Goutam RS et al. (2025), Bone morphogenetic protein-mediated Ventx3.2 re...

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Biochem Biophys Res Commun 2025 Sep 10;784:152623. doi: 10.1016/j.bbrc.2025.152623.

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Bone morphogenetic protein-mediated Ventx3.2 regulates mesendoderm patterning and gut morphogenesis during Xenopus embryogenesis.

Goutam RS , Kumar V , Lee U , Kim J .

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Bone morphogenetic protein (BMP) signaling plays a pivotal role in germ layer specification and organogenesis during vertebrate development. In Xenopus laevis, BMP signaling activates the Ventx family of homeobox transcription factors, which are essential regulators of mesodermal and endodermal (mesendoderm) fate. Within the Ventx family members, the specific role of Ventx3.2 in mesendoderm development and organogenesis remains poorly defined. Here, we elucidate the role of Ventx3.2 using gain- and loss-of-function approaches in X. laevis. We show that Ventx3.2 is preferentially expressed in the vegetal hemisphere during gastrulation and later in the ventral trunk and tail at tailbud stages. Functional assays reveal that Ventx3.2 contributes to the specification and morphogenesis of mesendoderm-derived tissues, particularly the gastrointestinal tract. Gene expression profiling and morphological analyses indicate that optimal Ventx3.2 expression is critical for normal gastrointestinal tract development and coiling morphology. Collectively, our findings identify Ventx3.2 as a crucial downstream target of BMP signaling, linking early mesendoderm patterning to gastrointestinal tract organogenesis.

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Species referenced: Xenopus laevis
Genes referenced: bmp2 bmp4 cdx1 cdx2 cdx4 cer1 darmin eomes fabp2 foxd4l1 hhex hnf1b hoxa9 irx1 myod1 not otx2 pdx1 sox17b tbxt vegt ventx3.2

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	Graphical Abstract
	Fig. 1. Ventx3.2 induces mesendodermal gene expression and rescues inhibition of BMP/Smad1 signaling. (A) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of whole embryos injected with dominant-negative BMP receptor (dnBR) mRNA (500 pg/embryo or uninjected controls, cultured to stage 11–11.5. (B) RT-qPCR of animal cap explants (ACEs) injected with ventx3.2-HA mRNA (100 pg) or uninjected controls, cultured to stage 11–11.5. (C) RT-qPCR of ACEs injected with dnBR mRNA (500 pg), co-injected with ventx3.2-HA mRNA (100 pg), or uninjected controls, cultured to stage 11–11.5. (D) RT-qPCR of ACEs injected with Smad1-MO (17 ng), co-injected with ventx3.2-HA mRNA (100 pg), or uninjected controls, cultured to stage 11–11.5. Gene expression was normalized to odc and quantified using the ΔΔCt method. Fold enrichment is shown relative to uninjected controls. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns, not significant. Data represent three independent biological replicates (n = 3–5 whole embryos or 10–12 ACEs per condition per replicate).
	Fig. 2. Ventx3.2 in vegetal cells promotes posterior endoderm formation. (A) Schematic of animal hemisphere (AH) and vegetal hemisphere (VH) explant isolation from stage 8 embryos, cultured to stage 11.5 for RT-qPCR. (B) RT-qPCR of AH and VH explants at stage 11–11.5. (C) Schematic of head, trunk, and tail dissections from stage 25–26 embryos. (D) RT-qPCR analysis of head, trunk, and tail tissues. (E) RT-qPCR analysis of dorsal versus ventral trunk tissues. (F) Schematic of animal cap (AC) assay. (G) RT-qPCR analysis of ACEs injected with ventx3.2 mRNA (100 pg) or uninjected controls, cultured to stage 22. Gene expression was quantified by the ΔΔCt method and normalized to odc. Fold change is shown relative to uninjected controls.∗p ≤ 0.05; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns, not significant. Data represent three independent biological replicates (n = 5–10 explants or 10–12 ACEs per condition per replicate). A, anterior; P, posterior; D, dorsal; V, ventral.
	Fig. 3. Ventx3.2 depletion impairs mesendodermal gene expression and posterior patterning. (A) Representative morphology of control and Ventx3.2-MO (20 ng)-injected embryos fixed at the tailbud stage. Scale bar = 500 μm. (A′) Quantification of phenotypic severity (normal, mild, severe) in embryos injected with Ventx3.2-MO into either the animal or vegetal hemisphere. (B) RT-qPCR of vegetal explants (VEs) from stage 10.5–11.5 embryos injected with Ventx3.2-MO (20 ng) or uninjected controls. (C) RT-qPCR of animal cap explants (ACEs) injected with bmp4 mRNA (500 pg), co-injected with bmp4 and Ventx3.2-MO (20 ng), or uninjected controls, harvested at stage 11–11.5. (D) RT-qPCR of stage 22 VEs injected with Ventx3.2-MO (20 ng) or uninjected controls. Gene expression was normalized to odc using the ΔΔCt method and presented as fold change relative to control. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns = not significant. Data represent three independent biological replicates (n = 5–10 explants or 10–12 ACEs per condition per replicate). A, animal; V, vegetal.
	Fig. 4. Ventx3.2 is essential for gut development. (A) Gut morphology (green box) in uninjected controls and embryos injected with increasing doses of Ventx3.2-MO (13–26 ng/embryo), fixed at stages 42–45. Scale bar = 1 mm. (A′) Quantification of gut abnormalities (normal, mild, severe) in control, 13 ng, and 26 ng Ventx3.2-MO injected embryos. (B) Morphology of dissected gut tissues from uninjected controls and Ventx3.2-MO (20 ng) injected embryos. Scale bar = 500 μm. (C) RT-qPCR analysis of dissected gut tissues from uninjected controls and Ventx3.2-MO injected embryos. (D) Rescue of the linear gut phenotype by MO-insensitive ventx3.2∗ mRNA. Embryos were uninjected (control), injected with Ventx3.2-MO (20 ng), or co-injected with 3FLAG-ventx3.2∗ mRNA (50–75 pg), and fixed at stage 42–45 for gut morphology (green box). Scale bar = 1 mm. (D′) Quantification of gut phenotypes (normal coiled gut, linear tubular gut) from embryos shown in panel D. Gene expression was normalized to odc using the ΔΔCt method and presented as fold change relative to uninjected controls. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001; ns = not significant. Data represent three independent biological replicates (n = 3–5 gut tissues per replicate). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Supplementary Figure 1. Ventx3.2 is sufficient to induce mesendodermal gene expression downstream of BMP signaling. (A) RT-qPCR data of ventx3.2 transcript level in animal cap explants (ACEs) injected with dominant negative BMP receptor (dnBR) mRNA (500 pg) compared to non-injected (NI)-ACEs at stage 11.5. (B) RT-qPCR analysis of Eomes and sox17-β in ACEs injected with dnBR mRNA (500 pg) alone and in combination with ventx3.2 mRNA (100 pg). Ventx3.2 restored mesendodermal gene expression in the BMP-inhibited condition. (C) RT-qPCR analysis of Eomes and sox17-β in ACEs injected with Smad1-MO (17 ng) alone and in combination with ventx3.2 mRNA (100 pg). Ventx3.2 restored mesendodermal gene expression independent of BMP/Smad1 signaling. Gene expression levels were quantified using ∆∆Ct method and normalized to ornithine decarboxylase (odc) as the internal control. Data are presented as fold enrichment relative to the control. Statistical significance * p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 and ***p ≤ 0.0001 are the significant, values assigned in panels A-C. Data represent three independent biological replicates (n= 10-12 ACEs per condition per replicate).
	Supplementary Figure 2. Ventx3.2 morpholino inhibits translation of ventx3.2 mRNA. (A) Schematic showing the mechanism of Ventx3.2 translation inhibition by Ventx3.2-MO. The pCS4-ventx3.2-3HA construct was utilised to generate ventx3.2 mRNA with a C-terminally tagged 3HA epitope. Ventx3.2-MO binding to the 5’ translation initiation site inhibits initiation of translation, thereby prevent protein synthesis. (B) Western blot analysis confirming the efficacy of Ventx3.2-MO, with α-tubulin used as a loading control.
	Supplementary Figure 3. pCS4-3FLAG-ventx3.2* mRNA (MO-insensitive) rescues protein translation. (A) Schematic showing the pCS4-3FLAG-ventx3.2 construct used to generate ventx3.2-insensitive mRNA with an N-terminally tagged 3FLAG epitope. The addition of 5’ 3FLAG sequence allows the initiation of translation, displacing the MO and enabling FLAG-Ventx3.2 protein production. (B) Western blot analysis confirms the successful translation of 3FLAG-Ventx3.2 protein in embryos injected with pCS4-3FLAG-ventx3.2 mRNA (100 pg) alone and in combination with Ventx3.2-MO (20 ng). α-tubulin used as a loading control.