Rankin SA , Zorn AM .

P01 HD093363 NICHD NIH HHS , 1R01DK123092 NIDDK NIH HHS

             respiratory system development

	Figure 1. Expression of ventx2 in developing Xenopus tropicalis embryos. (a–m) In situ hybridization was performed at the indicated stages using a full-length ventx2.1 probe (GenBank sequence CR848273.2) predicted to detect both ventx2.1/ventx2.2 due to 92% sequence identity. Section in f' is through the pharynx; section in g' is through the posterior foregut, where ventx2 is detected in both midline and medial-lateral ventral foregut cells. Abbreviations: AE, anterior endoderm; PE, posterior endoderm; dors-ant, dorsal-anterior; vent-post, ventral-posterior; de, dorsal eye; hg, hindgut; im, intermediate mesoderm; mg, midgut; nmp, neuromesodermal progenitors; nc, neural crest; ov, otic vesicle; dfg, dorsal foregut; v fg, ventral foregut; v ph, ventral pharynx; tra, trachea; lb, lung bud; es, esophagus. Dashed black lines in (f), (g), and (j)–(m) indicate the plane of section for those sections shown. Dashed yellow lines in (g') and (j2) outline the endoderm layer.
	Figure 2. BMP signaling acts prior to Wnt/β-catenin signaling to directly activate ventx2 expression in the foregut endoderm. (a) BMP signaling is active and ventx2-1 is expressed in respiratory progenitor endoderm prior to nkx2-1 and Wnt/β-catenin signaling. Comparison of in situ hybridization for ventx2 and nkx2-1 in foregut sections of stage NF31/32 and NF34/35 embryos shows expression of ventx2 in the ventral foregut at NF31/32 prior to onset of nkx2-1 expression. Note the ventx2 sections in this comparison are reused from Figure 1. Immunostaining of transgenic Wnt/β-catenin reporter X. tropicalis embryos (transgene carries seven Tcf/Lef binding sites driving eGFP expression; Tran et al., 2010) with antibodies indicative of active BMP (phosphorylated Smad1/5/9 [pSMAD1/5/9]; red) and Wnt/β-catenin (eGFP; green) signaling shows that pSMAD1/5/9 is detectable in the ventral foregut endoderm at stage NF31/32 coincident with ventx2, whereas active Wnt/β-catenin (eGFP) in the endoderm is detected later at stage NF34/35 coincident with nkx2-1. Scale bar = 100 μm. (b) BMP signaling is required for foregut expression of ventx2 at NF31/32, whereas both BMP and Wnt/β-catenin are necessary for robust foregut ventx2 expression at NF35. Embryos were cultured in DMSO (vehicle control), 20 μM DMH-1 (BMP type I receptor inhibitor), or 25 μM XAV939 (Tankyrase inhibitor that triggers β-catenin degradation) during NF25–31 or during NF25–35 and assayed by in situ hybridization for ventx2. (c) Direct BMP/Wnt target assay in X. laevis foregut endoderm explants. Foregut endoderm was isolated at stage NF25 and pre-treated for 2 h with cycloheximide (CHX) prior to further exposure to CHX and either 50 ng/ml BMP4 protein or 500 nM Bio, and gene expression was assayed by RT-qPCR after 8 h of total culture. The combination of 0.2% BSA + ethanol (EtOH) served as vehicle control treatment. BMP4 robustly induced expression of both ventx2.1 and ventx2.2 in the presence of CHX, demonstrating direct activation. foxa1 is a pan-endoderm gene not significantly affected by BMP or Wnt/β-catenin stimulation. Each black dot in the graphs represents a biological replicate (pool of n = 3 explants). *p < .05 relative to vehicle-treated explants, parametric two-tailed t-test.
	Figure 3. Data mining of published ChIP-seq and ATAC-seq data suggests multiple enhancers around ventx2.1 and ventx2.2 are re-utilized during development to regulate ventx2 expression. (a) IgV browser shot of the genomic locus around X. tropicalis ventx2.1/2.2 spanning approximately 55 kb. Tracks display the indicated ChIP-seq or ATAC-seq binding or accessibility data at the indicated NF stage of development. Gene models are drawn in black below and black arrows indicate the 5′–3′ direction of the genes, which lie in opposite orientations. Data were from the following sources: FoxA2 NF10.25: GSE85273 (Charney et al., 2017); phosphorylated Smad1/5/9 NF10: GSE113186 (Gentsch et al., 2019); β-catenin NF10: GSE113186 (Gentsch et al., 2019); p300 NF10.5, NF16, and NF30: GSE67974 (Hontelez et al., 2015); and ATAC-seq NF10.5 and NF16: GSE145619 (Bright et al., 2021). Blue shading and red asterisks indicate regions of possible differential p300 binding over developmental time (b) IgV browser shot of the genomic locus around X. laevis ventx2.1/2.2 spanning approximately 55 kb. Tracks display the indicated ChIP-seq data at the indicated NF stage of development. Gene models are drawn in black below and black arrows indicate the 5′–3′ direction of the genes, which lie in opposite orientations. Data were from the following sources: Smad1 NF20: GSE87652 (Stevens et al., 2017); β-catenin NF20 foregut explants: GSE87652 (Stevens et al., 2017); p300 NF20 foregut and hindgut explants: GSE87652 (Stevens et al., 2017); and p300 NF10.5: GSE76059 (Session et al., 2016)
	Figure 4. Ventx2 regulates respiratory system development. (a) Microinjection strategy to knock down Ventx2 in the developing foregut. Embryos at the 16-cell stage were injected in dorsal-anterior vegetal blastomeres to target the future foregut. A representative lineage trace of the injected cells is shown in an NF36/37 embryo via pink-gal staining (red signal) resulting from activity of the injected β-galactosidase mRNA (lineage tracer). (b) In situ hybridization for the lung epithelium marker sftp-c in NF42 embryos (lateral and dorsal views) revealing larger lung buds and expanded sftp-c in the pharynx (green arrow) in Ventx2 foregut morphants as compared to control MO injected embryos. The total numbers of embryos analyzed by sftp-c in situ hybridization that had the observed staining pattern are indicated. (c) Ventx2 is required for tracheal–esophageal separation and lung bud luminal structure formation. Confocal optical sections of NF42 embryos through the foregut immunostained for the endoderm transcription factor FoxA2 (purple), the splanchnic mesoderm ECM marker Fibronectin (green), and the mitotic nucleus marker phospho-Histone H3 (pHH3, red) reveal failed separation of the foregut tube into a distinct dorsal esophagus and ventral trachea in Ventx2 foregut morphants. Abbreviations: eso, esophagus; ph, pharynx; tra, trachea; lb, lung bud. Scale bar = 100 μm. (d) The dorsoventral pattern of the foregut is normal at stage NF33/34 in Ventx2 morphants. Confocal z-stacks (50 μm) showing immunostaining of the ventral respiratory progenitor marker Sox9 (red) and the dorsal esophageal progenitor marker Sox2 (green) are shown. Abbreviations: dfg, dorsal foregut; v fg, ventral foregut. Scale bar = 100 μm. (e,f) Elevated progenitor cell number in the foregut endoderm domain of NF36/37 Ventx2 foregut morphants. Imaris software was used to generate the cropped endoderm foregut region from 50-μm confocal z-stacks that were immunostained (e), and the numbers of nuclei positive for the pan-foregut endoderm transcription factor FoxA2 (purple), the ventral respiratory progenitor marker Sox9 (green), and mitotic marker phospho-Histone H3 (pHH3, red) were quantified (f). Scale bar = 100 μm. Each black dot in the graphs in (f) is a separate embryo 50-μm cropped endoderm confocal z-stack (n = 6 quantitated per condition). *p < .05,**p < .01, parametric two-tailed t-test; ns = not significant.
	Figure 5. Ventx2 regulates the timing and level of surfactant protein gene expression. (a) Expression of the lung epithelial marker sftp-c was precociously activated at NF33/34 in Ventx2 foregut morphants, which have larger lung buds at NF40. In situ hybridization of nkx2-1 and sftp-c at NF33/34 and NF40. (b) Schematic of foregut explant and culture assay to assess temporal kinetics of respiratory marker gene expression by RT-qPCR. (c) RT-qPCR analysis of control MO injected foregut explants demonstrating that gene expression in the fg explant culture system mirrors normal embryonic development, with ventx2.1 and ventx2.2 expression (white and red bars, respectively) increasing during NF25–35, but being lower at NF40. sftp-c expression (green bars) is not detectable prior to NF35 and follows nkx2-1 (gray bars). (d) RT-qPCR analysis of control and Ventx2 loss of function, rescued, or gain of function fg explants. Progenitor markers nkx2-1 and sox9 were not prematurely expressed during NF20–30 in response to Ventx2 depletion; however, the expression levels of differentiation markers sftp-b and sftp-c were prematurely elevated. Ventx2 gain of function (blue bars; GR-ventx2 construct was DEX-activated at NF25 and gene expression was assayed at NF35 and NF40) resulted in suppression of the respiratory differentiation markers sftp-b and sftp-c but had no effect on the respiratory progenitor markers nkx2-1 and sox9. Each black dot in the graphs in (c) and (d) represents a biological replicate (pool of n = 3 explants). *p < .05 relative to uninjected stage NF20 fg explants, parametric two-tailed t-test.
	Figure 6. Summary of ventx2 expression and possible mechanistic functions in developing Xenopus respiratory progenitor cells.

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