XB-ART-45492Genes Dev June 15, 2012; 26 (12): 1351-63.
fus/TLS orchestrates splicing of developmental regulators during gastrulation.
Here we investigated the function of the atypical RNA-binding protein fus/TLS (fused in sarcoma/translocated in sarcoma) during early frog development. We found that fus is necessary for proper mRNA splicing of a set of developmental regulatory genes during early frog development and gastrulation. Upon fus knockdown, embryos fail to gastrulate and show mesodermal differentiation defects that we connect to intron retention in fgf8 (fibroblast growth factor 8) and fgfr2 (fgf receptor 2) transcripts. During gastrulation, the animal and marginal regions dissociate, and we show that this is caused, at least in part, by intron retention in cdh1 transcripts. We confirm the specificity of splicing defects at a genomic level using analysis of RNA sequencing (RNA-seq) and show that 3%-5% of all transcripts display intron retention throughout the pre-mRNA. By analyzing gene ontology slim annotations, we show that the affected genes are enriched for developmental regulators and therefore represent a biologically coherent set of targets for fus regulation in embryogenesis. This shows that fus is central to embryogenesis and may provide information on its function in neurodegenerative disease.
PubMed ID: 22713872
PMC ID: PMC3387662
Article link: Genes Dev
Genes referenced: acvr1b adamts1 adap1l admp admp2 ag1 aggf1 akap2 alcam aldh1a2 alpl amotl2 api5 arid4a arl4a arl5c armc7 arnt arrdc2 asb7 asxl1 atf3 atg16l1 atp12a atp1b1 atp1b2 avp azin2 bambi bcor bend3 bix1.1 bix1.2 bmp2 bmp4 bmp7.2 bmpr1a brd2 btbd10 btg5.2 camlg cass4 cd81 cdc14b cdc16 cdc25b cdc42ep4 cdca3 cdca4 cdca5 cdca7 cdh1 cdkn2aip cdx1 cdx2 cdx4 celsr1 cenpf cep19 chrd.1 ciz1 clk1 clk2 clk3 creb3l2 cxcr4 eomes fgf8 fgfr2 fus gnao1 hpdl mapk1 notch1 nt5c3a odc1 pfkfb3 shh sox17b.1 tbxt tfap2a
Morpholinos: fus MO1 fus MO2 fus MO3 fus MO4 fus MO5
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|Figure 1. fus is expressed in the animal region, and knockdown results in gastrulation defects and animal dissociation. All pictures show a lateral view with the animal region up, except for D and H, which show vegetal view. (A–C) Normal embryos bisected and subjected to ISH with fus antisense probe. (A) Initial fus staining at stage 9 is confined to the animal region. (B) At mid-gastrulation, fus is expressed strongly throughout the prospective mesodermal and ectodermal regions but is excluded from the endoderm. (C) This pattern persists through gastrulation, although some weak staining can be observed in vegetal cells. (D–K) fus knockdown results in lack of blastopore formation and cell dissociation. (D,E) Normal embryos at mid-gastrulation, with prospective mesendoderm involuting at the blastopore. (H,I) fus morphants show complete lack of blastopore formation and gastrulation movements. (F) Normal embryo showing intact epithelia. (J) Individual cells detach from the animal and marginal regions in fus morphants. (G,K) Magnified view of the yellow box in F and J, respectively. (L–O) Absence of apoptosis during gastrulation in fus morphants. Control (L,M) and fusMO (N,O) embryos TUNEL-stained for apoptotic cells at cell dissociation (L,N) or after cell dissociation occurs (M,O). No apoptotic cells were detected at stage 12, when cell dissociation happens. At stage 13, some apoptotic cells that were detected in fus have dissociated and therefore mostly likely show a secondary effect. (P) Single-embryo RT–PCR spanning the first four exons of fus on embryos injected with fusMO4 showing aberrant splicing of the first intron in fus transcripts. Normal transcripts of both homeologs (fus-a and fus-b) in X. laevis are significantly reduced relative to control odc transcripts.|
|Figure 2. fus morphants form all germ layers, but have mesodermal differentiation defects. All pictures show lateral view with animal region up, except for A and E, which show an animal view, and C and G, which show a dorso-vegetal view. (A–H) Normal zygotic and localized gene expression in fus morphants. (A,E) Zygotic ectodermal tfap2 expression is normal in fus morphants. (B,F) Zygotic endodermal sox17b expression is normal in fus morphants. (C,G) Normal dorsal mesoderm expression of chd in fus morphants. Notice the superficial staining in fus morphants caused by lack of invagination of the mesoderm. (D,H) Normal marginal zone fgf8 expression in fus morphants. (I–N) Mesodermal differentiation defects in fus morphants. (I,J) bra is expressed circumferentially in the marginal zone in normal embryos but is essentially absent in fus morphants. (L,M) eomes expression is restricted to a narrow region of the dorsal mesoderm in normal embryos but is significantly elevated in fus morphants. (K) Rescue of bra expression in fus morphants by injection of MO-resistant fus mRNA. (N) Partial rescue of blastopore formation and eomes repression upon fus mRNA injection.|
|Figure 3. Defective FGF-dependent mesodermal gene expression and aberrant splicing of fgf8 and fgfr2. (A–H) Activin can induce eomes but not bra in fus morphants. (A,B) Normal expression of bra and eomes at stage 10.5. (C,D) activin mRNA injection induces ectopic bra and eomes at the site of injection (red staining) in normal embryos. (E,F) fusMO alone represses bra and, at this stage, modestly enhances eomes expression. (G,H) activin mRNA in combination with fusMO fails to induce ectopic bra expression, whereas activin potently induces ectopic eomes in combination with fusMO. (I) Defective splicing of fgf8 transcripts in fus morphants. RT–PCR showing fgf8a and fgf8b transcripts. In the fusMO lane, an aberrant transcript, fgf8b-in1ret, retains intron 1. (J) Selective decrease of fgfr2 transcript in fusMO morphants. RT–PCR using primers to amplify full-length fgfr1–4 transcripts. (K) RT–PCR using primers for individual splice forms of fgfr2 shows selective absence of the fgfr2c splice form in fus morphants, whereas fgfr2b is unaffected. odc was used as RT–PCR control. (L–N) Rescue of bra expression pattern by fgf8b and fgfr2c mRNA injection in fus morphants. (L) Normal bra expression at stage 10.5. (M) fus knockdown represses bra expression. (N) Coinjection of fgf8b and fgfr2c mRNA into two blastomeres at the two-cell stage of fus morphants partly restores endogenous bra expression.|
|Figure 4. Missplicing of cdh1 underlies the cell adherence defect in fus morphants. (A) RT–PCR of full-length cadherin transcripts showing significant and specific reduction of cdh1 in fus morphants, whereas bcad and ccad levels are normal. odc was used as RT–PCR control. (B) Intron retention of cdh1 in fus morphants causes a decrease in properly spliced transcripts. RT–PCR using primers covering the first five exons of cdh1 detects two aberrant transcripts retaining intron 3 (cdh1-in3ret) or introns 3 and 4 (cdh1-in3+4ret), respectively. (C–J) Restoration of cell adherence by cdh1 and fus mRNA in fusMO-injected animal caps. (C) Control animal cap forms an epidermal ball (24 of 24 caps had epithelial integrity). (D) The fusMO-injected animal cap completely dissociates into individual cells (zero of 22 showed integrity). (E) Epithelial integrity in animal cap coinjected with fusMO and cdh1 mRNA (14 of 19). (F) Epithelial integrity in animal cap coinjected with fusMO and MO-resistant fus mRNA (13 of 16). (G–J) Red fluorescence emission from coinjected mCherry mRNA used as tracer in E and F.|
|Figure 5. Pervasive intron retention in fus morphants detected by RNA-seq. In all panels, the top yellow and gray track shows the JGI v7.1 gene models for reference. The black tracks show the control read profile, and the red tracks show read profile for fus morphants. (A) Two splice forms, fgfr2b and fgfr2c, are detected in control lanes at stage 10, whereas only fgfr2b is detected in fus morphants. The alternatively spliced region is boxed in blue. At stage 12, the control read distribution is similar to that at stage 10, whereas the fusMO lane shows a complete flattening of the transcript profile, indicating that all introns are retained. (B) Normal exon profile of cdh1 in control lane at stage 10; dramatic intron retention is apparent in fus morphants. (C) The bcad transcript is normal in fus morphants at stage 10.|
|Figure 6. Intron retention in fus morphants affects a small fraction of all introns and transcripts. (A) Brief outline of the strategy for identifying affected introns. Scatter plot showing stage 10 (B) and stage 12 (C) introns with an adjusted P-value of <10−5. The distribution of the first pass introns (gray) indicates that the P-value is not sufficient to identify retained introns in fus morphants, as a significant number of the passed introns display only small changes. Introns changed more than fourfold and those that passed validation are shown in black. As a reference, introns from cdh1 at stage 10 (B) and from fgfr2 at stage 12 (C) are plotted as red dots. Pie charts in the bottom right of the scatter plots show the proportion of introns that pass the first filter (gray slice) and the final validated set of fus-affected introns (black slice). The white slice represents unchanged introns from expressed genes. Unchanged introns (P > 10−5) are not plotted in the scatter plots. Orange lines indicate the cutoff P-value = 10−5, and green and blue lines indicate thresholds for fourfold increase and decrease, respectively. The right panel shows relative kernel density plots of first-pass introns (gray line) and the final curated set (black line). (D) Area-proportional Venn diagrams showing the number of retained introns in fus morphants at stages 10 (red) and 12 (green), showing significant overlap between the stages. The number in parentheses indicates the total number of introns retained at the given stage. (E) Venn diagram similar to D, but showing the number of genes with retained introns in fus morphants. A significant majority (85%) of genes that are affected at stage 10 are also affected later, whereas almost half (42%) are only affected at stage 12.|
|Figure 7. GO term analysis of fus-affected genes. (A) Intron-retained genes in fus morphants are enriched for developmental regulators. Heat map showing GO slim terms annotated to intron-retained genes in fus morphants and expressed genes at stage 10 and stage 12. GO slim categories that are significantly enriched in fus-affected genes are indicated in red, and categories that are depleted are in blue. GO slim categories that contain terms associated with embryo development, morphogenesis, and differentiation (anatomical structure morphogenesis, cell differentiation, and embryonic development) are enriched in fus-affected genes. Also enriched are terms associated with transcription (nucleic acid binding and transcription factor activity) and signal transduction (signal transducer activity). (Inset) Values are expressed as a fraction of genes that are assigned the GO slim term, with rarely represented terms in yellow and more common terms in red. (***) P < 0.001; (**) P < 0.01 at both stage 10 and stage 12. (*) P < 0.05 at stage 12 only. Only GO slim terms from levels 3 and 4 in the categories “molecular function” and “biological process,” respectively, are shown. (B) All major developmental signaling pathways are affected in fus morphants. Bar graph showing the representation of GO signal categories in fus-affected genes expressed as fold change compared with expressed genes. The major developmental pathways regulated by either activin, adhesion, bone morphogenetic protein (bmp), fgf, hedgehog (hh), MAP kinase (mapk), notch, retinoic acid (ra), smad, tgf-β (tgfb), and wnts are enriched at stages 10 and 12. In contrast, GO terms associated with transfer RNA (tRNA), telomere, and intracellular vesicles (vesicle) are depleted in fus-affected genes. The red line indicates no change.|
|Supplemental Figure 1. The C-terminal tail of fus is dispensable for splicing. (A) fus genomic locus showing the full gene model in the top panel, and a close up of the last four exons in the bottom. The C-terminal region contains eight conserved amino acid residues (orange) that have been linked to fALS in humans and seven of these are encoded in the last exon 15. The splice blocking MO fus-fALS-MO5 (red) targets the donor splice site in exon 14. (B) Single embryo RT-PCR on two control embryos or fus-fALS morphants at stage 25. (B, Top panel) Missplicing of the last splice junction of fus resulting either in intron inclusion (fus-fALS-C), causing premature stop or use of two cryptic splice donor sites in exon 14 (fus-fALS-B and fus-fALS-A) that cause a frame shift. In all cases the result is C-terminal truncated forms of fus without the C-terminal region containing the residues mutated in fALS mutations. (B, lower panels) normal splicing and expression of fgfr2 and fgf8 in fus-fALS morphants. Internal control was odc. (C) fus-fALS morphants develop normally at least until stage 41. (D) fus-fALS morphants are immobile after hatching (stage 25, left barplot). As development proceeds fus-fALS morphants gain mobility, but are still impaired compared to normal embryos (stage34-38, right barplot). Normal swimming was scored as ability to swim for at least one second after being prodded. Swimming for shorter duration was scored as impaired.|
|Supplemental Figure 5. Nuclear localization of fus affected transcripts. ISH for fgf8 (A-B) and chd (C-D) in control (A,C) and fus morphants (B,D). Transcripts localize predominantly to the nucleus in fus morphants as shown in the close up pictures (Band D whereas in control embryos the transcripts are uniformly distributed in the cells (Aand C. Red brackets indicate section of image magnified.|
|fus (fused in sarcoma) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 9, dorsal right, anterior up.|
|fus (fused in sarcoma) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 11, dorsal right, anterior up.|
|fus (fused in sarcoma) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 11.5, dorsal right, anterior up.|