Suppression of Bmp4 signaling by the zinc-finger repressors Osr1 and Osr2 is required for Wnt/β-catenin-mediated lung specification in Xenopus

Abstract

Embryonic development of the respiratory system is regulated by a series of mesenchymal-epithelial interactions that are only partially understood. Mesenchymal FGF and Wnt2/Wnt2b signaling are implicated in specification of mammalian pulmonary progenitors from the ventral foregut endoderm, but their epistatic relationship and downstream targets are largely unknown. In addition, how wnt2 and wnt2b are regulated in the developing foregut mesenchyme is unknown. We show that the Odd-skipped-related (Osr) zinc-finger transcriptional repressors Osr1 and Osr2 are redundantly required for Xenopus lung specification in a molecular pathway linking foregut pattering by FGFs to Wnt-mediated lung specification and RA-regulated lung bud growth. FGF and RA signals are required for robust osr1 and osr2 expression in the foregut endoderm and surrounding lateral plate mesoderm (lpm) prior to respiratory specification. Depletion of both Osr1 and Osr2 (Osr1/Osr2) results in agenesis of the lungs, trachea and esophagus. The foregut lpm of Osr1/Osr2-depleted embryos fails to express wnt2, wnt2b and raldh2, and consequently Nkx2.1(+) progenitors are not specified. Our data suggest that Osr1/Osr2 normally repress bmp4 expression in the lpm, and that BMP signaling negatively regulates the wnt2b domain. These results significantly advance our understanding of early lung development and may impact strategies to differentiate respiratory tissue from stem cells.

Fig. 1

. osr1 and osr2 are dynamically expressed in the developing Xenopus foregut and are required for lung development. (A-G″′) In situ hybridization of osr1 (A-D) and osr2 (E-H) expression in Xenopus tropicalis embryos and isolated gut tubes (D,H) at the indicated stages (st), anterior leftwards, dorsal upwards. Stage 15 embryos (A,E) are bisected and stage 33 embryos (C′-C[H11630],G′-G[H11630]) are sectioned to show the endoderm (outlined in yellow in A,E). fg, foregut; hg, hindgut; de, dorsal endoderm; pe, posterior endoderm; e, esophagus; l/t, presumptive lung and trachea; lv, liver; lpm, lateral plate mesoderm; k, kidney; s, stomach. (I-N″) surfactant protein c (sftpc) (I-L) and nkx2.1 (M,N″) expression in embryos injected on one side with Osr1/Osr2-MOs (10 ng each) or negative control mutated oligos MM-MO (20 ng) shows lung agenesis on the Osr1/Osr2-depleted side. (M’-N″) Sections. Numbers of embryos with the shown phenotype are indicated. Dorsal view of an isolated gut tube (K,L) shows the lung bud absent on the injected side (red arrow).

Fig. 2.

Osr1/Osr2 are required for respiratory specification and tracheal-esophageal separation. (A-L′) Embryos were co-injected with control MM-MOs or Osr1/Osr2-MOs and GR-mOsr1 RNA (250 pg) in both dorsal-vegetal blastomeres at the eight-cell stage (bilateral injection) and then cultured ±dexamethasone from stage 20 onwards. (A-C,G-I) Analysis by in situ hybridization of nkx2.1 at stage 34/35 (A-C) and sftpc at stage 42 (G-I). (D-F′,J-L′) Confocal immunofluorescence of Nkx2.1 (green) and Sox2 (red) at stage 35/36 (D-F) and stage 42 (J-L). es, esophagus; tr, trachea; lb, lung buds; st, stomach, Scale bars: 100 μm.

Fig. 3.

Osr1/Osr2 are required for wnt2b and raldh2 expression in the lpm. (A-X) Analysis of stage 34/35 unilateral-injected embryos shows that Osr1/Osr2 are required for expression of nkx2.1 (A-C) and sox2 (D-F) in the epithelium, whereas shh (G-I) is unaffected. In the lpm, wnt2b (J-L) and raldh2 (M-O) are absent, while foxf1 is unaffected (P-R). Black arrows indicate normal expression on the control side and red arrows indicate lack of expression on the Osr1/Osr2-MO-injected side (right). Endoderm outlined by a yellow line. Numbers of embryos with the shown phenotype are indicated. fg, foregut; hg, hindgut; de, dorsal endoderm; pe, posterior endoderm; e, esophagus; l/t, presumptive lung and trachea; lv, liver; lpm, lateral plate mesoderm; k, kidney; s, stomach.

Fig. 4.

FGF, Wnt/β-catenin and RA signaling regulate Xenopus lung development in a pathway with Osr1/Osr2. (A-D) Embryos were treated from stages 25-35 with DMSO vehicle (A), 100 μM PD173074 (B), 50 μM XAV939 (C) or 10 μM BMS493 (D), and assayed at stage 35 with the indicated probes. (E). Embryos were treated between stages 28 and 35 with vehicle (DMSO), 10 μM BIO, 80 μM SU5402, a combination of 80 μM SU5402 + 10 μM BIO, 10 μg/ml cycloheximide (CHX) or CHX +10 μM BIO, and analyzed for nkx2.1 at stage 35 and sftpc at stage 40. Black arrows indicate normal expression, red arrows indicate absent expression, yellow arrows indicate reduced expression and green arrows indicate expanded or rescued expression.

Fig. 5.

Osr1/Osr2 act upstream of Wnt-mediated pulmonary specification and RA-regulated lung bud growth. (A-G) Osr1/Osr2-MO- and MM-MO-injected embryos were treated at stages 25-35 with DMSO vehicle, 10 μM BIO or 1 μM all-trans-retinoic acid (RA), and analyzed for nkx2.1, sox2, wnt2b and raldh2 at stage 35 (A-F) and sftpc at stage 42 (dorsal view) (G). Black arrows indicate normal expression, red arrows show absent expression, green arrows and lines indicate expanded expression and white arrows indicate rescued expression.

Fig. 6.

Osr1/Osr2 promotes lung specification and growth by restricting BMP signaling. (A). Analysis of Osr1/Osr2-MO and control MM-MO stage 35 embryos indicated that expression of bmp4 and BMP-target genes msx1 and hand1 are expanded into the ‘wnt2b-domain’ of the lpm on the Osr1/Osr2-depleted side. Black arrows indicate normal expression, red arrows indicate absent expression and green arrows indicate enhanced expression. (B) Confocal immunostaining of stage 35 embryos for phospho-Smad1/5/8 (green) and fibronectin (red). Scale bars: 50 μm. White arrows in B indicate ectopic pSMAD1 expression in dorsal foregut endoderm. (C) Analysis of nxk2.1, sox2, wnt2b and raldh2 at stage 35 or sftpc at stage 41 in embryos injected with Osr1/Osr2-MOs or MM-MOs and treated at stages 25-35 with either DMSO (vehicle control) or 10 μM LDN193189. Insets show ventral views of wnt2b expression. For analysis of sftpc expression at stage 41, embryos were removed from the LDN193189 at stage 35 and further cultured in DMSO vehicle until stage 41. Black arrows indicate normal expression, red arrows indicate absent expression, green arrows indicate expanded expression and white arrows indicate rescued expression.

Fig. 7.

Osr1/Osr2 can repress bmp4 expression. (A) GR-mOsr1 RNA (750 pg) was injected into both dorsal-vegetal cells at the eight-cell stage and embryos were dexamethasone treated at stage 20 to induce the construct and analyzed at stage 34/35 for expression of the indicated genes. Insets show ventral views of wnt2b expression. (B) The X. laevis bmp4 promoter construct, showing two putative odd-skipped-binding sites. Green sequence indicates consensus odd skipped-binding site, red sequence is the exact bmp4 promoter sequence, and mutations to these sites are shown below indicated by ‘Δ’ and in black. (C) RNA (750 pg) encoding Xenopus, mouse or Drosophila Osr factors was co-injected with the –2123 bmp4: luciferase reporter at the 32-cell stage and luciferase activity was assayed at stage 12. Average relative activity ±s.d. (D) Xenopus Osr1 or Osr2 RNA (750 pg) was co-injected with the wild-type bmp4 reporter or the mutated reporter constructs indicated in B, and the resulting average fold repression ±s.d., of the luciferase reporter from three independent experiments was determined. ^*P<0.005.

Fig. 8.

Model of the molecular pathway regulating Xenopus lung development. Osr1/Osr2 are key components of a molecular pathway regulating respiratory specification in Xenopus. Blue lines indicate relationships tested in this study and black lines indicate relationships predicted from published mouse studies.