Fbrsl1 is required for heart development in Xenopus laevis and de novo variants in FBRSL1 can cause human heart defects

Abstract

De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.

Keywords: AUTS2; Congenital malformation syndrome; Fbrsl1; Heart development.

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: 500 µm. 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.

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 10 ng control (Co) MO or 7.5 ng fbrsl1 sp MO together with 100 pg lacZ RNA as lineage tracer in one dorsal blastomere at the four-cell stage. mhcα (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: 500 µm. (D) Graph summarizing mhcα heart patterning defects of three independent experiments. Data are mean±s.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 7.5 ng morpholino oligonucleotides in one dorsal blastomere at the four-cell stage, combined with 50 pg mGFP RNA (CT3) or 100 pg lacZ RNA (MF20) as lineage tracer. Graph summarizing heart defects of CT3- or MF20-immunostained embryos from at least three independent experiments. Data are mean±s.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 7.5 ng of the respective morpholino oligonucleotides in combination with 50 pg 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 0 µm (red) to 50-120 µm (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 µm in F-I; 100 µm in F′-I′; 200 µm in N-Q″.

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 mhcα (FHF) and isl1 (SHF). (B-I) Embryos were injected at the four-cell stage into one dorsal blastomere with 5 ng-7.5 ng (B-G) or 7.5 ng-10 ng (H,I) control (Co) MO or fbrsl1 splice blocking (sp) MO and 80 pg lacZ RNA for lineage tracing, cultured to the respective stages and analyzed by in situ hybridization. Asterisks indicate the injected side. Scale bar: 500 µm. Embryos are shown from the ventral (B-F) or the anterior (H) side. (B) Stage 28 embryos analyzed by mhcα 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 mean±s.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.

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).