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Fig. 1.. Xenopus fbrsl1 spatial expression pattern analyzed by whole-mount in situ hybridization. (A,B) An embryo at the two-cell stage: animal view (A); lateral view (B). (C) Embryo at the blastula stage. (D) Embryo at blastula stage, hybridized with the fbrsl1 sense probe. (E) Embryo at the neurula stage, anterior view. Asterisk indicates the crescent-shaped expression domain. (F) Embryo at the neurula stage stained using a nkx2.5 antisense probe, anterior view. (G,H) An embryo at stage 19: anterior view (G); dorsal view (H). (I) Embryo at stage 23, lateral view. (J) Embryo at stage 23 hybridized using the fbrsl1 sense probe. (K) Embryo at stage 27, lateral view. (L) Embryo at stage 34, lateral view. (M) Embryo at stage 38, lateral view. (N) Embryo at stage 38, lateral view, hybridized with the fbrsl1 sense probe. (O) Transverse section of an embryo at stage 38. (P) Schematic indicating the section plane of the stage 38 embryo (Zahn et al., 2022) in O. Scale bars: 500m. an, animal; anp, anterior neural plate; b, brain; ba, branchial arches; bl, blastoporus; fb, forebrain; h, heart; nc, neural crest; nt, neural tube; ov, otic vesicle; pd, proctodeum; v, ventricle; ve, vegetal.
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Fig. 2. Fbrsl1 loss of function leads to cardiac defects in Xenopus embryos. (A) Schematic showing morpholino-binding sites at the 5 region of Xenopus laevis fbrsl1 (full-length X. laevis fbrsl1 consists of 19 exons). The fbrsl1 translation blocking (tb) MO targets the translational start site, whereas the fbrsl1 splice blocking (sp) MO targets the exon 1/intron 1 boundary (Ufartes et al., 2020). (B,C) Embryos were injected with either 10ng control (Co) MO or 7.5ng fbrsl1 sp MO together with 100pg lacZ RNA as lineage tracer in one dorsal blastomere at the four-cell stage. mhc (myh6) in situ hybridization marks the heart and facial muscles in wild-type and morpholino-injected embryos. (B) Wild-type and Co MO-injected embryos. (C) Embryos injected with fbrsl1 sp MO. Scale bar: 500m. (D) Graph summarizing mhc heart patterning defects of three independent experiments. Data are means.e.m.; the numbers of embryos are indicated, **P=0.0041 (one-way ANOVA with Tukey's post hoc test). (E) Embryos were injected with 7.5ng morpholino oligonucleotides in one dorsal blastomere at the four-cell stage, combined with 50pg mGFP RNA (CT3) or 100pg lacZ RNA (MF20) as lineage tracer. Graph summarizing heart defects of CT3- or MF20-immunostained embryos from at least three independent experiments. Data are means.e.m.; the numbers of embryos analyzed (n) are indicated, ****P<0.0001 (one-way ANOVA with Tukey's post hoc test). (F-I) Four-cell stage embryos were injected in one dorsal blastomere with 7.5ng of the respective morpholino oligonucleotides in combination with 50pg mGFP RNA as lineage tracers. Heart morphology was analyzed at stage 44; embryos are shown from the ventral side. (F-I) Higher magnification of the areas outlined in F-I. (F-G) Wild-type (F,F) and Co MO-injected (G,G) embryos show normal heart morphology. Ventricle (v) and outflow tract (oft) are marked by dashed white lines. (H-I) Injection of either the fbrsl1 sp MO (H,H) or the fbrsl1 tb MO (I,I) leads to defects in heart morphology. (N-Q) Z-stack images of Xenopus hearts immunostained for cardiac muscle troponin (CT3). (N,O) The heart ventricle is oval in wild-type (N) or Co MO-injected (O) embryos. (P,Q) Heart shape and morphology are disturbed after injection of fbrsl1 sp MO (P) or the fbrsl1 tb MO (Q). (N-Q) Depth color-coding profile indicates the extension of the heart ventricles from 0m (red) to 50-120m (blue). The endpoints are different in N-Q. (N-Q) xz views of heart ventricles. (J-M) Embryos from the experiments shown in F-I were used to measure the length of the OTF (a), the width of the OFT (b) and the ventricular area (c), as indicated in J. (K-M) Box and whiskers plots summarize the length (K) and the width (L) of the OFT as well as the ventricular area (M). The number of hearts measured and the median are indicated. The boxes extend from the 25th to the 75th percentiles with whiskers ranging from minimum to maximum values. ns, not significant. **P<0.01, ****P<0.0001 (one-way ANOVA with Tukey's post hoc test). Scale bars: 500 m in F-I; 100m in F-I; 200m in N-Q.
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Still from Movie 1. (Control)
The beating heart of a wild-type Xenopus laevis embryo. Wild-type embryos show a dynamic beating of the heart and blood flow through the ventricle and outflow tract. A representative embryo is shown from the ventral side.
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Still from Movie 3
The beating heart of a Xenopus embryo injected with fbrsl1 sp MO. Four-cell-stage embryos were injected in one dorsal blastomere with 7.5 ng fbrsl1 sp MO in combination with 50 pg mGFP RNA as a lineage tracer. The embryos were anesthetized with 0.1x MBS (modified Barth's saline) containing 0.01% benzocaine. Beating hearts were recorded at stage 44. A representative embryo is shown from the ventral side. The heart is malformed and has a smaller ventricle, but is able to beat. The blood flow is not visible.
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Still from Movie 4
The beating heart of a Xenopus embryo injected with fbrsl1 tb MO. Four-cell-stage embryos were injected in one dorsal blastomere with 7.5 ng fbrsl1 tb MO in combination with 50 pg mGFP RNA as a lineage tracer. The embryos were anesthetized in 0.1x MBS (modified Barth's saline) containing 0.01% benzocaine. Beating hearts were recorded at stage 44. A representative embryo is shown from the ventral side. The heart is severely malformed and the blood flow is not visible.
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Still from Movie 2.
The beating heart of a Xenopus embryo injected with Co MO. Four-cell-stage embryos were injected in one dorsal blastomere with 7.5 ng Co MO in combination with 50 pg mGFP RNA as a lineage tracer. The embryos were anesthetized with 0.1x MBS (modified Barth's saline) containing 0.01% benzocaine. Beating hearts were recorded at stage 44. A representative embryo is shown from the ventral side. The embryo shows a dynamically beating heart; the blood flow through the ventricle and outflow tract is visible.
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Fig. 3. Fbrsl1 loss of function disrupts the development of the first heart field, but not the cardiac progenitor cells. (A) Schematic illustration of the key steps in Xenopus heart development. At stage 15, cardiac progenitor cells are located anterior in a crescent-like structure (marked by isl1 expression), which gives rise to two separate populations: the first heart field (FHF) and the second heart field (SHF). At stage 24, nkx2.5 is expressed in both heart fields, whereas at stage 28 the two cell populations can be distinguished using the markers mhc (FHF) and isl1 (SHF). (B-I) Embryos were injected at the four-cell stage into one dorsal blastomere with 5ng-7.5ng (B-G) or 7.5ng-10ng (H,I) control (Co) MO or fbrsl1 splice blocking (sp) MO and 80pg lacZ RNA for lineage tracing, cultured to the respective stages and analyzed by in situ hybridization. Asterisks indicate the injected side. Scale bar: 500m. Embryos are shown from the ventral (B-F) or the anterior (H) side. (B) Stage 28 embryos analyzed by mhc whole-mount in situ hybridization. fbrsl1 sp MO-injected embryos show a reduction of the first heart field on the injected side (minor defect is shown). (C) Graph summarizing the defects in first heart field formation of three independent experiments. (D) Stage 28 embryos analyzed by isl1 in situ hybridization. Expression is visible in the second heart field. The fbrsl1 sp MO-injected side shows normal expression. (E) Graph summarizing the defects in second heart field formation of two (wild type) or three independent experiments. (F) Embryos analyzed by nkx2.5 whole-mount in situ hybridization at stage 24. Expression can be observed in the first and second heart field. The fbrsl1 sp MO-injected side shows reduced expression (minor defect is shown). (G) Graph summarizing the results of three independent experiments shown in F. (H) Whole-mount in situ hybridization showing isl1 expression in the cardiac progenitor cells. The fbrsl1 sp MO-injected side shows normal expression. (I) Graph summarizing the defects of cardiac progenitor cells of three independent experiments. Data are means.e.m. The numbers of evaluated embryos are indicated in all graphs. ns, not significant. ****P<0.0001 (one-way ANOVA with Tukey's post hoc test). a, anterior; d, dorsal; p, posterior; v, ventral.
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Fig. 4. Defects in mhcα patterning are rescued by the human short N-terminal FBRSL1 isoform 3.1. (A) Schematic of the FBRSL1 isoform 1 and the short N-terminal isoform 3.1. Red arrows indicate the position of the three patient variants. (B) 7.5 ng of the respective morpholino oligonucleotides and 150 pg of the plasmids were injected in one dorsal blastomere at the four-cell stage. 100 pg lacZ RNA was co-injected as lineage tracer. Representative embryos at stage 28 are shown from the ventral side; injected constructs are indicated. Asterisks indicate the injected side; blue β-galactosidase staining is visible. Scale bar: 500 µm. (C) The graph summarizes the percentage of mhcα patterning defects of three independent experiments. Data are mean±s.e.m. The numbers of embryos analyzed are indicated. **P<0.01; ns, not significant (P=0.06) (one-way ANOVA with Tukey's post hoc test)
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fbrsl1 ( fibrosin-like 1) gene expression in Xenopus laevis embryo, analyzed by whole-mount in situ hybridization, stage NF 2, animal view (A) and lateral view (B, animal pole up) .
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fbrsl1 ( fibrosin-like 1) gene expression in Xenopus laevis, analyzed by whole-mount in situ hybridization, stage NF 19, anterior view ( G) and dorsal view (H).
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fbrsl1 ( fibrosin-like 1) gene expression in Xenopus laevis, analyzed by whole-mount in situ hybridization, stage NF 23, lateral view, anterior left, dorsal up.
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fbrsl1 ( fibrosin-like 1) gene expression in Xenopus laevis, analyzed by whole-mount in situ hybridization, stage NF 27, lateral view, anterior left, dorsal up.
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myh6 (myosin, heavy chain 6, cardiac muscle, alpha; aka mhc-alpha) gene expression in X. laevis embryo, NF stage 44, assayed via in situ hybridization, ventral view of head region.
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Fig. S1. A stage 16 embryo analyzed using a sense fbrsl1 in situ hybridization probe.
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Fig. S2. fbrsl1 tb MO efficiently inhibits the translation of a Xenopus GFP-tagged Fbrsl1 isoform (Fbrsl1_A-eGFP). A One-cell stage embryos were injected with 10 ng Co MO, fbrsl1 sp MO or fbrsl1 tb MO together with 100 pg fbrsl1_A-eGFP RNA, a shorter Fbrsl1 transcript, isolated from wild-type Xenopus embryos. Protein expression was analyzed by western blotting using an -GFP antibody. The Fbrsl1_A-eGFP protein is detected in embryos injected with the fbrsl1_A-eGFP in combination with Co MO or fbrsl1 sp MO. However, Fbrsl1_A-eGFP expression is strongly inhibited in embryos co-injected with the fbrsl1 tb MO. B The ratio of signal intensity levels of Fbrsl1_A-eGFP to actin were normalized to the Co MO + Fbrsl1_A-eGFP levels and plotted. The graph shows the mean relative signal intensity (AU, arbitrary units) of Fbrsl1_A-eGFP expression of four independent experiments, s.e.m. are shown, **P< 0.01 (one-way ANOVA).
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Fig. S3. Heart morphology analyzed by antibody staining. Embryos were injected in one dorsal blastomere at the four-cell stage with 7.5 ng Co MO, fbrsl1 sp MO or fbrsl1 tb MO and 50 pg mGFP RNA (CT3) or 100 pg lacZ RNA (MF20) as lineage tracer. A, B CT3 antibody staining. C, D MF20 antibody staining. B, D Higher magnification of the heart region shown in the images in A and C, respectively. Asterixs marks the injected side. Scale bar in A and C 500 m, in B and D 100 m. v, ventricle.
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Fig. S4. Categorization of mhc-alpha and isl1 patterning defects. For statistical analyses of mhc-alpha and isl1 patterning defects, the embryos were categorized as having no, minor or severe defects. Representative embryos for each category are shown. Asteriks marks the injected side.
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Fig. S5. fbrsl1 sp MO and fbrsl1 tb MO binding sites on Xenopus laevis fbrsl1.L and fbrsl1.S
A The schematic shows the fbrsl1 tb MO and fbrsl1 sp MO binding sites on exon 1 and the exon1/intron1 boundary. The corresponding morpholino sequences are indicated in the dashed square. B shows a section of the fbrsl1.L and the fbrsl1.S sequences with the corresponding morpholino binding sites marked. Both morpholino oligonucleotides were designed to target the fbrsl1.L form. The fbrsl1 tb MO has 4 mismatches with fbrsl1.S and the fbrsl1 sp MO has 1 mismatch. ATG triplet is shown in bold.
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