EXPRESSions THE ANATOMICAL RECORD PART A 271A:189 –191 (2003) Slug Expression During Organogenesis in Mice KATHLEEN F. ORAM, ETHAN A. CARVER, AND THOMAS GRIDLEY* Jackson Laboratory, Bar Harbor, Maine ABSTRACT The vertebrate Slug gene encodes a zinc finger-containing transcriptional repressor. Here we report expression of the mouse Slug gene during organogenesis and late fetal development using histochemical detection of ␤-galactosidase expressed from a targeted SluglacZ knock-in allele. The Slug gene is highly expressed in the mesenchymal or stromal component of numerous organs. It is also highly expressed in craniofacial mesenchyme, in bone of both mesodermal and neural crest origin, and in the outflow tract and the endocardial cushions of the heart. Anat Rec Part A 271A:189 –191, 2003. © 2003 Wiley-Liss, Inc. Key words: bone; Slug; heart; lacZ reporter; zinc finger protein The Slug gene encodes a zinc finger-containing transcriptional repressor homologous to the product of the Drosophila Snail gene (Hemavathy et al., 2000; Nieto, 2002). Evidence in tissue culture cells suggests that Slug expression may regulate epithelial–mesenchymal transitions (Savagner et al., 1997). In several vertebrate species, expression of the Slug gene is implicated in regulating the formation and delamination of the mesoderm and neural crest, both of which are formed by an epithelial–mesenchymal transition (Nieto, 2001). The Slug gene has also been identified as a downstream target of the E2A-HLF fusion oncoprotein, and evidence in this system indicated that Slug acts as an anti-apoptotic agent in mammalian pro-B cells (Inukai et al., 1999). We previously described the phenotype of a targeted null mutation of the mouse Slug gene (Jiang et al., 1998). Slug mutant mice exhibited postnatal growth deficiency, but, surprisingly, Slug was not required for either mesoderm or neural crest generation, apparently because of differences in the early expression pattern of Slug in mice as compared to other vertebrates (Jiang et al., 1998; Sefton et al., 1998). In this report we describe Slug expression during organogenesis in mice, utilizing sensitive histochemical detection of ␤-galactosidase protein expressed from a targeted SluglacZ knock-in allele (Jiang et al., 1998). MATERIALS AND METHODS The official allele designation for the SluglacZ targeted mutant allele (Jiang et al., 1998) is Slughtm2Grid. Embryos were collected from timed matings of a Slughtm2Grid/⫹ heterozygous male with wild-type females from a mixed C57BL/6J X 129S1/SvImJ background. Embryos were dissected in ice-cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde and suspended in 20% sucrose over© 2003 WILEY-LISS, INC. night at 4°C. Embryos were mounted in OTC medium, frozen in liquid nitrogen, and cryosectioned at 18 m. Sections were stained for ␤-galactosidase activity, as described by Hogan et al. (1994). Sections were counterstained with eosin. RESULTS AND DISCUSSION In order to analyze expression of the Slug gene during later stages of embryogenesis and organogenesis in mice, we used histochemical detection of ␤-galactosidase protein expressed from a targeted SluglacZ knock-in allele. We demonstrated previously that expression of the SluglacZ knock-in allele faithfully reflects the expression pattern of Slug RNA during early embryogenesis (Jiang et al., 1998). However, histochemical detection of the ␤-galactosidase fusion protein was more sensitive and had less background compared to detection of Slug RNA by in situ hybridization. Therefore, we used histochemistry to assess expression of the Slug gene during organogenesis and late fetal development in mice. Overall, SluglacZ expression levels were highest during the early stages of organogenesis and declined markedly by embryonic days 17 (E17) and E18 (Fig. 1A–C). We Grant sponsor: NIH; Grant numbers: RO1 HD34883; P60 DE13078; T32 CA09217; P30 CA34196. *Correspondence to: Thomas Gridley, Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. Fax: (207) 288-6077. E-mail: email@example.com Received 10 October 2002; Accepted 25 October 2002 DOI 10.1002/ar.a.10027 Published online 27 January 2003 in Wiley InterScience (www.interscience.wiley.com). 190 ORAM ET AL. Figures 1–3. 191 SLUG EXPRESSION DURING ORGANOGENESIS IN MICE lacZ previously showed that at E10, the Slug gene was highly expressed in craniofacial mesenchyme, particularly in the frontonasal region (Jiang et al., 1998). The frontonasal region continued to be a major site of SluglacZ expression through E16 (Fig. 1A–D). The SluglacZ gene was highly expressed in developing bone, including bones undergoing endochondral or intramembranous ossification (Fig. 1D and E). In developing long bones, SluglacZ expression was highest in proliferating chondrocytes, and was downregulated in hypertrophic chondrocytes (Fig. 1E). Expression was also observed in both vibrissae (Fig. 1D) and hair follicles. SluglacZ expression was observed in the outflow tract of the heart, in the endocardial cushions, and later in the heart valves (Fig. 2). Slug expression was previously observed in the endocardial cushions of the avian heart, where it is proposed to regulate the epithelial–mesenchymal transformation involved in development of the endocardial cushions (Romano and Runyan, 1999, 2000; Carmona et al., 2000). lacZ Very high levels of Slug expression were observed in the mesenchymal or stromal component of many developing organs. These included the kidney (Fig. 3A), lung (Fig. 3B), and submandibular gland. High levels of SluglacZ expression were observed in the wall of the stomach and intestines, although expression was excluded from the epithelial lining of the gut (Fig. 3C). SluglacZ expression was notably excluded from the central and peripheral nervous systems. In the brain, the only SluglacZ expression observed was in mesenchymal cells in the choroid plexus (Fig. 3D). In summary, SluglacZ expression levels were highest during the early stages of organogenesis and declined markedly by E17 and E18. The highest levels of SluglacZ expression during organogenesis were observed in craniofacial mesenchyme, proliferating chondrocytes in bone of both neural crest and mesodermal origin, intestinal and stomach walls, and mesenchyme of the lung and kidney. LITERATURE CITED Fig. 1. SluglacZ expression during organogenesis stages and in developing bones. A–C: SluglacZ expression is highest at E13–E15, and then begins to decline. The regions of most intense ␤-galactosidase activity include the frontonasal region, developing bone, lung, kidney, and gut. SluglacZ expression is notably excluded from the central nervous system. D: Expression in the frontonasal region. SluglacZ is expressed in the nasal cartilages (nc), craniofacial mesenchyme/connective tissue, and vibrissae follicles (arrowheads). SluglacZ expression is excluded from the olfactory sensory epithelium (arrows) and the vomeronasal organ (vo). E: In the developing long bones, SluglacZ is expressed in proliferating chondrocytes (pc) and the perichondrium (arrows). SluglacZ expression is downregulated in hypertrophic chondrocytes (hc). SluglacZ is also expressed in isolated cells (arrowheads) in the bone matrix. All sections in all figures were stained for ␤-galactosidase activity. A–C, and E: Parasagittal sections. D: Transverse section. Fig. 2. SluglacZ expression during heart development. A: SluglacZ is expressed in the atrioventricular cushions (arrowhead) at E13.5. B: SluglacZ expression is observed in the outflow tract (ot). C: SluglacZ expression is observed in the mitral (mt) and tricuspid (tv) valves at E15.5. All sections are parasagittal. Fig. 3. SluglacZ expression in mesenchyme of numerous organs. A: SluglacZ is expressed in kidney mesenchyme, but is not expressed in epithelial cells of developing tubules or glomeruli (arrowheads). Expression is also observed in the perichondrial fibroblasts of the ribs (arrows). B: SluglacZ is highly expressed in lung mesenchyme, but not in alveolar epithelium. C: SluglacZ expression was observed in the intestinal wall (arrow), but is not expressed in the epithelial lining of the gut. D: Expression is observed in mesenchyme of the choroid plexus. E: SluglacZ expression is observed in the tongue in a subepithelial layer (arow) and in isolated cells in the tongue epithelium (arrowheads). A–D: Parasagittal sections. 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