Fibronectin is required for proper extracellular matrix organization and cardiac outflow tract elongation in Xenopus laevis

Figures

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  FIGURE 1. Comparison of the OFT elongation during cardiac development in Mus musculus and Xenopus laevis. (A–I) Three-dimensional (3D) reconstructions (A–C), magnified views of the boxed regions (D–F), and representative sagittal sections (G–I) of the embryonic heart in Mus musculus at E9.5 and in Xenopus laevis at NF35 and NF42 stages. Nuclei were labeled with DAPI or Hoechst (blue), and myocardium was visualized with MF20 (green). In Mus musculus E9.5 embryos (D), white arrows indicate the pOFT and dOFT regions of the OFT. In Xenopus laevis, comparable cardiac structures are observed at NF42 (F). Sagittal sections highlight the relative arrangement of the OFT, V, and A. Abbreviations: V, ventricle; A, atrium; OFT, outflow tract (red); dOFT, distal outflow tract (yellow); pOFT, proximal outflow tract (purple); DPW, dorsal pericardial wall. (J) Schematic representation of OFT and measurement strategy. At NF35, the OFT was quantified as a single undifferentiated domain, whereas at NF42, dOFT and pOFT regions were measured separately. (K) Quantification of OFT length at NF35 and NF42. At NF35, the total OFT length was measured; at NF42, the distal and proximal segments were quantified…

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  FIGURE 2. Stage-dependent stratification of the DPW during Xenopus laevis heart development. (A–D1) Xenopus laevis embryos at NF35 (A–B1) and NF42 (C–D1) were processed by whole-mount immunostaining to detect ISL1 (white) and nuclei (blue). The regions outlined in (B–D) are shown at higher magnification in (B1,D1), white arrows indicate the aDPW and pDPW regions. Cyan dashed lines indicate the boundary between endoderm and DPW. DPW thickness is indicated by a white bracket (E–F1). NF35 (E–E1) and NF42 (F–F1) embryos were processed by whole-mount immunostaining to detect E-cadherin (magenta) and nuclei (blue). The boxed regions in (E,F) are shown at higher magnifications in (E1,F1), white arrows indicate the aDPW and pDPW regions. (G,F) Quantification of ISL1+ nuclei across the thickness of the aDPW (G) and pDPW (H) at NF35 and NF42. Each dot represents an independent embryo (n = 3 embryos at NF35 and 3 embryos at NF42). Data are presented as mean ± SD. Statistical significance was determined using t-test. (I,J) Schematic representations summarizing the stage-dependent transition of the DPW from a predominantly monolayered organization at NF35 (I) to a multilayered structure at NF42 (J),…

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  FIGURE 3. Stage-dependent changes in epithelial organization, cell shape, and polarity of SHF-associated cells within the DPW. (A,B) Whole-mount immunostaining of Xenopus laevis embryos at NF35 (A) and NF42 (B), labeled for β1-integrin (cyan) and nuclei (blue). Dashed boxes in A and B highlight the DPW regions shown at higher magnification in (A1,B1) β1-integrin prominently delineates the boundaries of DPW cells (white arrows). Yellow arrows indicate integrin-enriched interfaces facing the pharyngeal endoderm. (C,D) Quantification of DPW cell circularity (4πA/P2) (C) and area (D) at NF35 and NF42. Each larger dot in a different color represents the average value for an individual embryo, whereas the smaller dots represent the values of individual cells (n = 50 cells from 3 embryos at NF35 and 3 embryos at NF42). Bars indicate mean ± SD. Statistical significance was determined using t-test. p < 0.05; ns, not significant. (E–H) Analysis of epithelial polarity using Par3 immunostaining (white) with DAPI (blue). Representative images at NF35 (E, E1) and NF42 (G,G1) are shown. Panels F and H display thermal LUT representations of Par3 signal intensity (n = 3 embryos at NF35 and 3 embryos at…

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  FIGURE 4. Stage-dependent remodeling of ECM composition and spatial distribution in the DPW during Xenopus laevis heart development. (A) Representative Western blot analysis of Fn1, TnC, and Col I (α1 and α2 chains) in whole-embryo protein extracts at developmental stages NF14, NF30, NF35, and NF42. α-tubulin was used as a loading control. (B–D) Densitometric quantification of Fn1 (B), TnC (C), and Col I (α1+α2) (D), protein levels normalized to α-tubulin. Each dot represents an individual biological replicate (N = 4). Data are presented as mean ± SD. Statistical significance was determined using t-test p < 0.05 (*), p < 0.01 (**), ns: not significant. (E–G) Whole-mount immunostaining of NF42 embryos showing Fn1 (green), TnC (magenta), and merged signal, with nuclei labeled with Hoechst (blue). Dashed boxes indicate the DPW region adjacent to the pharyngeal endoderm and OFT. The yellow arrowhead shows Endoderm-DPW limits. (E1–G1) Higher-magnification views of boxed regions illustrating Fn1 enrichment along tissue boundaries and TnC localization within the DPW. (H) Representative line-scan intensity profiles across the DPW at NF42 comparing normalized Fn1 and TnC fluorescence signals,…

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  FIGURE 5. Fn1 depletion impairs OFT elongation and ventricular morphogenesis in Xenopus laevis. (A) Western blot analysis of Fn1 protein levels in embryos injected with Co-MO or Fn1-MO at 48 and 72 h post-fertilization (hpf). Embryos were injected with 10 or 20 ng of MO, as indicated. α-tubulin was used as a loading control. (B,C) Densitometric quantification of Fn1 protein levels normalized to α-tubulin at 48 hpf (B) and 72 hpf (C). Each dot represents an individual biological replicate (N = 4). Data are presented as mean ± SD. Statistical significance was determined using t-test. p < 0.05 (*), p < 0.01(**), p < 0.001(***), p < 0.0001 (****), ns: not significant. (D–G) Whole-mount immunostaining for MF20 (green) with nuclear labeling (blue) showing cardiac morphology in control (D,E) and Fn1-depleted (F,G) embryos at NF42. Representative images of the right (D,F) and left (E,G) views are shown. Dashed outlines indicate the ventricle, and the arrows mark the dOFT and pOFT regions. (H,I) Quantification of distal (H) and proximal (I) OFT length normalized to the otic vesicle area. (J) Quantification of ventricular area normalized to the otic vesicle area. Abbreviations: V, ventricle; A,…

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  FIGURE 6. Fn1 depletion alters ECM composition. (A) Representative Western blot of TnC and ColI (α1 and α2 chains) protein levels in embryos injected with Co-MO or Fn1-MO at 48 and 72 h post-fertilization (hpf). Embryos were injected with 10 or 20 ng of MO as indicated. α-tubulin was used as a loading control. (B) Densitometric quantification of TnC protein levels at 48 hpf (left) and 72 hpf (right), expressed in arbitrary units (a.u.) and normalized to α-tubulin. (C) Densitometric quantification of Col I α1+ α2 chain levels normalized to α-tubulin at 48 hpf (left) and 72 hpf (right). Each dot represents an individual biological replicate (N = 3). Data are presented as mean ± SD. Statistical significance was determined using t-tests; p < 0.05 (*), p < 0.01 (**); ns, not significant.

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  FIGURE 7. Fn1 depletion decreases TnC expression in the DPW. (A–F1) Whole-mount immunofluorescence of NF42 embryos injected with Co-MO (A–C1) and Fn1-MO, showing Fn1(green), TnC (magenta), and nuclei labeled with Hoechst (blue). Fn1 and TnC are detected in the DPW, adjacent to the pharyngeal endoderm and the OFT. (C1,F1) Higher-magnification views of the boxed regions in (C,F), highlighting intercellular localization of TnC within the DPW (arrows). (G–I) Representative line-scan intensity profiles across the DPW illustrating the spatial distribution of Fn1 (G), TnC (H), and DAPI (I) in control and Fn1-depleted embryos. (n = 3 Co-MO embryos, 4 Fn1-MO embryos). The analyzed region is indicated by a dashed yellow rectangle in panels (C1–F1). (J) Schematic representation of the regions analyzed within the DPW, including total DPW, DPW-endoderm interface (DPW-End Limit), medial DPW, and outer DPW limit. (K,L) Quantification of Fn1 (K) and TnC (L) fluorescence intensity across defined DPW regions, normalized to DAPI signal. Each dot represents a different embryo (n = 3 Co-MO embryos, 4 Fn1-MO embryos). Data are presented as mean ± SD. Statistical significance was determined using t-test, <0.05…

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  FIGURE 8. Fn1 depletion reduces DPW tissue area without affecting SHF-associated cells number or individual cell morphology. (A,B) Whole-mount immunofluorescence of NF42 embryos injected with Co-MO (A) or Fn1-MO (B) stained for ISL1 (white) and nuclei labeled with DAPI (blue). Dashed boxes indicate the DPW region located between the pharyngeal endoderm and the developing OFT. (A1,B1) Higher-magnification views of the boxed regions showing ISL1+ nuclei within the DPW. (C,D) Quantification of the number of ISL1+ nuclei within a specific ROI and within the whole DPW. (E,F) Whole-mount immunofluorescence of NF42 embryos stained for β1-integrin (cyan) and DAPI (blue) in control (D) and Fn1-depleted (E) embryos. Arrows indicate β1-integrin labeling at DPW cell boundaries. n = 4, different slices were evaluated in 4 Co-MO and 4 Fn1-MO embryos. (E1,F1) Higher-magnification views of the boxed regions. (G,H) Quantification of DPW cell circularity (G) and cell area (H) based on β1-integrin-defined cell boundaries. A total of N = 3 independent experiments were performed, each analyzing 2–3 different control (Co-Mo) and Fn1-depleted (Fn1-Mo) embryos. (I,J) Whole-mount immunofluorescence of NF42 embryos…

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  FIGURE 9. Comparative model of ECM organization in the DPW during OFT development, and the Fn1 role, in Mus musculus and Xenopus laevis. (A) Model of Mus musculus (E8.5–E9.5) DPW organization. Both aDPW and pDPW display a predominantly epithelial organization, with Fn1 (green) and TnC (blue) primarily localized at the interface between the DPW and the adjacent endoderm. (B) Model of Xenopus laevis (NF35–NF42) DPW organization. DPW undergoes a transition toward a multilayered organization, accompanied by increased intercellular accumulation of Fn1 and TnC within the tissue. (C) Model of Fn1 depletion effect over OFT. Functional depletion of Fn1 leads to shortening of the OFT and reduced ventricular size, in both Mus musculus and Xenopus laevis, highlighting that the requirement of Fn1 for proper cardiac morphogenesis is conserved.