A retinoic acid–dependent network in the foregut controls formation of the mouse lung primordium (original) (raw)
Dkk1 is aberrantly expressed in RA-deficient foreguts. In a previous effort to investigate the molecular mechanisms of lung agenesis, we analyzed global transcriptional profiles of mouse embryonic foreguts from a pharmacological and a genetic model of RA deficiency. RA-deficient specimens consisted of E8.5 WT foreguts cultured for 24 hours with the RA receptor antagonist BMS493 (referred to herein as BMS) or E8.5 _Raldh2_-null foreguts cultured in control medium; both showed lung agenesis. RA-sufficient specimens were either control WT or _Raldh2_-null foreguts in which lung formation was rescued by exogenous RA (7–9, 11). Among the genes differentially expressed, we found that Dkk1, an endogenous Wnt signaling antagonist, was significantly upregulated in RA-deficient foreguts from both models (BMS model, 3.5-fold, P = 1.46 × 10–9; _Raldh2_-null model, 2.8-fold, P = 1.95 × 10–8). To confirm this observation, we assessed Dkk1 expression by real-time PCR in RA-deficient and RA-sufficient foreguts and found a highly statistically significant increase in the levels of Dkk1 mRNA in BMS-treated versus untreated control foreguts (P = 0.002; Figure 1A). Consistent with this, we also observed a dramatic increase in levels of Dkk1 in _Raldh2_-null foreguts compared with the respective null foreguts rescued by exogenous RA (P = 0.0005).
Dkk1 is a target of RA at the onset of lung morphogenesis. (A) Real-time PCR. Dkk1 upregulation in RA-deficient foreguts (BMS-treated WT and unrescued Raldh2–/–) compared with respective controls (Ctr). (B and C) Dkk1 ISH in WT and Raldh2–/– embryos (12S, top) coimmunostained with Sox2 (bottom), depicting (B) no Dkk1 signals in the foregut (Fg, boxed regions) of WT embryo and (C) strong Dkk1 (blue arrowheads) in both endoderm (red arrowheads, Sox2-labeled brown nuclei) and mesoderm of Raldh2–/– embryo. Original magnification, ×10 (top); ×100 (bottom). (D–I) Reciprocal pattern of Dkk1 expression (D–F, WMISH) and RARElacZ activity (G–I) in the foregut, head, and tail (arrows) in RA-sufficient (D and G, 16S–18S; E and H, 11S–12S), and RA-deficient (F and I, 11S–12S) embryos. (J–M) WMISH of Dkk1 in 24-hour cultured foreguts revealed increased signals in RA-deficient conditions in which the lung failed to form (K and M, asterisks). Below, diagrams depict morphology of each condition after 72 hours. Ht, heart; Lu, lung; St, stomach; Th, thyroid. Scale bar: 450 μm (D–I); 300 μm (J–M).
To learn about the distribution of Dkk1 transcripts in vivo, we performed in situ hybridization (ISH) analysis in embryos prior to and at the onset of lung development. Analysis of E8.5–E9.5 WT embryos revealed Dkk1 signals at restricted sites in the anterior foregut, such as the branchial arches (data not shown), but not in the prospective lung field (Figure 1B; 12 somite-stage [12S]), consistent with previous reports (12, 13). In contrast, Dkk1 expression was strong throughout the foregut of Raldh2–/– mutants at a similar stage (compare boxed regions in Figure 1, B and C). Double ISH (Dkk1) and immunohistochemistry (Sox2, which labels the foregut endoderm) showed that in the absence of RA signaling, Dkk1 was ectopically expressed in both mesodermal and endodermal layers (Figure 1, B and C, bottom).
To further investigate the relationship between Dkk1 and RA, we compared the patterns of Dkk1 expression by whole-mount ISH (WMISH) and X-gal staining of a RA reporter mouse in vivo under RA-sufficient (RARElacZ) and -deficient (Raldh2–/–RARElacZ) conditions. Interestingly, we found that overall Dkk1 and β-galactosidase were expressed in a reciprocal pattern in the E8.5–E9.5 embryo. For example, in the RA-sufficient embryos, Dkk1 transcripts were strong in the head and tail regions, where RARElacZ was inactive (Figure 1, D, E, G, and H). Conversely, Dkk1 expression was nearly absent in the trunk, including the midforegut region, which showed strong RARElacZ signals (Figure 1, D, E, G, and H, boxed regions). Remarkably, we found widespread increase in Dkk1 expression in Raldh2–/–RARElacZ embryos, an RA-deficient line that carries a copy of the RA reporter transgene, which confirmed the absence of RA activation in most structures (Figure 1, F and I).
Next, we used WMISH to determine how Dkk1 expression was altered in the foregut explants in which we modulated RA signaling in vitro. Figure 1, J–M, depicts Dkk1 expression under the different experimental conditions (top, 24 hours; bottom, corresponding morphological effects at 72 hours, as previously reported; refs. 8, 9, 11). Dkk1 signals were consistently stronger in both endodermal and mesodermal components of BMS-treated WT and Raldh2–/– foreguts than in their respective RA-sufficient counterparts. Dkk1 upregulation was not restricted to the presumptive lung field, but was consistently prominent at this site (Figure 1, J–M, boxed regions). The data strongly suggest that Dkk1 expression is regulated by endogenous RA during foregut organogenesis and that this regulation may be functionally relevant for the emerging lung primordium.
Dkk1 is a direct target of RA in lung mesenchymal cells. To gain insights into the mechanism by which RA influences Dkk1 expression, we investigated a potential direct effect of RA in gene transcription. Analysis of mouse Dkk1 genomic sequences revealed 1 RA-responsive element (RARE) 1,212 bp upstream of the translation start site. The Dkk1 genomic fragment was cloned into a luciferase reporter–containing plasmid and transfected into lung mesenchymal (MLg) cells. These cells are derived from neonatal murine lung; upon RA activation, they show features — such as upregulation of Tgfβ targets and Fgf10 downregulation — that mimic the RA responses of our foregut culture system (11, 14). Consistent with this, real-time PCR analysis of nontransfected MLg cells growing in control medium showed low levels of endogenous Dkk1; levels were further reduced by treatment with RA (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI40253DS1). RA treatment of MLg cells transfected with the WT _Dkk1_-promoter-luciferase construct significantly decreased luciferase activity compared with controls. The decrease was obliterated by mutating the RARE in the construct (Supplemental Figure 1, B and C). Thus, the data strongly suggest that RA directly represses transcription of Dkk1.
RA regulates Wnt signaling during the initiation of lung morphogenesis. The above findings prompted us to investigate whether the changes in Dkk1 expression result in changes in Wnt signaling in the foregut. To assess canonical Wnt activity in our system, we used BATgal mice, a well-characterized TCF binding element–based LacZ reporter model (15). Previous studies using BATgal or TOPgal, another canonical Wnt reporter line, showed reporter activity in the endoderm associated with the early lung (15–17). We did not detect BATgal activity in the foregut at E8.5, prior to the emergence of the lung buds (data not shown). However, signals were identified in both epithelial and mesenchymal layers of the trachea and lung primordium (Figure 2, A and B). BATgal activity was also present in the emerging lung buds from RA-sufficient cultured foreguts (Figure 2C).
RA regulation of Wnt during lung formation. (A and B) E10.0 BATgal lung (A, whole mount; B, section) displaying lacZ activity in both epithelium (Ep) and mesenchyme (Me; red arrow). (C–F) Control cultured foregut revealed BATgal activity in the lung and thyroid regions (C) that was suppressed in the lung field of BMS-treated BATgal (D) and Raldh2–/–BATgal (E) foreguts. This activity was preserved in the thyroid region (above yellow lines) of Raldh2–/–BATgal mice (D and E), a domain known to be devoid of RA activity, as seen in RARElacZ reporter mice (F). Li, liver; Pa, pancreas. Original magnification, ×40 (B); ×20 (C, right). (G) Real-time PCR of 24-hour cultured foreguts showing significant downregulation of Axin2 and Lef1 in RA-deficient conditions. *P < 0.05.
Next, we examined how disruption of RA influences Wnt signaling. To help monitor canonical Wnt activity, we generated Raldh2–/– mice carrying the BATgal transgene (Raldh2–/–BATgal mice). Interestingly, no LacZ signals were detected in the presumptive lung region of BMS-treated BATgal or Raldh2–/–BATgal foreguts. However, signals were readily identified in more anterior regions of these explants (Figure 2, D and E, area above the yellow line). This suggested selective regulation of Wnt activity by RA in the midforegut (Figure 2F), consistent with the domain of RA activity previously described in the foregut of RARElacZ mice.
Furthermore, we assessed expression of known targets of Wnt, such as Axin2 and Lef1 (18, 19), by real-time PCR and found significant downregulation of these genes in RA-deficient foreguts from both models (Figure 2G). We concluded that canonical Wnt is regulated by RA signaling at the initiation of lung morphogenesis.
Lung bud formation is abrogated by Wnt inhibition. To what extent does disruption of Wnt signaling contribute to the failure to form the lung primordium in RA-deficient foreguts? We reasoned that interfering primarily with Wnt signaling in RA-sufficient foreguts could reproduce the phenotype of RA-deficient foreguts. Two independent approaches were used to disrupt Wnt signaling. First, WT foreguts were treated with mouse recombinant Dkk1, which binds to the Wnt coreceptor Lrp5/6, making it unavailable to bind to Wnt (20). Alternatively, foreguts were treated with quercetin, a flavanoid known to inhibit the interaction between Tcf and β-catenin in the nucleus (21). Lung progenitors arise from the primitive foregut as a group of endodermal cells posterior to the thyroid that expresses NK2 homeobox 1 (Nkx2-1). Nkx2-1 is the earliest known marker of lung progenitor cell fate, although it is also expressed in the thyroid (22, 23). Figure 3, A and B, show a typical control foregut culture with emerging _Nkx2-1_–expressing lung buds.
Wnt disruption leads to lung bud agenesis in RA-sufficient foreguts. (A–F) Nkx2-1 WMISH (A, C, and E) and corresponding H&E (B, D, and F) of cultured foreguts. Control foreguts showed 2 emerging lung buds labeled by Nkx2-1 (A and B). In Dkk1-treated WT foreguts, Nkx2-1 still marked the lung field, but no buds were present (C and D), reminiscent of BMS-treated WT foreguts (E and F). (G–I) X-gal staining demonstrated strong RARElacZ signals in both control and Dkk1-treated foreguts (G and H), in contrast to the lack of signals in the BMS-treated foreguts (I). (J–L) BATgal activity was abolished with Dkk1 (K) or quercetin (Que; L) treatment compared with the control (J). Red asterisks in C–F, H, I, K, and L mark the prospective lung field. Original magnification, ×10 (A, C, E, and G–L). Scale bar: 250 μm (B, D, and F). (M) Real-time PCR revealed downregulation of Axin2 and Lef1 in 24-hour cultured foreguts treated with Dkk1 or quercetin. *P < 0.05 versus control.
Treatment with Wnt inhibitors had a dose-dependent negative effect in lung budding. Dkk1 had no effect on lung bud formation in the foreguts at 50 ng/ml. At 100–200 ng/ml, Dkk1-treated foreguts developed only a single lung bud (data not shown). Similarly, quercetin had no effect on lung formation in the foregut explants at 5 μm (data not shown). At the higher doses, however, 400 ng/ml Dkk1 (n = 15) and 10 μM quercetin (n = 9) suppressed formation of the lung primordium completely, so that the prospective lung field was identifiable only by local endodermal expression of Nkx2-1. The decreased intensity of Nkx2-1 signals was consistent with the requirement of Wnt signaling to maintain lung endodermal cell fate in the foregut (17, 24). The Dkk1 phenotype was strikingly similar to that previously reported in BMS-treated foreguts (n = 23; Figure 3, C–F, and refs. 8, 11). Thus, Wnt inhibition in WT foreguts led to lung agenesis. This effect was not caused by disruption of RA signaling or overall toxicity, since the RARElacZ foreguts cultured with recombinant Dkk1 or quercetin maintained strong lacZ signals in contrast to BMS-treated foreguts (Figure 3, G–I, and data not shown). Inhibition of Wnt activity by Dkk1 or quercetin treatment was confirmed by loss of BATgal activity (Figure 3, J–L) and downregulation of the Wnt targets Axin2 and Lef1, as assessed by real-time PCR (Figure 3M).
Wnt regulates Fgf10 expression during lung formation. Lung progenitor cell expansion and bud induction is critically dependent on the local activation of Fgfr2b signaling in the foregut endoderm by Fgf10 in the adjacent mesoderm (23, 25). There is evidence that canonical Wnt directly regulates Fgf10 expression in the developing heart and limb (26, 27). We asked whether the mechanism by which Wnt inhibition influences lung morphogenesis also involves interactions with Fgf10. During organogenesis, Fgf10 was locally expressed in the mesoderm associated with foregut derivatives, such as the lung and thyroid, consistent with the pattern seen in controls (Figure 4A). Analysis of explants in which Dkk1 treatment led to lung bud agenesis revealed no Fgf10 signals in the presumptive lung field (n = 5; Figure 4B), again similar to RA-deficient foreguts (n = 7; Figure 4C). To further consolidate the relationship between Wnt signaling and Fgf10 expression, we performed a gain-of-function experiment by treating the foreguts with a widely used Gsk3b inhibitor, SB216763. This compound inhibits the ability of Gsk3b to phosphorylate β-catenin, thereby allowing the nuclear translocation of β-catenin and the transcription of canonical Wnt targets (20). Hyperactivation of Wnt signaling was demonstrated by widespread BATgal expression in these foreguts and by upregulation of Axin2 and Lef1 (compare Figure 4, D and E, with Figure 3J). As predicted, this led to a significant increase in Fgf10 signals and expansion of its domains (Figure 4, compare A and F). Interestingly, analysis of morphology and Nkx2-1 expression revealed the presence of ectopic buds in the lung primordium and the neighboring stomach (7 of 11; Figure 4G). The effect of hyperactivation of Wnt signaling in Fgf10 expression and bud formation was similarly observed upon engraftment of a Wnt3a-soaked bead in a WT control foregut (Figure 4H and data not shown).
Wnt regulates Fgf10 in the foregut mesoderm. (A–C) WMISH of Fgf10 showed local expression in the mesoderm associated with lung buds (A, arrowheads). Dkk1 inhibition of Wnt signaling abolished Fgf10 in the prospective lung field (B, asterisk; enlarged at right), an effect strikingly similar to that of BMS treatment in the foregut (C, asterisk). (D) Hyperactivation of Wnt by Gsk3b inhibitor (Gski) resulted in widespread LacZ activity in BATgal foreguts (red arrowheads). (E) Real-time PCR revealed upregulation of Axin2 and Lef1 in 24-hour cultured foregut treated with Gsk3b inhibitor or Wnt3a. *P < 0.05 versus control. (F) WMISH of Gsk3b inhibitor–treated foregut exhibiting stronger and broader Fgf10 signals (yellow arrowheads) compared with the control (A). (G and H) WMISH of Nkx2-1 in Gsk3b inhibitor–treated (G) or Wnt3a bead–engrafted (H) WT foreguts demonstrated ectopic buds in the lung and stomach (yellow arrowheads). Scale bar: 300 μm (B, right, C, and G).
Wnt activator or FGF10 rescues Dkk1-induced lung agenesis in RA-sufficient foreguts. To test whether the Dkk1-mediated disruption of lung morphogenesis is caused by insufficient local activation of Wnt signaling, we provided exogenous recombinant Wnt3a on beads to Dkk1-treated WT foreguts. A Wnt3a-soaked bead allowed formation of both lung buds in the presence of exogenous Dkk1 (5 of 5), whereas PBS-soaked control beads did not (7 of 7), as demonstrated by morphology and Nkx2-1 expression (Figure 5, A–C). Dkk1-mediated lung agenesis was similarly prevented by induction of endogenous canonical Wnt signaling using the Gsk3b inhibitor (4 of 4; Figure 5D).
Wnt rescues lung agenesis in Dkk1-treated and RA-deficient foreguts. (A–D) Engraftment of beads soaked in Wnt3a, but not PBS, rescued lung buds bilaterally in Dkk1-treated foreguts (A–C), as confirmed by strong Nkx2-1 expression (arrowheads). Bilateral rescue was also observed when Gsk3b inhibitor was added to Dkk1-treated foreguts (D). (E and F) Exogenous FGF10 (F10) beads induced buds expressing Nkx2-1 (arrows) in Dkk1-treated foreguts (E), a response similarly elicited in BMS-treated foreguts (F). (G–P) Engraftment of Wnt3a beads onto BMS-treated foreguts induced only 1 lung bud, labeled by Nkx2-1 (G and H, arrowheads), although BATgal activity (I) and Fgf10 expression (J) were clearly present on both sides of the lung endoderm (arrows). Similar phenotypes were seen in foreguts treated simultaneously with BMS and Gsk3b inhibitor (K–N) and in Raldh2–/– foreguts treated with Wnt3a beads (O–P). Red asterisks indicate sites of unilateral bud agenesis. Original magnification, ×10 (A, B, D–G, I–K, and M–P); ×20 (C, H, and L).
We found that the inability to induce Fgf10 in the presence of Dkk1 was the ultimate reason for the failure to form lung buds. Dkk1-treated foreguts in which FGF10-soaked beads were engrafted showed bud outgrowth and Nkx2.1 expression in the epithelium associated with the bead (3 of 3; Figure 5E). The response to FGF10 was similar to that in BMS-treated foreguts (6 of 7; Figure 5F). From these data, we concluded that loss of Wnt signaling, even in the presence of normal RA levels, prevents local induction of Fgf10 and disrupts lung formation. However, this seemed to be fully prevented by providing exogenous Wnt or by inducing the endogenous Wnt pathway.
Lung formation is only partially rescued by activating Wnt signaling in an RA-deficient background. Next we tested whether preventing the downregulation of Wnt signaling in RA-deficient foreguts could rescue lung agenesis. We cultured foregut explants in the presence of the RA antagonist BMS and a Wnt3a-soaked bead (400 ng/ml). Surprisingly, only a single bud was induced in the lung field, as confirmed by the strong local expression of Nkx2-1 (11 of 11). This was puzzling, since assessment of BATgal and Fgf10 expression showed signals of apparently equal intensity at both sides of the prospective lung endoderm, consistent with bilateral rescue (3 of 3; Figure 5, G–J). Bilateral expression of Fgf10 was also seen in _Raldh2_-null foreguts cultured with the Wnt3a bead (3 of 3; Figure 5, O and P). The discrepancy between the unilateral rescue of the lung bud and the bilateral induction of BATgal and Fgf10 was similarly observed when Gsk3b inhibitor was provided, instead of Wnt3a, in conjunction with BMS (5 of 5; Figure 5, K–N). It appears that in the setting of RA deficiency, activation of Wnt signaling can only elicit 1 bud formation despite bilateral expression of Fgf10.
Both Wnt activation and Tgfβ inhibition are required to rescue lung bud formation in RA-deficient foreguts. We have previously reported that disruption of RA signaling results in hyperactivation of the Tgfβ pathway in the foregut mesoderm. We also showed that antagonizing Tgfβ in RA-deficient foreguts only rescued 1 lung bud, similar to RA-deficient foregut treated with Wnt activators (11).
We reasoned that correcting the changes in Wnt and Tgfβ signaling of RA-deficient foreguts could rescue lung morphogenesis at both sides. Thus, we analyzed RA-deficient foreguts in which we simultaneously activated Wnt by using either the recombinant Wnt3a in beads or the Gsk3b inhibitor, while inhibiting Tgfβ signaling by using either the Tgfβ receptor 1 antagonist SB431542 (28) or a pan-specific Tgfβ blocking antibody (200 μg/ml; refs. 11, 29). Strikingly, _Nkx2-1_–expressing lung buds were seen bilaterally in the foregut in both the genetic and the pharmacologic models of RA deficiency (Figure 6, A and E).
Tgfβ inhibition and Wnt activation fully rescue lung buds in RA-deficient conditions. (A–H) Addition of Tgfβ blocking antibody (TAb) and Wnt3a bead to BMS-treated foreguts elicited bilateral lung bud formation (A and B, arrowheads), which was associated with local rescue of Fgf10 (C and D, arrowheads and circled regions). Similar rescue of local budding (E–G) and Fgf10 expression (H) was observed in Raldh2–/– foreguts treated with the Tgfβ receptor 1 antagonist SB4 and Gsk3b inhibitor. Corresponding H&E staining is shown in B and G. Original magnification, ×20 (A, right, B, and F); ×10 (D and H). Scale bar: 250 μm.
The rescue was most efficient when Tgfβ blocking antibody and Wnt3a were combined. This combination resulted in clearly distinct foci of Fgf10 expression and well-elongated lung buds (11 of 11; Figure 6, A–D). Bud elongation was less prominent when Gsk3b inhibitor was used to activate Wnt (5 of 7; Figure 6, E–H). This could be explained by less effective inhibition of the Tgfβ pathway, and thus less growth, in the presence of Gsk3b inhibitor. Indeed, endogenous Gsk3b can act as a negative regulator of Tgfβ signaling (30). Thus, RA appears to be critical in the foregut in controlling at least 3 major pathways, Tgfβ, Wnt, and Fgf, to allow proper formation of the lung primordium.