Neural Crest Expression of Cre Recombinase Directed bythe Proximal Pax3 Promoter in Transgenic MiceJun Li, Fabian Chen, and Jonathan A. Epstein*Cardiovascular Division, Department of Medicine, University of PennsylvaniaHealth System, Philadelphia, Pennsylvania

Received 19 November 1999; Accepted 19 November 1999

Pax3 encodes a transcription factor expressed duringearly to midgestation in the dorsal neural tube andsomite (Goulding et al., 1991). Mutations in humans andmice indicate that Pax3 is required for neural crest andhypaxial muscle development (Auerbach, 1954; Epsteinet al., 1991; Tassabehji et al., 1992). Homozygous Pax3deficient Splotch embryos die during midgestation withcardiac and muscular defects. Cardiac abnormalities aresimilar to those seen in chick embryos after ablation ofpremigratory neural crest cells (Kirby et al., 1983).Hence, Pax3 has been implicated in migration or func-tion of cardiac neural crest (Conway et al., 1997). Mu-tant embryos have other neural crest defects includingabsence or deficiencies of dorsal root and sympatheticganglia.

Pax3 is normally expressed in the dorsal neural tube,the region from which neural crest cells emerge, begin-ning at E8.5 (Goulding et al., 1991). Shortly afterward, itis expressed in presomitic mesoderm, and is later re-stricted to the lateral dermomyotome, which gives riseto hypaxial musculature (Tajbakhsh et al., 1997). ByE14.5, expression abates, though it later re-emerges inmature Schwann cells (Kioussi et al., 1995).

We have recently described a 1.6-Kb proximal Pax3upstream region that directs expression of lacZ in dorsalneural tube and in neural crest derived dorsal root gan-glia (drg), but not in the somite (Li et al., 1999). Whenthis promoter element directs expression of Pax3 pro-tein in transgenic mice, it is capable of rescuing cardiacdevelopment and formation of drg in Splotch embryos.Transgenic Splotch embryos survive until birth, but lacka muscular diaphragm due to absence of Pax3 in myo-genic precursors. Hence, the 1.6-Kb proximal Pax3 pro-moter regulates expression of Pax3 in neural crest butnot somitic domains.

We have used the proximal 1.6 Kb Pax3 promoter todirect expression of Cre recombinase in transgenic miceusing a B6/SJL genetic background (Fig. 1). In situ hy-bridization analysis for Cre mRNA confirmed appropriateexpression in dorsal neural tube (not shown). In multi-ple lines, expression in the somite or lateral mesodermwas not detected. P3Pro-Cre mice were crossed withR26R reporter mice and tissue specific recombinationwas detected by b-galactosidase staining.

At E10.5 b-galactosidase expression was detected inthe dorsal neural tube in a pattern similar to that seen inP3Pro-lacZ embryos (Li et al., 1999; Natoli et al., 1997)recapitulating normal Pax3 expression in the neural tubeexcept that expression in the cervical region was dimin-ished (Fig. 2A and B). Dorsal root and sympathetic gan-glia expressed b-galactosidase (Fig. 2C). Unlike P3Pro-lacZ animals (Li et al., 1999; Natoli et al., 1997),b-galactosidase expression was also detected in cellsemigrating from the dorsal neural tube and populatingthe pharyngeal arches (Fig. 2A, black arrow). Stainingwas also detected in cells invading the outflow tract ofthe heart (Fig. 2D) whereas P3Pro-lacZ and Pax3 are notnormally expressed in this region suggesting that thesecells represent neural crest derivatives of Pax3-express-ing precursors.

By E14.5, the dermis was populated by b-galactosidaseexpressing cells that likely represent neural crest-derivedmelanocytes (Fig. 2E). The drg remained intenselystained (Fig. 2F). The aortic arch and proximal headvessels were b-galactosidase positive as was the ductusarteriosus and the pulmonary artery, though the de-scending aorta distal to the ductus arteriosus was devoidof blue cells (Fig. 2G). Transverse sections through theaortic arch indicated that b-galactosidase-expressingcells were located in the media of the great vesselswhere smooth muscle cells are located (Fig. 2H). Someportions of the intestine were also b-galactosidase posi-tive suggesting that Pax3-expressing neural crest progen-

* Correspondence to: Jonathan A. Epstein, 954 BRB II, 421 Curie Blvd.,Philadelphia, PA 19104.

E-mail: epsteinj@mail.med.upenn.eduGrant sponsor: NIH (to J.A.E.); Grant numbers: HL62974, HL61475.

FIG. 1. The P3Pro-Cre transgene was composed of the proximal1.6 Kb of the murine Pax3 promoter, derived from P3Pro-lacZ (Li etal., 1999), upstream of Cre and a bovine growth hormone polyad-enylation site (BGH polyA). A 3-Kb NarI/NotI fragment (shown) wasgel purified and used for injection of fertilized eggs.

© 2000 Wiley-Liss, Inc. genesis 26:162–164 (2000)

itors populate the gut (not shown). Surprisingly, popu-lations of cells in the cartilaginous portions of the ribsexpressed b-galactosidase (Fig. 2I).

These results suggest that P3Pro-Cre mice can func-tion to direct tissue-specific recombination in neuralcrest cells. This line should be useful for neural crest-specific gene inactivation in future studies.

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FIG. 2. Tissue-specific Cre-mediated recombination results in b-galactosidase expression in P3Pro-Cre, R26R embryos at E10.5 (A–D) andE14.5 (E–I). At E10.5, b-galactosidase is expressed in the dorsal neural tube (nt) and dorsal root ganglia (drg) (A, B), like Pax3 (not shown),and also in pharyngeal arches (A, arrow) and cells migrating to the drg (B, arrows). Transverse section (C) reveals expression in sympatheticganglia (sg). Expression is seen in the cardiac outflow tract (ot) (D). By E14.5, the skin is populated by b-galactosidase expressing cells (E)consistent with the neural crest origin of melanocytes. Transverse section reveals expression in drg (F). Dorsal view of an explanted heart(G) reveals labeling of the aortic arch (aa), and ductus arteriosus (da), but no blue cells are seen in the descending aorta (dAo) beyond thejunction of the ductus arteriosus (arrowhead, G). A transverse section through the ascending aorta shown in G reveals labeled cells in themedia of the artery (H). Blue cells are also found in the cartilaginous portions of the ribs (I). lv, left ventricle; la, left atrium; c, carotid; ao,aorta.

163Pax3 PROMOTER IN TRANSGENIC MICE

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164 LI ET AL.