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Expression of Pbx1b during mammalian organogenesis
Mechanisms of Development 100 (2001) 131±135
www.elsevier.com/locate/modo
Gene expression pattern
Expression of Pbx1b during mammalian organogenesis Catherine A. Schnabel, Licia Selleri, Yakop Jacobs, Roger Warnke, Michael L. Cleary* Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA Received 27 July 2000; received in revised form 12 October 2000; accepted 13 October 2000
Abstract Mammalian Pbx genes (Pbx1±3) encode a family of TALE homeodomain proteins that function as transcriptional regulators in numerous cell types (Curr. Opin. Genet. Dev. 8 (1998) 423). The present study highlights distinctive features of Pbx1b expression during mouse embryonic development as a framework to understand its biological functions. Immunohistochemical analyses demonstrate extensive expression of Pbx1b throughout post-implantation development, with highest levels observed during early to mid-gestation. Its initial distribution is predominantly associated with condensing mesoderm, however, Pbx1b displays dynamic expression patterns in derivatives of all principal germ layers. In particular, Pbx1b localizes to sites of mesenchymal±epithelial interactions during periods of active morphogenesis in tissues such as the lung, kidney, tooth buds and vibrissae follicles. Furthermore, BrdU labeling studies reveal that Pbx1b expression domains partially overlap with regions of cellular proliferation. Taken together, these data suggest that Pbx1b contributes to multiple cellular processes during embryogenesis, which may include roles in cell-autonomous regulation as well as in the mediation of tissue interactions. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pbx; Homeodomain; Mouse; Embryogenesis; Mesenchymal±epithelial; Proliferation; Organogenesis
1. Results and discussion Pbx1, the prototypic Pbx family member, encodes the alternatively spliced Pbx1a and Pbx1b isoforms that exhibit characteristic biochemical properties (DiRocco et al., 1997; Asahara et al., 1999). Immunohistochemical analyses reveal that Pbx1b is the predominant isoform expressed during mouse embryogenesis (this study), whereas Pbx1a expression is primarily restricted to developing neural tissues (data not shown). Here, Pbx1b expression pro®les were examined to elucidate its potential function during embryogenesis. Pbx1b expression is evident as nuclear staining throughout the development of multiple systems, however, particular features of its tissue and organ speci®c distribution are highlighted here. Initial patterns of Pbx1b expression are detected in the developing paraxial mesoderm and de®nitive endoderm (Fig. 1B±D). In emerging axial structures, Pbx1b is expressed in the somitic epithelium and the enclosed mesenchyme, and is concentrated in the sclerotomes and its skeletal derivatives as differentiation proceeds (Figs. 1C and 2A). In the branchial arches at embryonic day E9.5, nascent Pbx1b expression is observed in the surface ectoderm (data not shown) and progresses into both the * Corresponding author. Fax: 11-650-498-6222. E-mail address: [email protected] (M.L. Cleary).
epithelial and mesenchymal compartments at E10.5 (Fig. 1B). Pbx1b is present in the visceral mesoderm as well as in the endodermal epithelium during gut tube formation, and later, is prevalent in the mesenchymal components of associated organ primordia (Fig. 1D±I). In the primitive lung, Pbx1b is expressed in the mesenchyme that surrounds the branching epithelium during the initial stages of budding and outgrowth (Fig. 1E). As the bronchial tree continues to form, lung mesenchyme exhibits strong Pbx1b immunoreactivity (Fig. 1F), which persists in the pulmonary interstitium to adulthood (data not shown). Sagittal sections at the level of the posterior foregut at E11.5 reveal intense Pbx1b staining in both the undifferentiated epithelium and the associated mesenchyme of the stomach as well as in the mesenchyme of the outgrowing pancreatic bud (Fig. 1G). In subsequent stages, distribution of Pbx1b in the developing stomach is progressively restricted. Namely, expression is decreased as the mucosal epithelium differentiates whereas the submucosa and outer layers of connective tissue show continued expression at late gestation (Fig. 1H,I). Pbx1b displays striking expression patterns in several epidermal organs. At E12.0, intense staining is detected in the dental lamina during the early stages of odontogenesis (Fig. 1J). As the tooth buds form, Pbx1b expression is evident in the dental lamina as they grow into the underlying
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00516-5
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C.A. Schnabel et al. / Mechanisms of Development 100 (2001) 131±135
Fig. 2. Pbx1b expression overlaps with regions of cellular proliferation. Comparative immunostaining of Pbx1b (A,C,E,G) and BrdU (B,D,F,H) in adjacent tissue sections of E12.0 embryos. Colocalization is seen in vertebral elements in a sagittal view (A,B, arrows) and in transverse sections of the developing neural retina (C,D, arrows), lung (E,F, arrows) and diencephalon (G,H, arrows). Arrowheads in A,C,E,G indicate regions where Pbx1b expression does not overlap with BrdU-positive areas. Scale bars: 0.2 mm.
ectomesenchyme (Fig. 1K). By E16.5, Pbx1b expression persists in the oral epithelium and in the outer epithelium of the growing tooth, but is undetectable as the cells stop dividing to form the enamel knot (Fig. 1L). Distinct patterns of Pbx1b expression are also present during the develop-
133
ment of the facial integument. Before the onset of hair follicle development, Pbx1b is strongly expressed in the basal layer of the epidermis (Fig. 1M). More notably, when primary follicles are initiated, a focal aggregation of Pbx1b-positive mesenchymal cells can be seen in the dermis and in the invaginating epithelium as components of the epidermal placode (Fig. 1M). In transverse sections at E14.5, Pbx1b immunoreactivity is seen in concentric layers comprised of mesenchymal and sheath cells that surround the developing follicle (Fig. 1N). At more advanced stages of folliculogenesis, Pbx1b expression continues in mesenchymal components around the hair shaft and in the dermal papilla, and is also apparent in the root sheath (Fig. 1O). Pbx1b is present in structures derived from the intermediate mesoderm and throughout various stages of urogenital system ontogeny. In sections through mid-stage embryos, high levels of Pbx1b are visible in the mesenchyme associated with the mesonephric tubules and the genital ridge (Fig. 1P,Q). At E14.5 in both male and female embryos, Pbx1b epithelial expression distinguishes the MuÈllerian duct, whereas surrounding mesenchyme of the descending WoÈlf®an and MuÈllerian ducts exhibits strong Pbx1b staining (Fig. 1Q and data not shown). Early in metanephric development, Pbx1b expression is detected in the blastema as it is induced by the ureter (data not shown). At later stages, Pbx1b is absent in ureter-derived structures, but pervades throughout the nephrogenic mesenchyme and stroma, and is detected in glomerular cells (Fig. 1R). Since previous work implicates a neoplastic role for Pbx proteins in altered cellular proliferation (Krosl et al., 1998), further analysis of Pbx1b expression was performed in conjunction with BrdU labeling studies to examine its localization in relation to proliferating cells. As shown in Fig. 2, subregions of Pbx1b immunoreactivity colocalize with that of BrdU in a variety of tissues. In adjacent sections through E12.0 embryos, Pbx1b staining is associated with skeletal bodies and the primordial neural retina in areas where cells are actively cycling (Fig. 2, compare A and B, C and D). During lung formation, discrete regions of proliferation are apparent at the distal tips of the mesenchyme that overlap
Fig. 1. Pbx1b expression during mouse embryogenesis. (B±R) Immunohistochemical analysis of Pbx1b expression performed on tissue sections from different days of gestation showing transverse or sagittal ®elds as schematized in (A). (B,C) Pbx1b expression at E10.5 is present in the surface ectoderm (arrowhead) and mesenchyme (arrow) of the branchial arches (B) and in the dermamyotome (arrowhead) and the sclerotome (arrows) of the somite (C). (D) Pbx1b is expressed in both the endodermal (arrowhead) and mesenchymal (arrow) components of the fore- and midgut of an E9.5 embryo. At E11.5 (E), Pbx1b expression is detected in the mesenchyme (arrow) but not the epithelium (arrowhead) of the lung anlage, and persists in the interstitial mesenchyme at E16.5 (F). (G±I) Pbx1b expression is found in the mesenchyme and the endoderm of the stomach at E11.5 (G) and E13.5 (H) but is restricted to the submucosal tissues at E16.5 (I). Pbx1b is detected in the oral epithelium (arrows) during tooth morphogenesis at E12.0 (J), E13.5 (K) and E16.5 (L). Note additional expression of Pbx1b in the mesenchyme of the palatal shelves and tongue (arrowheads in K). (M) Pbx1b expression in the forming whiskers is seen in the epidermis (arrowheads) and in the underlying mesenchyme (arrow) at E13.0. Later stages of vibrissae folliculogenesis at E14.5 (N) and 16.5 (O) show Pbx1b expression in the root sheath (arrowheads), surrounding mesenchyme (arrow in N) and the dermal papillae (arrow in O). Panels (P±R) display Pbx1b mesenchymal expression in the developing urogenital system. (P) Pbx1b is visible in mesonephric mesenchyme (arrow) and in the peripheral mesenchyme and coelemic epithelium of the genital ridge (arrowhead). (Q) At E14.5, Pbx1b is seen in the surrounding mesenchyme of the both the MuÈllerian (arrowhead) and WoÈlf®an (arrow) ducts, and in the interstitial cells (concave arrowheads) surrounding the testicular cords. Note Pbx1b expression in the MuÈllerian duct. (R) Expression of Pbx1b in the metanephric kidney at E16.5 is evident in the nephrogenic mesenchyme (arrowheads) and in glomerular cells (arrows). Scale bars: (D,H,R), 0.05 mm; (B,C,E±G,I±Q), 0.1 mm.
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with the uniform expression pattern of Pbx1b (Fig. 2, compare E and F). In neural tissues, Pbx1b-positive domains are evident which extend through a region of proliferation in the developing diencephalon (Fig. 2, compare G and H). Thus, Pbx1b expression at mid-gestation partially overlaps with proliferative zones in various tissues. However, although the presence of Pbx1b in cycling cells is apparent, these data clearly reveal that its expression domains also include areas that are BrdU-negative. For instance, Pbx1b expression is detected in mesenchymal regions, surrounding the forming vertebrae and in the central portion of the budding lung, which do not exhibit BrdU immunoreactivity (Fig. 1, compare A and B, E and F). In other tissues, Pbx1b expression is evident in the transitional zone of the neuroretina and along the apical surface of the neuroepithelium where BrdU staining is undetected (Fig. 2, compare C and D, G and H). Therefore, it is likely that Pbx1b participates in diverse regulatory pathways, some of which may play a role in cellular proliferation and tissue expansion. Insight into the potential role of Pbx genes in developmental processes has been provided by studies with its Drosophila homolog, extradenticle (exd), which demonstrate a function in parallel with members of the Hox gene family to regulate axial patterning events (Rauskolb et al., 1993). Consistent with the notion that Pbx and Hox homeoproteins function cooperatively to regulate speci®c target genes (Mann and Affolter, 1998), expression of Pbx1b is detected in many tissues previously reported to express Hox genes during mouse embryogenesis (reviewed in Krumlauf, 1994). Early in development, overlapping regions of expression include segmented structures such as the branchial arches and somites, and later during organogenesis, tissues such as the lung, stomach and skeletal elements (Krumlauf, 1994; Yokouchi et al., 1995; Volpe et al., 1997). In anterior structures, however, Pbx1b is expressed in tissues where Hox gene expression has not been reported, such as the eyes and teeth, which suggest its participation in Hox-independent developmental pathways. Whereas Pbx1b expression early in development suggests its involvement in embryonic patterning and regionalization, its expression during organogenesis indicates a potential function in cell fate determination in a variety of tissues that depend on mesenchymal±epithelial interactions for their coordinated morphogenesis (Sharpe and Ferguson, 1988; Birchmeier and Birchmeier, 1993; Yasugi, 1993; Thesleff et al., 1995; Dassule and McMahon, 1998; Oro and Scott, 1998; Hogan, 1999; Roberts et al., 1999; Warburton et al., 2000). In general, Pbx1b expression is not strictly associated with either the mesenchymal or epithelial compartments, but varies in regards to tissue type. Pbx1b is selectively expressed within the mesenchyme (lung, urogenital system) or epithelia (teeth) in certain regions and is detected in both layers in others (stomach, whiskers). More interestingly, Pbx1b localizes to these apposed layers during critical periods of organogenesis. In summary, these
data suggest that Pbx1b serves multiple functions during embryogenesis, and implicate its participation in the regulation of cellular differentiation and tissue expansion. 2. Materials and methods 2.1. Tissue preparation and Immunohistochemistry C57BL/6 embryos were collected from timed-pregnancies at the days indicated. Dissected embryos were ®xed in 10% buffered formalin for 6 h to overnight at 48C and dehydrated through increasing concentrations of ethanol prior to embedding in paraf®n wax. Tissue sections (4 mm) were de-paraf®nized and treated for epitope unmasking in 500 mM Tris (pH 10) by microwave heating before incubation with anti-Pbx1b monoclonal antibody. Monoclonal antibody production and demonstration of antibody speci®city have been described previously (Chang et al., 1997). After washing in PBS, sections were incubated with biotinconjugated goat anti-mouse IgG and horseradish peroxidase-conjugated streptavidin (Jackson Immunoresearch) and detected with DAB (Sigma). Tissues were counterstained with hematoxylin and slides were mounted with Clearmount media (American Master Tech Scienti®c Inc.). 2.2. BrdU labeling Pregnant C57BL/6 mice were injected intravenously with bromodeoxyuridine (BrdU) (Sigma) at 50 mg/g of body weight 3 h prior to sacri®ce. E12.0 embryos were processed and immunostained as above using an anti-BrdU monoclonal antibody (Neomarkers) on adjacent sections as those stained with anti-Pbx1b. Detection was performed with the Vector Nova-Red substrate kit (Vector Laboratories) according to manufacturer's instructions. Acknowledgements We gratefully acknowledge Eva Pfendt for her expertise in immunohistochemistry, Bich-Tien Rouse for antibody production, Carmencita Nicolas and Maria Ambrus for technical assistance, Phil Verzola for photographic help, and Greg Barsh and Anthony Oro for critical reading of the manuscript. This work was supported by grants from the NIH (CA70404, CA42971 and CA34233). C.A.S. is a fellow of the Leukemia and Lymphoma Society. References Asahara, H., Dutta, S., Kao, H.Y., Evans, R.M., Montminy, M., 1999. Pbx± Hox heterodimers recruit coactivator-corepressor complexes in an isoform-speci®c manner. Mol. Cell. Biol. 19, 8219±8225. Birchmeier, C., Birchmeier, W., 1993. Molecular aspects of mesenchymal± epithelial interactions. Annu. Rev. Cell. Biol. 9, 511±540. Chang, C.P., Jacobs, Y., Nakamura, T., Jenkins, N.A., Copeland, N.G., Cleary, M.L., 1997. Meis proteins are major in vivo DNA binding
C.A. Schnabel et al. / Mechanisms of Development 100 (2001) 131±135 partners for wild type but not chimeric Pbx proteins. Mol. Cell. Biol. 17, 5679±5687. Dassule, H., McMahon, A.P., 1998. Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the developing tooth. Dev. Biol. 202, 215±227. DiRocco, G., Mavilio, F., Zappavigna, V., 1997. Functional dissection of a transcriptionally active, target-speci®c Hox-Pbx complex. EMBO J. 16, 3644±3654. Hogan, B.L.M., 1999. Morphogenesis. Cell 96, 225±233. Krosl, J., Baban, S., Krosl, G., Rosenfeld, S., Largman, C., Sauvageau, G., 1998. Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with Pbx1. Oncogene 16, 3403± 3412. Krumlauf, R., 1994. Hox genes in vertebrate development. Cell 78, 191± 201. Mann, R.S., Affolter, M., 1998. Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423±429. Oro, A.E., Scott, M.P., 1998. Splitting hairs: dissecting roles of signaling systems in epidermal development. Cell 95, 575±578. Rauskolb, C., Peiper, M., Wieschaus, E., 1993. Extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene, pbx1. Cell 74, 1101±1112.
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Roberts, L.M., Hirokawa, Y., Nachtigal, M.W., Ingraham, H.A., 1999. Paracrine-mediated apoptosis in reproductive tract development. Dev. Biol. 208, 110±122. Sharpe, P.M., Ferguson, M.W., 1988. Mesenchymal in¯uences on epithelial differentiation in developing systems. J. Cell Sci. Suppl. 10, 195±203. Thesleff, I., Vaahtokari, A., Partanen, A.-M., 1995. Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int. J. Dev. Biol. 39, 35±50. Volpe, M.V., Martin, A., Vosatka, R.J., Mazzoni, C.L., Nielsen, H.C., 1997. Hox b-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate. Histochem. Cell. Biol. 108, 495±504. Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K.D., Cardoso, W., 2000. The molecular basis of lung development. Mech. Dev. 92, 55±81. Yasugi, S., 1993. Role of epithelial-mesenchymal interactions in the differentiation of epithelium of vertebrate digestive organs. Dev. Growth Differ. 35, 1±9. Yokouchi, Y., Sakiyama, J., Kuroiwa, A., 1995. Coordinated expression of Abd-B subfamily genes of the HoxA cluster in the developing digestive tract of chick embryo. Dev. Biol. 169, 76±89.
www.elsevier.com/locate/modo
Gene expression pattern
Expression of Pbx1b during mammalian organogenesis Catherine A. Schnabel, Licia Selleri, Yakop Jacobs, Roger Warnke, Michael L. Cleary* Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA Received 27 July 2000; received in revised form 12 October 2000; accepted 13 October 2000
Abstract Mammalian Pbx genes (Pbx1±3) encode a family of TALE homeodomain proteins that function as transcriptional regulators in numerous cell types (Curr. Opin. Genet. Dev. 8 (1998) 423). The present study highlights distinctive features of Pbx1b expression during mouse embryonic development as a framework to understand its biological functions. Immunohistochemical analyses demonstrate extensive expression of Pbx1b throughout post-implantation development, with highest levels observed during early to mid-gestation. Its initial distribution is predominantly associated with condensing mesoderm, however, Pbx1b displays dynamic expression patterns in derivatives of all principal germ layers. In particular, Pbx1b localizes to sites of mesenchymal±epithelial interactions during periods of active morphogenesis in tissues such as the lung, kidney, tooth buds and vibrissae follicles. Furthermore, BrdU labeling studies reveal that Pbx1b expression domains partially overlap with regions of cellular proliferation. Taken together, these data suggest that Pbx1b contributes to multiple cellular processes during embryogenesis, which may include roles in cell-autonomous regulation as well as in the mediation of tissue interactions. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pbx; Homeodomain; Mouse; Embryogenesis; Mesenchymal±epithelial; Proliferation; Organogenesis
1. Results and discussion Pbx1, the prototypic Pbx family member, encodes the alternatively spliced Pbx1a and Pbx1b isoforms that exhibit characteristic biochemical properties (DiRocco et al., 1997; Asahara et al., 1999). Immunohistochemical analyses reveal that Pbx1b is the predominant isoform expressed during mouse embryogenesis (this study), whereas Pbx1a expression is primarily restricted to developing neural tissues (data not shown). Here, Pbx1b expression pro®les were examined to elucidate its potential function during embryogenesis. Pbx1b expression is evident as nuclear staining throughout the development of multiple systems, however, particular features of its tissue and organ speci®c distribution are highlighted here. Initial patterns of Pbx1b expression are detected in the developing paraxial mesoderm and de®nitive endoderm (Fig. 1B±D). In emerging axial structures, Pbx1b is expressed in the somitic epithelium and the enclosed mesenchyme, and is concentrated in the sclerotomes and its skeletal derivatives as differentiation proceeds (Figs. 1C and 2A). In the branchial arches at embryonic day E9.5, nascent Pbx1b expression is observed in the surface ectoderm (data not shown) and progresses into both the * Corresponding author. Fax: 11-650-498-6222. E-mail address: [email protected] (M.L. Cleary).
epithelial and mesenchymal compartments at E10.5 (Fig. 1B). Pbx1b is present in the visceral mesoderm as well as in the endodermal epithelium during gut tube formation, and later, is prevalent in the mesenchymal components of associated organ primordia (Fig. 1D±I). In the primitive lung, Pbx1b is expressed in the mesenchyme that surrounds the branching epithelium during the initial stages of budding and outgrowth (Fig. 1E). As the bronchial tree continues to form, lung mesenchyme exhibits strong Pbx1b immunoreactivity (Fig. 1F), which persists in the pulmonary interstitium to adulthood (data not shown). Sagittal sections at the level of the posterior foregut at E11.5 reveal intense Pbx1b staining in both the undifferentiated epithelium and the associated mesenchyme of the stomach as well as in the mesenchyme of the outgrowing pancreatic bud (Fig. 1G). In subsequent stages, distribution of Pbx1b in the developing stomach is progressively restricted. Namely, expression is decreased as the mucosal epithelium differentiates whereas the submucosa and outer layers of connective tissue show continued expression at late gestation (Fig. 1H,I). Pbx1b displays striking expression patterns in several epidermal organs. At E12.0, intense staining is detected in the dental lamina during the early stages of odontogenesis (Fig. 1J). As the tooth buds form, Pbx1b expression is evident in the dental lamina as they grow into the underlying
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00516-5
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C.A. Schnabel et al. / Mechanisms of Development 100 (2001) 131±135
C.A. Schnabel et al. / Mechanisms of Development 100 (2001) 131±135
Fig. 2. Pbx1b expression overlaps with regions of cellular proliferation. Comparative immunostaining of Pbx1b (A,C,E,G) and BrdU (B,D,F,H) in adjacent tissue sections of E12.0 embryos. Colocalization is seen in vertebral elements in a sagittal view (A,B, arrows) and in transverse sections of the developing neural retina (C,D, arrows), lung (E,F, arrows) and diencephalon (G,H, arrows). Arrowheads in A,C,E,G indicate regions where Pbx1b expression does not overlap with BrdU-positive areas. Scale bars: 0.2 mm.
ectomesenchyme (Fig. 1K). By E16.5, Pbx1b expression persists in the oral epithelium and in the outer epithelium of the growing tooth, but is undetectable as the cells stop dividing to form the enamel knot (Fig. 1L). Distinct patterns of Pbx1b expression are also present during the develop-
133
ment of the facial integument. Before the onset of hair follicle development, Pbx1b is strongly expressed in the basal layer of the epidermis (Fig. 1M). More notably, when primary follicles are initiated, a focal aggregation of Pbx1b-positive mesenchymal cells can be seen in the dermis and in the invaginating epithelium as components of the epidermal placode (Fig. 1M). In transverse sections at E14.5, Pbx1b immunoreactivity is seen in concentric layers comprised of mesenchymal and sheath cells that surround the developing follicle (Fig. 1N). At more advanced stages of folliculogenesis, Pbx1b expression continues in mesenchymal components around the hair shaft and in the dermal papilla, and is also apparent in the root sheath (Fig. 1O). Pbx1b is present in structures derived from the intermediate mesoderm and throughout various stages of urogenital system ontogeny. In sections through mid-stage embryos, high levels of Pbx1b are visible in the mesenchyme associated with the mesonephric tubules and the genital ridge (Fig. 1P,Q). At E14.5 in both male and female embryos, Pbx1b epithelial expression distinguishes the MuÈllerian duct, whereas surrounding mesenchyme of the descending WoÈlf®an and MuÈllerian ducts exhibits strong Pbx1b staining (Fig. 1Q and data not shown). Early in metanephric development, Pbx1b expression is detected in the blastema as it is induced by the ureter (data not shown). At later stages, Pbx1b is absent in ureter-derived structures, but pervades throughout the nephrogenic mesenchyme and stroma, and is detected in glomerular cells (Fig. 1R). Since previous work implicates a neoplastic role for Pbx proteins in altered cellular proliferation (Krosl et al., 1998), further analysis of Pbx1b expression was performed in conjunction with BrdU labeling studies to examine its localization in relation to proliferating cells. As shown in Fig. 2, subregions of Pbx1b immunoreactivity colocalize with that of BrdU in a variety of tissues. In adjacent sections through E12.0 embryos, Pbx1b staining is associated with skeletal bodies and the primordial neural retina in areas where cells are actively cycling (Fig. 2, compare A and B, C and D). During lung formation, discrete regions of proliferation are apparent at the distal tips of the mesenchyme that overlap
Fig. 1. Pbx1b expression during mouse embryogenesis. (B±R) Immunohistochemical analysis of Pbx1b expression performed on tissue sections from different days of gestation showing transverse or sagittal ®elds as schematized in (A). (B,C) Pbx1b expression at E10.5 is present in the surface ectoderm (arrowhead) and mesenchyme (arrow) of the branchial arches (B) and in the dermamyotome (arrowhead) and the sclerotome (arrows) of the somite (C). (D) Pbx1b is expressed in both the endodermal (arrowhead) and mesenchymal (arrow) components of the fore- and midgut of an E9.5 embryo. At E11.5 (E), Pbx1b expression is detected in the mesenchyme (arrow) but not the epithelium (arrowhead) of the lung anlage, and persists in the interstitial mesenchyme at E16.5 (F). (G±I) Pbx1b expression is found in the mesenchyme and the endoderm of the stomach at E11.5 (G) and E13.5 (H) but is restricted to the submucosal tissues at E16.5 (I). Pbx1b is detected in the oral epithelium (arrows) during tooth morphogenesis at E12.0 (J), E13.5 (K) and E16.5 (L). Note additional expression of Pbx1b in the mesenchyme of the palatal shelves and tongue (arrowheads in K). (M) Pbx1b expression in the forming whiskers is seen in the epidermis (arrowheads) and in the underlying mesenchyme (arrow) at E13.0. Later stages of vibrissae folliculogenesis at E14.5 (N) and 16.5 (O) show Pbx1b expression in the root sheath (arrowheads), surrounding mesenchyme (arrow in N) and the dermal papillae (arrow in O). Panels (P±R) display Pbx1b mesenchymal expression in the developing urogenital system. (P) Pbx1b is visible in mesonephric mesenchyme (arrow) and in the peripheral mesenchyme and coelemic epithelium of the genital ridge (arrowhead). (Q) At E14.5, Pbx1b is seen in the surrounding mesenchyme of the both the MuÈllerian (arrowhead) and WoÈlf®an (arrow) ducts, and in the interstitial cells (concave arrowheads) surrounding the testicular cords. Note Pbx1b expression in the MuÈllerian duct. (R) Expression of Pbx1b in the metanephric kidney at E16.5 is evident in the nephrogenic mesenchyme (arrowheads) and in glomerular cells (arrows). Scale bars: (D,H,R), 0.05 mm; (B,C,E±G,I±Q), 0.1 mm.
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with the uniform expression pattern of Pbx1b (Fig. 2, compare E and F). In neural tissues, Pbx1b-positive domains are evident which extend through a region of proliferation in the developing diencephalon (Fig. 2, compare G and H). Thus, Pbx1b expression at mid-gestation partially overlaps with proliferative zones in various tissues. However, although the presence of Pbx1b in cycling cells is apparent, these data clearly reveal that its expression domains also include areas that are BrdU-negative. For instance, Pbx1b expression is detected in mesenchymal regions, surrounding the forming vertebrae and in the central portion of the budding lung, which do not exhibit BrdU immunoreactivity (Fig. 1, compare A and B, E and F). In other tissues, Pbx1b expression is evident in the transitional zone of the neuroretina and along the apical surface of the neuroepithelium where BrdU staining is undetected (Fig. 2, compare C and D, G and H). Therefore, it is likely that Pbx1b participates in diverse regulatory pathways, some of which may play a role in cellular proliferation and tissue expansion. Insight into the potential role of Pbx genes in developmental processes has been provided by studies with its Drosophila homolog, extradenticle (exd), which demonstrate a function in parallel with members of the Hox gene family to regulate axial patterning events (Rauskolb et al., 1993). Consistent with the notion that Pbx and Hox homeoproteins function cooperatively to regulate speci®c target genes (Mann and Affolter, 1998), expression of Pbx1b is detected in many tissues previously reported to express Hox genes during mouse embryogenesis (reviewed in Krumlauf, 1994). Early in development, overlapping regions of expression include segmented structures such as the branchial arches and somites, and later during organogenesis, tissues such as the lung, stomach and skeletal elements (Krumlauf, 1994; Yokouchi et al., 1995; Volpe et al., 1997). In anterior structures, however, Pbx1b is expressed in tissues where Hox gene expression has not been reported, such as the eyes and teeth, which suggest its participation in Hox-independent developmental pathways. Whereas Pbx1b expression early in development suggests its involvement in embryonic patterning and regionalization, its expression during organogenesis indicates a potential function in cell fate determination in a variety of tissues that depend on mesenchymal±epithelial interactions for their coordinated morphogenesis (Sharpe and Ferguson, 1988; Birchmeier and Birchmeier, 1993; Yasugi, 1993; Thesleff et al., 1995; Dassule and McMahon, 1998; Oro and Scott, 1998; Hogan, 1999; Roberts et al., 1999; Warburton et al., 2000). In general, Pbx1b expression is not strictly associated with either the mesenchymal or epithelial compartments, but varies in regards to tissue type. Pbx1b is selectively expressed within the mesenchyme (lung, urogenital system) or epithelia (teeth) in certain regions and is detected in both layers in others (stomach, whiskers). More interestingly, Pbx1b localizes to these apposed layers during critical periods of organogenesis. In summary, these
data suggest that Pbx1b serves multiple functions during embryogenesis, and implicate its participation in the regulation of cellular differentiation and tissue expansion. 2. Materials and methods 2.1. Tissue preparation and Immunohistochemistry C57BL/6 embryos were collected from timed-pregnancies at the days indicated. Dissected embryos were ®xed in 10% buffered formalin for 6 h to overnight at 48C and dehydrated through increasing concentrations of ethanol prior to embedding in paraf®n wax. Tissue sections (4 mm) were de-paraf®nized and treated for epitope unmasking in 500 mM Tris (pH 10) by microwave heating before incubation with anti-Pbx1b monoclonal antibody. Monoclonal antibody production and demonstration of antibody speci®city have been described previously (Chang et al., 1997). After washing in PBS, sections were incubated with biotinconjugated goat anti-mouse IgG and horseradish peroxidase-conjugated streptavidin (Jackson Immunoresearch) and detected with DAB (Sigma). Tissues were counterstained with hematoxylin and slides were mounted with Clearmount media (American Master Tech Scienti®c Inc.). 2.2. BrdU labeling Pregnant C57BL/6 mice were injected intravenously with bromodeoxyuridine (BrdU) (Sigma) at 50 mg/g of body weight 3 h prior to sacri®ce. E12.0 embryos were processed and immunostained as above using an anti-BrdU monoclonal antibody (Neomarkers) on adjacent sections as those stained with anti-Pbx1b. Detection was performed with the Vector Nova-Red substrate kit (Vector Laboratories) according to manufacturer's instructions. Acknowledgements We gratefully acknowledge Eva Pfendt for her expertise in immunohistochemistry, Bich-Tien Rouse for antibody production, Carmencita Nicolas and Maria Ambrus for technical assistance, Phil Verzola for photographic help, and Greg Barsh and Anthony Oro for critical reading of the manuscript. This work was supported by grants from the NIH (CA70404, CA42971 and CA34233). C.A.S. is a fellow of the Leukemia and Lymphoma Society. References Asahara, H., Dutta, S., Kao, H.Y., Evans, R.M., Montminy, M., 1999. Pbx± Hox heterodimers recruit coactivator-corepressor complexes in an isoform-speci®c manner. Mol. Cell. Biol. 19, 8219±8225. Birchmeier, C., Birchmeier, W., 1993. Molecular aspects of mesenchymal± epithelial interactions. Annu. Rev. Cell. Biol. 9, 511±540. Chang, C.P., Jacobs, Y., Nakamura, T., Jenkins, N.A., Copeland, N.G., Cleary, M.L., 1997. Meis proteins are major in vivo DNA binding
C.A. Schnabel et al. / Mechanisms of Development 100 (2001) 131±135 partners for wild type but not chimeric Pbx proteins. Mol. Cell. Biol. 17, 5679±5687. Dassule, H., McMahon, A.P., 1998. Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the developing tooth. Dev. Biol. 202, 215±227. DiRocco, G., Mavilio, F., Zappavigna, V., 1997. Functional dissection of a transcriptionally active, target-speci®c Hox-Pbx complex. EMBO J. 16, 3644±3654. Hogan, B.L.M., 1999. Morphogenesis. Cell 96, 225±233. Krosl, J., Baban, S., Krosl, G., Rosenfeld, S., Largman, C., Sauvageau, G., 1998. Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with Pbx1. Oncogene 16, 3403± 3412. Krumlauf, R., 1994. Hox genes in vertebrate development. Cell 78, 191± 201. Mann, R.S., Affolter, M., 1998. Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423±429. Oro, A.E., Scott, M.P., 1998. Splitting hairs: dissecting roles of signaling systems in epidermal development. Cell 95, 575±578. Rauskolb, C., Peiper, M., Wieschaus, E., 1993. Extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene, pbx1. Cell 74, 1101±1112.
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Roberts, L.M., Hirokawa, Y., Nachtigal, M.W., Ingraham, H.A., 1999. Paracrine-mediated apoptosis in reproductive tract development. Dev. Biol. 208, 110±122. Sharpe, P.M., Ferguson, M.W., 1988. Mesenchymal in¯uences on epithelial differentiation in developing systems. J. Cell Sci. Suppl. 10, 195±203. Thesleff, I., Vaahtokari, A., Partanen, A.-M., 1995. Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int. J. Dev. Biol. 39, 35±50. Volpe, M.V., Martin, A., Vosatka, R.J., Mazzoni, C.L., Nielsen, H.C., 1997. Hox b-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate. Histochem. Cell. Biol. 108, 495±504. Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K.D., Cardoso, W., 2000. The molecular basis of lung development. Mech. Dev. 92, 55±81. Yasugi, S., 1993. Role of epithelial-mesenchymal interactions in the differentiation of epithelium of vertebrate digestive organs. Dev. Growth Differ. 35, 1±9. Yokouchi, Y., Sakiyama, J., Kuroiwa, A., 1995. Coordinated expression of Abd-B subfamily genes of the HoxA cluster in the developing digestive tract of chick embryo. Dev. Biol. 169, 76±89.