction, a phenotype not seen in either individual mutant [8]. Tran- scriptional regulation of Pax genes in distinct domains has been partially evaluated. In many cases, regulation is com- plex with distantly located cis-acting enhancer regions. For instance, Pax6 contains at least three transcriptional start sites, and critical enhancers required for expression in a variety of tissues are located 5',3', and within introns of the Pax6 gene [9 –14]. In the case of Pax3, we have identified distinct neural crest [15] and myogenic enhancer regions located several kilobases apart upstream of the minimal promoter (C.B.B. and J.A.E., unpublished). Pax3 is the Pax family member most closely related to Pax7 and the two genes are thought to have arisen from gene duplication [6]. They share many aspects of genomic structure including * Corresponding author. Fax: +1-215-573-2094. E-mail address: epsteinj@mail.med.upenn.edu (J.A. Epstein). R Available online at www.sciencedirect.com Genomics 82 (2003) 553–560 www.elsevier.com/locate/ygeno 0888-7543/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0888-7543(03)00178-2