Bmp Expression and Signaling Is Dependent on Acsl4a

(A and B) Whole-mount mRNA in situ hybridization of bmp transcripts in control embryos and embryos injected with acls4a MO (500 fmol) or acsl4a mRNA (1.25 ng). In acls4a morphants, the ventral-to-dorsal gradient of (A) bmp2b and (B) bmp7a is attenuated (in 87% and 78%, respectively) while acls4a overexpression enhances bmp expression (in 77% and 75%, respectively). n = 4; 88–119 embryos. Animal pole view, dorsal to the right (30% epiboly).

(C) Western blot of C-terminally phosphorylated Smad1/5/8 (pSmad^C-Term) and total Smad1/5/8 (Smad^Total) from shield-stage control embryos and embryos injected with 750 fmol acsl4a MO (left). Quantification of three (sphere) or five (shield) experiments (right). Data are represented as mean ± SE. p < 0.05 p < 0.001; one sample t test with Bonferroni correction (sphere stage MO = 500 fmol, shield stage MO = 500–750 fmol).

(D) Whole-mount immunostain against pSmad^C-Term at 80% epiboly. Staining is reduced in morphants (500 fmol) when compared to control siblings (98%; n = 5; 155 embryos). Animal pole view, dorsal to the right (80% epiboly). Inset is a 2× magnification of the ventral-most region.

(E) Acsl4a is needed in the Bmp ligand-receiving cell for Smad activation. (Left) Schematic of transplantation experiment. (Right) pSmad^C-Term immunofluorescence in transplanted cells in ventral equatorial regions of host embryos (80% epiboly). Top panel: wild-type cells transplanted into wild-type embryos have normal pSmad^C-Term staining (26/26 embryos, three independent experiments). Middle panel: Morphant cells transplanted into wild-type embryos lack pSmad^C-Term staining, whereas adjacent cells have normal pSmad^C-Term staining (16/16 embryos, n = 3). Bottom panel: Wild-type cells transplanted into morphants have pSmad^C-Term staining, whereas adjacent morphant cells lack pSmad^C-Term signals (17/17 embryos, n = 3). Wild-type cells transplanted into morphants show reduced pSmad^C-Term staining compared to wild-type cells transplanted into wild-type hosts (compare bottom and top panels). FL, fluorescein dextran.

See also Figure S3.

Acsl4a Acts Downstream of Bmp Receptor Activation

(A) The extent of dorsalization is measured by adding the angles of the arcs of dorsal marker tbx6 expression on either side of the tbx6 negative midline at 80% epiboly. Embryos are considered completely dorsalized if the arcs meet on the ventral side.

(B and C) Constitutively active Bmp receptor does not rescue the phenotype caused by acsl4a depletion. (B) tbx6 expression in a control embryo, embryo injected with acsl4a MO alone (750 fmol), or acsl4a MO combined with constitutively active alk8 (alk8CA, 1.5 ng) mRNA. Vegetal pole view, dorsal to the right (80% epiboly). (C) Quantification of tbx6 expression domain. Data are represented as mean of experimental means ± pooled SE (n = 3–8; 12–80 embryos/experiment). ANOVA with Dunnett post hoc test; p < 0.0001, compared to control.

(D and E) Western blots (n = 3) of C-terminally phosphorylated Smad1/5/8 (pSmad^C-Term) from embryos injected with alk8CA mRNA (20-40 pg), acsl4a MO (500 fmol), or acsl4a MO and alk8CA mRNA combined. Anti-BAP/MAK32 is the loading control. (D) Lysates from sphere-stage embryos. (E) Lysates from shield-stage embryos. An extraneous lane between control and alk8CA is omitted.

See also Figure S4.

Acsl4a Acts via R-Smad Phosphorylation at MAPK Consensus Sites

(A) Model of R-Smad regulation by phosphorylation. (Top) The Bmp receptor positively regulates (green) R-Smad activity by phosphorylating the C terminus. Negative regulation (red) of R-Smad occurs in sequential steps: (1) MAPK phosphorylates the R-Smad linker. (2) GSK3 phosphorylates the linker upstream of MAPK phosphoresidues. (3) Smurf1/2 E3 ubiquitin ligase recognizes R-Smad after phosphorylation by MAPK and/or GSK3. (4) Smurf1/2 polyubiquitinates R-Smad, leading to its proteasomal degradation. (Bottom) Sequence alignment of zebrafish Smad1 and Smad5 linker regions with important residues highlighted.

(B) tbx6 expression in a control embryo, embryo injected with acsl4a MO alone (750 fmol), acsl4a MO combined with a C-terminal phosphomimic smad5 mRNA (smad5PM; 1.5 ng), and acsl4a MO combined with a MAPK-insensitive version of smad5 mRNA (smad5^ΔMAPK; 1.5 ng). Vegetal pole view, dorsal to the right (80% epiboly).

(C) Quantification of tbx6 expression domain. Data are represented as mean of experimental means ± pooled SE (n = 3–8, 12–80 embryos/experiment). ANOVA with Dunnett post hoc test; p < 0.01, p d 0.005, p < 0.0001; compared to control.

See also Figure S5.

Acsl4a Regulates p38 MAPK Activity and R-Smad Linker Phosphorylation

(A) An acsl4a MO-dependent increase in MAPK activity would result in phosphorylation of R-Smad and recruitment of Smurf ubiquitin ligase. An increase in MAPK activity can be assayed by antibodies specific to activated MAPK.

(B) p38 MAPK activity is upregulated in acsl4a morphants (750 fmol) at high stage. Western blot for phosphorylated (Thr¹⁸⁰/Tyr¹⁸²) p38 MAPK (active-p38 MAPK; n = 6).

(C) Smad5^ΔSmurf lacks the WW domain-binding box (PPPAY→ΔΔΔΔΔ) recognized by Smurf E3 ubiquitin ligase, thus it is prevented from linker-mediated degradation. An increase in MAPK phosphorylation of R-Smad can be assayed with a phosphospecific antibody, pSmad^MAPK.

(D) Smad5^ΔSmurf is phosphorylated at the MAPK consensus site (Ser²¹⁵) after acsl4a knockdown. Western blot for MAPK phosphorylated Smad (pSmad^MAPK; gift of de Robertis) at sphere stage. acsl4a MO injection (750 fmol) results in increased pSmad^MAPK staining. Alpha tubulin is the loading control.

See also Figure S6.

Acsl4a Regulates GSK3 Activity

(A) An acsl4a MO-dependent increase in GSK3 activity would result in phosphorylation of R-Smad and recruitment of Smurf ubiquitin ligase. There are inhibitory phosphorylation sites (GSK3α Ser²¹/GSK3β Ser⁹) that control GSK3 activity level, thus an antibody that recognizes the inhibitory phosphorylation (inactive GSK3) can assay GSK3 activity.

(B) GSK3 activity is increased in acsl4a morphants (500 fmol). (Left) Western blot for inhibitory phosphorylation (GSK3αSer²¹/GSK3β Ser⁹) of GSK3 (inactive GSK3). (Right) Western blot for total GSK3β. BAP/MAK32 is the loading control.

(C) GSK3 is inhibited by Akt. An acsl4a MO-dependent decrease in Akt activity would result in decreased inhibition (thus activation) of GSK3. A decrease in Akt activity can be assayed by an antibody specific to active (phosphorylated on Ser⁴⁷³) Akt.

(D) Akt activity is decreased in acsl4a morphants (500 fmol). Western blot for phosphorylated (Ser⁴⁷³) Akt (active AKT). BAP/MAK32 is the loading control.

(E) Quantification of blots from (B) and (D). Data are mean ± SE of three to four experiments and represented as percent of control (p < 0.05 p < 0.001; one sample t test with Bonferroni correction).

(F–H) Constitutively active, myristoylated Akt rescues the acsl4a MO’s dorsalized phenotype. (F) Constitutively active Akt (50–100 pg) decreases GSK3 activity (increase in inactive GSK3) in acsl4a MO-injected (333 fmol) embryos. Data are represented as mean ± SD (n = 2). (G) Expression of tbx6 in a control embryo, embryo injected with acsl4a MO alone (333 fmol), embryo injected with constitutively active akt mRNA (50–100 pg; aktCA), and acsl4a MO with aktCA. Vegetal pole view, dorsal to the right (80% epiboly). (H) Quantification of tbx6 expression domain. Data are represented as mean of experimental means ± pooled SE (n = 3; 20–24 embryos/experiment). ANOVA with Dunnett; p < 0.05, p < 0.0001.

Identification and embryonic expression of zebrafish acsl4a, which is necessary for proper dorsoventral patterning, related to Figure 1.
(A-D) Zebrafish Acsl4a is closely related to characterized ACSL4 proteins.
(A) Regions of conserved synteny demonstrate the relationship between human and zebrafish acsl4 orthologs. Syntenic analysis was performed using reciprocal BLASTN searches of the latest zebrafish (Ensembl Zv9) and human (Ensembl GRCh37) genomic assembly. These data were used to identify the genes adjacent to zebrafish acsl4 homologs and search for their human ortholog’s chromosomal locations. Genes surrounding both human ACSL4 and zebrafish acsl4 genes are shown.
(B) Amino acid sequence comparison of zebrafish (Danio rerio) Acsl4 proteins (Dr Acsl4a and Dr Acsl4l) to human ACSL4 splice isoforms (Hs ACSL4v1 and Hs ACSL4v2). Percentages shown are percent identity (ID) and homology (H) determined using BLOSUM62 matrix (Henikoff and Henikoff, 1992).
(C) Motif-II (Black et al., 1992; Kameda and Imai, 1985; Steinberg et al., 2000; Watkins et al., 2007) of ACSL4 homologs. Multiple sequence alignment of ACSL4 orthologs performed using the Jalview software (Clamp et al., 2004; Waterhouse et al., 2009). The MUSCLE algorithm was used with default settings (BLOSUM62). Acsl4l has an amino acid change from Glutamine to Glutamate at position 526 (arrowhead), an amino acid important for arachidonic acid specificity (Stinnett et al., 2007).
(D) Acsl4a contains the homologous N-terminal region of the longer human ACSL4 isoform. Multiple sequence alignment of zebrafish Acsl4 proteins and human ACSL4 splice isoforms performed using the Jalview with MUSCLE. Sequences are colored for percent identity.
(E) acsl4a expression in late stage embryonic development. Whole-mount mRNA in situ hybridizations using acsl4a sense control riboprobe (control) and antisense riboprobe (acsl4a). Lateral views, anterior to the left. ysl: yolk syncytial layer, cns: central nervous system.
(F-G) Whole-mount mRNA in situ hybridization of pax2a/erg2b/myod1 in 12-somite stage embryos either uninjected (control) or injected with 500 fmol acsl4a MO. pax2a marks m/h: midbrain/hindbrain boundary, o: otic vesicle. erg2b marks r3: rhombomere 3, r5: rhombomere 5. myod1 marks s: somites. (F) Lateral view, anterior to the left. (G) Dorsal view, anterior to the left.
(H) Whole-mount mRNA in situ hybridization of pax2a in 12-somite stage embryos either uninjected (control) or injected with 500 fmol acsl4a MO. Anterior view, dorsal to the right.
(I) Whole-mount mRNA in situ hybridization of ntl in 12-somite stage embryos either uninjected (control) or injected with 500 fmol acsl4a MO. Dorsal view, anterior to the left.

acsl4a MOs specifically inhibit maternal acsl4a transcripts essential for dorsoventral patterning defect, related to Figure 1.
(A-D) Two translation-blocking acsl4a MOs dorsalize zebrafish embryos.
(A) Whole-mount mRNA in situ hybridization of pax2a in 20 hpf embryos of control, acsl4a MO1-injected (750 fmol) or acsl4a MO2-injected (750 fmol) embryos. pax2a marks m/h: midbrain/hindbrain boundary, r: anterior retina. (top) Dorsal view, anterior to the left. (bottom) Lateral view, anterior to the left. White arrowhead indicates wound-up trunk.
(B) tbx6, a marker of dorsal presumptive paraxial mesoderm, is expanded ventrally in acsl4a MO1- and acsl4a MO2-injected embryos (750 fmol). Lateral view, dorsal to the right.
(C-D) acsl4a MOs dose dependently decreases ventral marker gata1a.
(C) Representative images of whole-mount mRNA in situ hybridization of gata1a expression in embryos injected with increasing doses of acsl4a MOs. gata1a expression was binned into one of three levels: wild-type (blue), decreased (yellow), or absent (red). 8-somite stage embryos. Dorsal view, anterior to the left.
(D) Percentages of embryos with wild-type (blue), decreased (yellow), or absent (red) levels of gata1a expression after injection of varying doses of acsl4a MOs (acsl4a MO1: n=31-50 embryos, from 3 separate experiments; acsl4a MO2: n=30-60 embryos, from 3 separate experiments).
(E-F) Injection of acsl4a mRNA attenuates the acsl4a morphant’s dorsalized phenotype.
(E) Representative images of whole-mount mRNA in situ hybridization of tbx6 expression in 80% epiboly stage embryos. Embryos were uninjected (control), injected with acsl4a MO1, or injected with acsl4a MO1 combined with acsl4a mRNA lacking the MO target sequence. Vegetal pole view, dorsal to the right.
(F) Quantification of the angle of tbx6 expression. Data are mean ± SE of a representative experiment with more than 20 embryos per condition. ANOVA with REGWQ post-hoc test was performed. * p<0.05
(G-H) acsl4a is required maternally for dorsoventral patterning.
(G) MOs that inhibit splicing of zygotic Acsl4a transcript do not yield dorsoventral patterning defect. Representative bright field images of control embryo and embryo injected with acsl4a splice-site blocking MO (30 hpf; spliceMO1 1000 fmol).
(H) Acsl4a is not required in the yolk syncytial layer for proper dorsoventral patterning. Representative combined bright-field and fluorescent images of embryos injected with a fluorescein-conjugated MO (control) to indicate injection site or fluorescein-conjugated MO plus acsl4a MO (translation blocking acsl4a MO1). 30 hpf, lateral view, anterior to the left. (Top) Embryos were injected at the 1-4 cell stage. (Bottom) Embryos were injected into the yolk syncytial layer (YSL) at 1K-cell stage. The MOs were restricted from the cells of the embryo proper. 99% of embryos injected at the 1-cell stage with acsl4a MO were dorsalized in comparison to 3% of those injected in the YSL (n=92-93 embryos from 4 experiments). Embryos injected with control MO into the YSL also had occasional dorsalization (4%, n=52 embryos from 3 experiments), whereas 0% of control embryos injected at the 1-cell stage were dorsalized.

Bmp signaling is dependent on Acsl4a, related to Figure 2.

Representative western blots of C-Terminal phosphorylated (pSmad^C-Term) and pan Smad1/5/8 (Smad^Total) from control embryos and embryos injected with alk8CA mRNA (20-40 pg), acsl4a MO (500 fmol). Anti-BAP/MAK32 is shown as a loading control. (A) Lysates from sphere-stage embryos. (B) Lysates from shield-stage embryos.

Acsl4a is epistatic to the Bmp receptor Alk8, related to Figure 3.
(A) Representative bright-field images of 24 hpf embryos injected with 500 fmol acsl4a MO (class C4), 50 fmol bmp2b MO (class C5), 20-40 pg constitutively active (CA) alk8 mRNA (class V3), a combination of acsl4a MO and alk8CA mRNA (class C4), or a combination of bmp2b MO and alk8CA mRNA (class V3). Lateral view, anterior to the left. (B) Percentages of phenotypic classes. Dorsalization: strong (C5) to weak (C1). Ventralization: strong (V4) to weak (V1). n=283-298 embryos, from 3 separate experiments.

Epistasis analysis of Bmp signaling cascade, related to Figure 4.
(A-B) Diagrams of Smad5 constructs, showing mutated residues in the linker region. (A) MAPK-insensitive Smad5: Smad5^ΔMAPK (B) Non-degradable Smad5: Smad5^ΔSmurf (C) Quantification of the angle of tbx6 expression from whole-mount in situ hybridization after injection with acsl4a MO (750 fmol) alone or combined with chordin morpholino (chdMO 20fmol), constitutively active alk8 mRNA (alk8CA 1.5 ng) mRNA, C-terminal phosphomimic smad5 (smad5PM 1.5 ng) mRNA, and MAPK-insensitive smad5 (smad5^ΔMAPK 1.5 ng) mRNA. Data are represented as mean of experimental means ± pooled SE. For acsl4a MO + chordin MO, data are represented as mean of experimental means ± pooled SD (n=2-8 experiments, 12-80 embryos/experiment). ANOVA with Dunnett post-hoc test was performed; acsl4a MO + chordin MO data was excluded from analysis. ** p d0.005, *** p<0.0001: compared to control. (D-E) Constructs used for rescue analysis in Figures 2B&C, 4B&C and S5A ventralize embryos to a similar extent. (D) Whole-mount in situ hybridization of ventral mesoderm marker eve1 after injection with chordin MO or mRNAs from Figures 2B&C, 4B&C and S5A. Injections were performed in parallel (i.e., with the same injection needle) with those in Figures 2B&C, 4B&C and S5A. Vegetal pole view, dorsal to the right (80% epiboly). (E) Data are represented as mean of experimental means ± pooled SD (n=2-7 experiments, 8-47 embryos/experiment). (F) Smad5 mutant constructs are phosphorylated by the Bmp receptor independent of Acsl4a. Representative western blot (n=4) of C-Terminal phosphorylated Smad (pSmadC-Term) from control embryos, embryos injected with acsl4a MO (750 fmol) or embryo injected with acsl4a MO combined with smad5^ΔMAPK (1.5 ng) or smad5^ΔSmurf (1.5 ng) mRNA. Anti-alpha tubulin is shown as a loading control. An extraneous lane between acsl4a MO and acsl4a MO + smad5^ΔMAPK is omitted.

Acsl4a does not activate Erk1/2 or Jnk MAPK cascades, related to Figure 5.

(A) Erk1/2 is not activated in acsl4a morphants. Representative western blot of phosphorylated Erk1/2 from control embryos and embryos injected with acsl4a MO (500 fmol) at sphere stage. Anti-BAP/MAK32 is shown as a loading control. An extraneous lane is omitted. pERK1/2 levels in acsl4a MO-injected embryos are 90±19% of control (not significant, n=3). (B) Jnk signaling cascade is not activated in acsl4a morphants. Representative western blot of phosphorylated MKK4 from control embryos and embryos injected with acsl4a MO (750 fmol) at sphere stage. Blots were stripped and probed with alpha-tubulin as a loading control. pMKK4 levels in acsl4a MO-injected embryos are 1.57 ± .82% of control (not significant, n=4). (C) Representative western blot (n=3) testing the antibody against MAPK phosphorylated hSmad1 (pSmad^”MAPK) (gift of Eddie de Robertis (Fuentealba et al., 2007)) on zebrafish Smad constructs (1 ng). An extraneous lane between smad1^”Smurf and smad1^{”Smurf”MAPK} is omitted.

Acsl4a does not significantly alter Smad2/3 signaling, related to Figure 7.
(A) Whole-mount mRNA in situ hybridization of ntl in 50% epiboly stage embryos, uninjected (control) or injected with 500 fmol acsl4a MO. Animal pole view. 96% of acsl4a MO-injected embryos appear wild-type (n=3 experiments; 127 embryos). (B) Sequence alignment of zebrafish Smad1/5 and Smad2/3 linker regions with important residues highlighted.