RESEARCH REPORT 4171
Development 137, 4171-4176 (2010) doi:10.1242/dev.056077 © 2010. Published by The Company of Biologists Ltd BMP signaling in the development of the mouse esophagus and forestomach Pavel Rodriguez1, Susana Da Silva1, Leif Oxburgh2, Fan Wang1, Brigid L. M. Hogan1 and Jianwen Que1,*,†
SUMMARY The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26CAG-loxpstoploxp-caBmprIa) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1aflox/flox embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach.
KEY WORDS: BMP signaling, Esophagus, Forestomach, Stratification, Differentiation, Mouse
INTRODUCTION Morrisey and Hogan, 2010; Roberts et al., 1998). Analysis of Nog- Similar to the stratified squamous epidermis, the epithelium of null mutants has shown that BMP signaling regulates the initial foregut-derived tissues, including the esophagus, arises from a dorsal-ventral patterning of the single foregut tube and the separation layer of simple columnar cells that stratify and differentiate. In the into dorsal esophagus and ventral trachea (Li et al., 2007; Que et al., epidermis, these processes are associated with multiple signaling 2006). Here, we show that Nog also regulates the transition from pathways, including those driven by bone morphogenetic protein simple columnar to stratified squamous epithelium in the developing (BMP) and Wnt (Botchkarev, 2003; Botchkarev and Sharov, 2004). esophagus. Combining gain- and loss-of-function studies, we further However, it remains unknown whether these pathways are also show that BMP signaling plays multiple roles in the development of active in the embryonic foregut during development and in the the esophagus and forestomach, the epithelium of which appears steady state. histologically as a continuation of the esophagus. The canonical BMP signaling pathway is activated by a secreted ligand (e.g. Bmp4, Bmp7) interacting with a complex of type I and MATERIALS AND METHODS type II transmembrane receptors. This initiates signal transduction Mouse strains by phosphorylation of Smads 1/5/8 (R-Smads), which interact with Heterozygous BRE-lacZ (Blank et al., 2008), NoglacZ/+ (Brunet et al., 1998) a co-Smad (Smad4) to regulate downstream target genes (Miyazono and Bmp7lacZ/+ (Godin et al., 1998) reporter mice were maintained on the et al., 2009). BMP signaling is modulated by extracellular C57Bl/6 ϫ 129/SvEv background. To conditionally activate BMP signaling in foregut endoderm, Shh-GFP-Cre (Shh-Cre) mice (Harfe et al., antagonists, including noggin (Nog), chordin (Chrd) and follistatin CAG-loxpstoploxp-caBmpr1a/+ CA/+ (Fst), that interfere with productive binding to receptors (Massague 2004) were crossed with Rosa26 (Bmpr1a ) mice. In this line, a constitutively active Bmpr1a (caBmpr1a) was inserted into et al., 2005). In the epidermis, conditional deletion of Bmpr1a leads the Rosa26 locus preceded by a CMV early enhancer/chicken -actin to increased epithelial proliferation (Kobielak et al., 2003; Qiao et (CAG) promoter and a floxed neo cassette (see Fig. S2A in the al., 2006; Yuhki et al., 2004). Deletion of Smad4 in the epithelium supplementary material). The caBmpr1a construct has a single amino acid also results in basal cell hyperproliferation and leads to squamous substitution (Q to D) within the glycine-serine (GS) domain, giving the cell cancer (Qiao et al., 2006). protein strong kinase activity even in the absence of ligand or type II The mouse esophagus separates from the foregut tube between receptor (Zou et al., 1997). To conditionally delete Bmpr1a, Shh- embryonic day (E) 9.5 and E11.5. This separation process is Cre;Bmpr1aflox/flox or Shh-Cre;Bmpr1aflox/null compound mutants were regulated by multiple signaling pathways, including Wnt, BMP and generated (Yuhki et al., 2004). Shh, which may act upstream of Bmp4 (Litingtung et al., 1998; In situ hybridization In situ hybridization was performed as previously described (Que et al., 2006; Que et al., 2007). Digoxigenin (DIG)-labeled antisense cRNA probes 1Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, against mouse Bmpr1a, Bmp7, Nog, Fst, Fstl1, Chrd, Grem1 and Cer1 2 USA. Department of Molecular Medicine, Maine Medical Center Research Institute, were synthesized using T3, T7 or SP6 RNA polymerases. Grem1 was a 81 Research Drive, Scarborough, ME 04074, USA. kind gift from Dr Clifford Tabin (Harvard University). *Present address: Department of Biomedical Genetics, University of Rochester, Rochester, NY 14642, USA Immunohistochemistry, immunostaining and X-gal staining †Author for correspondence ([email protected]) Tissues were fixed in 4% paraformaldehyde for 3-4 hours at 4°C and embedded in paraffin for sectioning. Cryosections were also used for
Accepted 6 October 2010 immunostaining with phospho (p) Smad1/5/8 antibody (1:1000; kind gift DEVELOPMENT 4172 RESEARCH REPORT Development 137 (24) from Dr Edward Laufer, Columbia University). The following primary antibodies were used on paraffin sections: mouse anti-p63 (1:200; Santa Cruz Biotechnology); mouse anti-Krt14 (1:200; Thermo Scientific); rat anti-Krt8 (1:100; DSHB); rat anti-phospho-histone H3 (1:1000, Upstate Biotechnology); rabbit anti-loricrin (1:100) and anti-involucrin (1:10,000) (kind gifts from Dr Terry Lechler, Duke University); and rabbit anti-Krt5 (1:100; Covance). Whole embryos, isolated esophagi and stomachs were stained with X-gal and processed as previously described (Que et al., 2006).
RESULTS AND DISCUSSION Dynamic BMP signaling in the developing mouse esophagus and forestomach The epithelium of the embryonic esophagus and forestomach starts to stratify at early E11.5. At E10.5, the esophagus is lined by keratin 8 (Krt8)-positive simple columnar epithelial cells, a subset of which express p63 (Trp63 – Mouse Genome Informatics) (Fig. 1A,B), a transcription factor that is required for epithelial stratification (Daniely et al., 2004). By E11.5, the epithelium is 1- 2 cells thick (Yu et al., 2005), with all cells expressing p63 and Krt8, but not Krt5 and Krt14, which only starts to be expressed from E14.5 (Fig. 1C,D; data not shown). Similarly, the epithelium in the forestomach starts to stratify between E10.5 and E11.5 (Fig. 1E-G). To investigate canonical BMP pathway activation in the developing esophagus and forestomach, we used the BRE-lacZ transgenic reporter line, which expresses -galactosidase (-gal) under the control of BMP response elements (BREs) from Id1 (Blank et al., 2008). At E9.5, -gal-positive cells were observed in the epithelium and mesenchyme of the ventral foregut (see Fig. S1A in the supplementary material), which generates the trachea at E11.5 (see Fig. S1B in the supplementary material). Although the simple columnar epithelium of the hindstomach and mesenchyme showed active BMP signaling at E13.5 (Fig. 1H), the epithelium of the esophagus and forestomach remained negative (Fig. 1H-L). By E14.5, -gal activity was seen in a limited number of suprabasal cells in both the esophagus and forestomach, and this increased by E15.5 (see Fig. S1C-H in the supplementary material). At postnatal Fig. 1. BMP signaling in mouse foregut development. day (P) 0, all suprabasal cells exhibited -gal activity (Fig. 1M-R) (A-G)Transverse sections of E10.5-11.5 esophagus (A-D) and and ~75% of these cells were also positive for pSmad1/5/8. longitudinal sections of E10.5-11.5 forestomach (E-G) stained with Interestingly, ~5% of basal cells were positive for both activities Hematoxylin and Eosin (H&E) (A,C,E,F) or antibodies against p63 and (Fig. 1O,R). Krt8 (B,D,G). (H-L)Longitudinal sections of E13.5 BRE-lacZ esophagus and forestomach. X-gal staining is present in the blood vessels Expression of BMP ligands and inhibitors in the (arrowheads in H) and hindstomach epithelium (arrow in H), developing esophagus and forestomach esophageal (I,J) and forestomach (K,L) mesenchyme. (M-R)At P0, BRE- In situ hybridization showed that between E9.5 and E15.5, Bmpr1a lacZ is active mostly in the suprabasal layers of the esophagus and is ubiquitously expressed, whereas Bmp7 transcripts are enriched in forestomach, whereas p63 is only found in the basal layer of the epithelium. BRE-lacZ reporter activity correlates with pSmad1/5/8 the esophageal and forestomach epithelium (Fig. 1S-X; data nuclear localization (O,R). The inset in Q shows that p63-positive basal not shown). The expression of Bmp7 in the epithelium of the cells are also positive for X-gal staining (arrowheads), and this correlates esophagus and forestomach was confirmed by analysis of the with pSmad staining in a subset of basal cells (arrows in R). (S-V)In situ Bmp7-lacZ ‘knock-in’ allele (see Fig. S1I,J in the supplementary hybridization shows that Bmp7 is present in the dorsal epithelium of material). At E15.5, low levels of Bmp6 were detected in the the E9.5 (S, transverse section) and E10.5 (T) foregut, E12.5 esophageal differentiating suprabasal cells (see Fig. S1K,L in the supplementary (U) and forestomach (V) epithelium. (W,X)Bmpr1a is ubiquitously material). expressed at E12.5 in the esophagus and forestomach. Nuclei in Although the epithelial cells express Bmp7 and Bmpr1a from at H,I,K,M,P,S-X are counterstained with Nuclear Fast Red. Dotted lines least E10.5 to E13.5, BRE-lacZ-positive cells were only observed indicate the basal membrane. ep, epithelium; me, mesenchyme; from E14.5. We hypothesized that the absence of BMP signaling fs, forestomach; hs, hindstomach; fg, foregut; d, dorsal; v, ventral; es, esophagus; tr, trachea. Scale bars: 25m in A-G; 50m in H-X. in the epithelium is due to extracellular BMP inhibitors that block ligand-receptor interaction. Nog is expressed in the dorsal E9.5 foregut (Li et al., 2007; Que et al., 2006). This expression pattern cells in the esophagus and in the dorsal forestomach (Fig. 2A-C). was maintained as the separation proceeds at ~E10.5-11.5, and Although at E13.5 Nog-lacZ expression was very low in the
Nog-lacZ expression was restricted to a subpopulation of epithelial esophageal epithelium, Nog transcripts could still be detected by in DEVELOPMENT BMP in esophagus/forestomach development RESEARCH REPORT 4173
of the E12.5 forestomach at a moderate level, which contrasts with the very low levels found in the esophagus (see Fig. S1O,P in the supplementary material).
Ectopic activation of BMP signaling in the epithelium Previously, we reported esophageal atresia and tracheoesophageal fistula (EA/TEF) in nearly 60% of Nog-null mutants (Que et al., 2006). In the remaining 40% of mutants, the foregut separates normally. However, in the E18.5 dorsal esophagus we found multiple abnormal gland-like pits lined with simple columnar epithelium (Fig. 2L-QЈ; n12). The epithelial cells lining the pits were positive for Alcian Blue staining, which is indicative of mucus production (Fig. 2M,MЈ), and the number of p63-positive cells was reduced compared with the wild type (Fig. 2P-QЈ). No expression of the tracheal transcription factor Nkx2.1 or the Clara cell marker Scgb1a1 was detected (data not shown), excluding the possibility that the cells have acquired a respiratory identity. It is noteworthy that there were no abnormalities in the forestomach of Nog-null mutants at any stage. Here, other BMP inhibitors in the forestomach, such as Fst, might play a compensatory role in the event of Nog deletion (see Fig. S1P in the supplementary material). Our results suggest that the formation of a multilayered epithelium from a simple columnar epithelium between E10.5 and E14.5 is inhibited by the loss of Nog and by the resultant increase in canonical BMP signaling. To further explore this idea, we used a combination of a new conditional Rosa26 allele that incorporates constitutively active Bmpr1a (Bmpr1aCA) (see Fig. S2A in the supplementary material) and a Shh-Cre allele that is preferentially expressed in the ventral esophagus and forestomach between E10.5 and E11.5 (Harris-Johnson et al., 2009) (Fig. 3A). By E13.5, most of the epithelial cells in these tissues of Shh-Cre;R26R embryos were labeled by X-gal staining, suggesting that the recombination Fig. 2. Expression of BMP inhibitors in the developing esophagus later extends dorsally (Fig. 3B). All of the Shh-Cre;Bmpr1aCA/+ and forestomach. (A-C)Transverse sections of X-gal-stained E10.5 and mutants died before P5, probably owing to respiratory distress (J.Q. E11.5 Nog-lacZ mouse embryos show that Nog is expressed in the and B.L.M.H., unpublished). The activation of BMP signaling was epithelium of the dorsal esophagus and forestomach (arrowhead). confirmed by pSmad1/5/8 staining, which showed nuclear signals (D)In situ hybridization shows that the Nog transcript is present in the in most epithelial cells of the E15.5 mutant esophagus and epithelium (arrow) and mesenchyme (arrowhead) of the E13.5 forestomach, as compared with the control (see Fig. S2B-E in the esophagus. (E)At E14.5, Nog-lacZ is active only in the muscle of the esophagus. (F-K) In situ hybridization of transverse sections of E10.0- supplementary material). CA/+ 11.5 embryonic foregut shows that Fst (F,G), Chrd (H,I) and Cer1 (J,K) Examination of the E15.5 Shh-Cre;Bmpr1a mutant revealed are transcribed in the foregut epithelium. (L-QЈ) Longitudinal sections clusters of simple columnar epithelial cells along the esophagus, of the E18.5 Nog+/– and Nog–/– esophagus stained with Alcian Blue preferentially in the ventral region (Fig. 3C-J; n9). These simple (L-MЈ), H&E (N-OЈ) and antibody to p63 (P-QЈ). The Nog–/– esophagus epithelial cells maintained a high level of Krt8, whereas the number shows multiple gland-like pits that are present dorsally and contain of p63-positive cells was apparently reduced (Fig. 3F). Moreover, simple columnar epithelium, which stain positive for Alcian Blue (M, the simple columnar epithelium was negative for Krt5 and Krt14, arrowheads). In these pits, the number of p63-positive cells is reduced. two cytokeratins that are characteristic of basal cells of lu, lung; st, stomach. Scale bars: 50m. stratified/multilayered epithelia (Fig. 3J). In the mutant forestomach, scattered regions of simple columnar (Krt8-positive, Krt5/14-negative, p63 low) epithelium were also observed at E15.5 situ hybridization (Fig. 2D). However, at E14.5, Nog-lacZ was only and P0 (Fig. 3K-R and see Fig. S2F,G in the supplementary detected in the inner circular muscle layer of the esophagus (Fig. material; n24). At E15.5, phospho-histone H3 staining revealed 2E). We also examined transcripts of other putative BMP inhibitors no significant differences in cell proliferation between the simple including Fst, follistatin-like 1 (Fstl1), Chrd, gremlin 1 (Grem1) columnar and stratified epithelium in the mutants and control (data and cerberus (Cer1) (reviewed by Umulis et al., 2009). Between not shown). E10.5 and E11.5, Fst, Chrd, Cer1 and Fstl1 were expressed in the The simple columnar epithelium was present only on the ventral epithelium, whereas Grem1 was found only in the mesenchyme side of the esophagus of Shh-Cre;Bmpr1aCA/+mutants, which (Fig. 2F-K; data not shown). After E12.5, only Cer1 expression correlated with the ventral activation of Shh-Cre before E12.5 (Fig. was maintained at a high level in the epithelium, whereas Chrd, 3A). At E13.5, even though Shh-Cre was expressed on the dorsal Grem1 and Fstl1 were expressed in the mesenchyme (see Fig. side of the esophagus (Fig. 3B), the resultant ectopic BMP S1M-R in the supplementary material; data not shown). signaling was incapable of reversing the stratified epithelium back
Interestingly, Fst was expressed in the epithelium and mesenchyme to a monolayer (Fig. 3D,H), suggesting that there is a limited time DEVELOPMENT 4174 RESEARCH REPORT Development 137 (24)
Fig. 3. Activation of BMP signaling in the Shh-Cre;Bmpr1aCA/+ mutant inhibits stratification of the esophageal and forestomach epithelium. (A,B)Longitudinal sections of X-gal-stained E11.5 and E13.5 Shh- Cre;R26R mouse esophagus and stomach. At E11.5, Cre activity is in the ventral esophageal epithelium (A), with non-uniform expression in the forestomach epithelium (arrowheads). By E13.5, the recombination pattern is more uniform throughout the esophagus and forestomach (B). (C,D)Transverse sections of E15.5 control and Shh-Cre;Bmpr1aCA/+ mutant esophagus stained with H&E. Note that the mutant esophagus contains epithelium that is non-stratified ventrally (arrowheads), but stratified dorsally (arrows). (E,F)The ventral columnar epithelium in the Shh- Cre;Bmpr1aCA/+ mutant has few p63-positive cells, whereas most are positive for Krt8. (G,H)Longitudinal sections of E15.5 mutant esophagus show non-stratified epithelium ventrally (arrowheads) and stratified epithelium dorsally. (I,J)The ventral columnar epithelium is negative for Krt5 and Krt14 (arrowheads), whereas the stratified dorsal epithelium is positive for both. (K-N)H&E staining shows that simple columnar epithelium is present (arrowheads) in the Shh-Cre;Bmpr1aCA/+ mutant but not in the control. M and N show the boxed regions in K and L, respectively, at high magnification. (O-R)The corresponding simple columnar epithelium present in the mutant (arrowheads) has few or no cells that are positive for p63, Krt5 and Krt14. Scale bars: 50m. period during which the fate of the epithelium can be influenced to a more obvious defect in the differentiation of suprabasal cells by ectopic signals. Similar observations have been reported in the (Fig. 4E-L). The affected regions remained p63 positive, but lacked chick gizzard, where ectopic Bmp2 can only switch the luminal to involucrin and loricrin (Fig. 4I-L). In addition, some of these p63- glandular epithelium within a certain time window (Yasugi and positive cells remained proliferative, as indicated by phospho- Mizuno, 2008). histone H3 staining (see Fig. S2L,M in the supplementary material). These results suggest that BMP signaling is required for Conditional deletion of Bmpr1a in the the differentiation of the suprabasal cells in the esophagus and forestomach epithelium forestomach. Interestingly, other studies have shown that the After E14.5 the BMP pathway is active in the differentiating inhibition of BMP signaling by overexpression of Nog under a Krt5 suprabasal cells of the esophagus and forestomach (see Fig. S1C- promoter in the epidermis also results in alterations in eyelid H in the supplementary material). To address whether BMP epithelial differentiation, and the levels of involucrin and loricrin signaling is necessary for the differentiation of the epithelium, we are reduced (Sharov et al., 2003). Conversely, the addition of Bmp4 deleted Bmpr1a by generating Shh-Cre;Bmpr1aflox/flox and Shh- to cultured human oral epithelium increases the expression of Cre;Bmpr1aflox/null mutants. Since similar phenotypes were involucrin (Kim et al., 2006). These studies suggest that BMP observed in the Shh-Cre;Bmpr1aflox/flox and Shh-Cre;Bmpr1aflox/null signaling regulates the differentiation of suprabasal cells in mutants, subsequent analyses employed the former. Most mutants stratified epithelium. died in utero or at birth, presumably owing to heart defects, but a In conclusion, our results suggest that BMP signaling has two few did survive to P9 (n3). The loss of Bmpr1a transcripts in the distinct functions in the development of the mouse esophagus and mutant esophagus and forestomach at E15.5 was confirmed by in forestomach (Fig. 4M). Initially, between E10.5 and E14.5, the situ hybridization (see Fig. S2H-K in the supplementary material). inhibition of BMP signaling by Nog is necessary for epithelial In the esophageal epithelium of the E18.5 and surviving P9 stratification to occur. The deletion of Nog or expression of mutants, expression of p63 persisted in the upper layers (Fig. 4A,B; constitutively active BMP receptor maintains a simple columnar data not shown), suggesting that these cells are not fully epithelium. Then, starting at ~E14.5, BMP signaling is required for differentiated. There was no involucrin or loricrin immunostaining the differentiation of the suprabasal cells. In the absence of in the mutant epithelium, but even the wild-type epithelium Bmpr1a, the epithelial stratification does occur but the suprabasal expressed low levels of these proteins at this stage (Fig. 4C,D; data cells do not fully differentiate. In this study, we have only
not shown). By contrast, the loss of Bmpr1a in the forestomach led investigated the role of BMP signaling in the embryonic and early DEVELOPMENT BMP in esophagus/forestomach development RESEARCH REPORT 4175
Milano et al., 2007; Wang et al., 2010). In the future, it will be important to determine the role of the BMP signaling pathway in the steady-state maintenance of the normal esophageal epithelium and in various injury and repair models.
Acknowledgements We thank members of the B.L.M.H. laboratory and Sarah Fausett, Chandra Davenport, Drs John Klingensmith and Terry Lechler of Duke University Medical Center for critical reading and helpful suggestions. We are also grateful to Drs Dan Vasiliauskas, Susan Morton, Tom Jessell and Edward Laufer for the phospho-Smad1/5/8 antibody. This work was supported by NIH K99/R00 (project number 1K99DK082650-01A2 to J.Q.) and a Howard Hughes Medical Institute Medical Research Training Fellowship (to P.R.). Deposited in PMC for release after 6 months.
Competing interests statement The authors declare no competing financial interests.
Supplementary material Supplementary material for this article is available at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.056077/-/DC1
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