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First published online November 26, 2007
doi: 10.1242/10.1242/dev.000026

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Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

Camila V. Esguerra^1,2,,*, Luc Nelles^3,4,, Liesbeth Vermeire^3,4, Abdelilah Ibrahimi^3,4,, Alexander D. Crawford^1,2,¶, Rita Derua⁵, Els Janssens^1,2, Etienne Waelkens⁵, Peter Carmeliet^1,2, Desiré Collen^1,2 and Danny Huylebroeck^3,4,*

¹ Center for Transgene Technology and Gene Therapy, VIB, Herestraat 49, B-3000 Leuven, Belgium.
² Department of Molecular and Cellular Medicine, KULeuven, Herestraat 49, B-3000 Leuven, Belgium.
³ Laboratory of Molecular Biology, Department of Molecular and Developmental Genetics, VIB, Herestraat 49, B-3000 Leuven, Belgium.
⁴ Department of Human Genetics, KULeuven, Herestraat 49, B-3000 Leuven, Belgium.
⁵ Division of Biochemistry, Department of Molecular Cell Biology, KULeuven, Herestraat 49, B-3000 Leuven, Belgium.

View larger version (77K): [in this window] [in a new window]   Fig. 1. Ttrap knockdown affects heart looping. Hearts were visualized via WISH for cmlc2 at 48 hpf. (A) Control embryo, normal heart looping. (B-D) TtrapMO embryos with reversed heart looping, no looping or cardia bifida. Arrowheads depict bilateral hearts in D. Frontal views; a, atrium; v, ventricle.

View larger version (110K): [in this window] [in a new window]   Fig. 2. Ttrap knockdown with higher MO dose induces gastrulation defects. (A,B) TtrapMO embryos (B) with thickened germ ring at shield stage and a less distinct shield (arrowheads) than control (A). Animal views, dorsal on top (gr, germ ring; sh, shield). (C-F) ttrap is essential for CE movements. papc/myod-marked paraxial mesoderm cells fail to converge close to the midline in TtrapMO embryos (D,F). Dorsal-posterior views, tailbud stage. Double-headed arrows depict width between cells spanning the midline. (G-I) ttrap is required during epiboly. (G) Control embryo (3 ss) showing blastopore closure (red arrowhead) and normal head with polster (blue). (H,I) TtrapMO embryos (3 ss) showing varying degrees of severity with respect to epibolic movements. (H) TtrapMO embryo displaying mild epiboly defect (red arrowheads), which shows only slightly open blastopore and relatively normal head morphology with polster (blue). (I) TtrapMO embryo with more severe epiboly defect and larger open blastopore (red arrowheads). Downward spread of blastoderm cells only covers 80% of yolk cell; many of these embryos lyse shortly after; head region severely reduced in size with polster missing (blue arrowhead). The combination of CE and epiboly defects leads to severely truncated embryos. Blue dashed arrows and semi-circle depict embryo length and angle between anterior-posterior (AP) ends. Lateral views, dorsal to the right. (J,K) Live controlMO versus TtrapMO embryo, 24 hpf. The morphant embryo displays AP-axis truncation, microcephaly and micropthalmia.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Randomized LR gene expression and organ laterality in TtrapMO embryos. (A-E) Left-sided, right-sided, bilateral or absent/reduced marker expression. For all panels, dorsal views, anterior at top; single arrows denote sidedness, double arrows bilateral expression. (A) bmp4 in cardiac primordium (22 ss). Dashed white line denotes midline (L, left; R, right). (B,C) pitx2 and spaw in LPM (22 ss). (D) foxA3, 48 hpf (orange arrow, liver, liv; red arrow, pancreas, pa). (E) lft1 in the diencephalon (22-24 ss). Dashed line indicates midline; arrows indicate the relevant altered expression domain. (F) Bar graphs showing percentage of embryos with sided expression of these markers within each phenotypic category (y-axis), in controlMO and TtrapMO embryos (x-axis). Blue bars, left-sided expression; red, right-sided; yellow, bilateral; purple, absent/reduced expression. n=total embryos from two experiments.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Expression of ttrap and its role during Kupffer's vesicle formation. (A) Transcripts are detectable by RT-PCR throughout the first 24 hpf (MBT, mid-blastula transition; epi, epiboly; ss, somite stage). β-actin sequences are shown as loading control (NC, negative control; -RT, no reverse transcriptase). (B-E) Whole-mount ISH (WISH) analysis of ttrap. (B) Maternal ttrap mRNA contribution during early cleavage stages. (C) At 30% epiboly, transcripts are distributed throughout the blastula. Lateral views, animal pole at top. (D) At 60% epiboly, expression in DFCs becomes detectable (arrowheads). Dorsal view, animal pole at top. (E) In addition to the uniform expression throughout the embryo, stronger expression within tailbud and surrounding Kupffer's vesicle (KV, arrow) from 5 ss was observed. (F-I) Absence of KV in TtrapDFCMO embryos. (F) KV is present in controlDFCMO embryo and not detectable in (G) TtrapDFCMO embryo (16 ng MO; arrowheads, KV). Embryos scored live (5-9ss). Posterior views; nc, notochord. (H,I) WISH for cxcr4a (5-9 ss) to confirm KV in (H) controlDFCMO embryo and (I) absence in TtrapDFCMO (arrowheads). Posterior views.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Ttrap associates with Smad3 and Alk4 in mammalian cells. (A-C) Co-immunoprecipitation of TTRAP with Smad3 but not Smad2/4. HA-tagged mouse Smad2, -3 or -4 were co-produced with Flag-TTRAP (+), or Flag-TTRAP-frameshift as control (-), in HEK293T cells and precipitated using anti-Flag antibody. Precipitates were immunoblotted and co-precipitated proteins detected with anti-Smad3 or anti-HA antibody for Smad2 or Smad4. Star indicates IgG and arrow shows lack of co-immunoprecipitation with Smad2/4. (D) TTRAP-Alk4 interaction. Myc-Alk4 and Flag-TTRAP (+) or control (-) were co-produced and precipitated using anti-Flag antibody. Precipitates were analyzed with anti-Myc (top panel). Star, IgG; arrow, co-immunoprecipitation of Alk4.

View larger version (73K): [in this window] [in a new window]   Fig. 6. ALK4 phosphorylates TTRAP. (A) SDS-PAGE showing in vitro phosphorylation of TTRAP (arrowhead) and ALK4 autophosphorylation (arrow), when incubated with [-32P]ATP and ALK4 kinase (+; - denotes no ALK4 added). (B) LC-MS/MS plot depicting in vitro phosphopeptides with T88(phos) and T92(phos). (C,D) Phospho-T88 and phospho-T92 are essential for Ttrap function. (C) mRNA injection, yielding overproduction of TTRAPT88A/T92A, is compatible with normal gastrulation. Live embryo at 80% epiboly showing normal germ ring (gr) and emerging dorsal axial structures (arrowhead). (D) Injection of TTRAPT88A/T92A is incapable of rescuing TtrapMO defects, as evidenced by thickened germ ring and lack of shield/axial structures. These embryos showed a severe delay in epiboly and appeared as if they had not passed germ ring stage even at 8 hpf (normally 80% epiboly). The defects observed in TtrapMO embryos were indistinguishable from TtrapMO+TTRAPT88A/T92A-injected embryos (not shown). Animal views, dorsal to the right.

View larger version (57K): [in this window] [in a new window]   Fig. 7. Ttrap knockdown modulates Nodal-Alk4 signaling. (A) Ttrap knockdown increases activity of ARE-luciferase reporter. ARE-lux plasmid (50 pg) was co-injected with TtrapMO (or controlMO) and embryo lysates assayed for luciferase at shield stage. Knockdown results in fivefold greater induction relative to control (4.6±2.5; P0.01; Student's unpaired t-test; eight independent experiments). No significant increase in luciferase was detectable for control pGL3. (B) TtrapMO potentiates ARE-lux by sqt or cyc. This experiment was performed as described in A, this time in combination with sqt or cyc mRNA injection (11 pg). The addition of TtrapMO induced ARE-lux an additional tenfold relative to induction by either one of the ligands [81.0±15.6 (ARE+sqt+TtrapMO) vs 9.0±2.6 (ARE+sqt); 38.0±8.6 (ARE+cyc+TtrapMO) vs 3.8±0.8 (are+cyc); P0.0001, One-way analysis of variance (ANOVA)]. y-axis, fold-induction of luciferase. (C-F) bon is visibly upregulated in TtrapMO and Smad3bOE embryos; WISH at 50% epiboly, animal views (asterisks). (G,H) TtrapMO-mediated increase in bon expression depends on intact alk4 activity. (G) TtrapMO embryo shows strong expression of bon, whereas (H) TtrapMO embryo treated with SB431542 no longer expresses bon. Animal views (asterisks). (I-N) Overexpression of Smad3b causes CE and epiboly defects. WISH at 90% epiboly, paraxial mesoderm marker expression in βgalOE (700 pg) and Smad3bOE (700 pg) embryos. Dorsal-posterior views, anterior at top. (I,L) papc cells fail to converge near the midline in Smad3bOE embryos compared with control βgalOE embryos. Arrowheads indicate blastopore opening, which is wider in Smad3bOE embryos. (J,M) Distance between myod cells is greater in Smad3bOE relative to control embryos (double-headed arrows). (K,N) Live observation of β-galOE and Smad3bOE embryos, 90% epiboly. (K) Control β-galOE embryo displaying normal epiboly and nearing blastopore closure. (N) Smad3bOE embryo showing severe delay in epiboly. Red arrowheads, edge of blastoderm margin. Lateral views, anterior at top. Note that the gastrulation defects depicted here are at an earlier timepoint than those shown for TtrapMO embryos (see Fig. 2). Importantly however, the same gastrulation defects were also observed for TtrapMO embryos at this earlier stage (i.e. 90% epiboly).

View larger version (48K): [in this window] [in a new window]   Fig. 8. Double Ttrap and Smad3b knockdown rescues CE defect in Ttrap knockdown. myod marks paraxial/adaxial mesoderm (8 ss). (A) Broadened somites and a wide distance between myod cells in Ttrap single knockdown embryo. (B) A combination of 16 ng TtrapMO and 1 ng Smad3bMO shows closer convergence of myod cells at midline. However, somitic expression is still broad relative to the fully rescued embryo co-injected with 2 ng Smad3bMO (C), which now displays the normal myod domain. Dorsal views, anterior at top.

View larger version (74K): [in this window] [in a new window]   Fig. 9. Aberrant DFC migration and clustering as a result of Ttrap-Smad3-mediated downregulation of cdh1. (A-D) cas/sox32 as DFC marker, 80-90% epiboly. (A,C) Tight clustering of DFCs (black arrowheads) in controlMO and βgalOE embryos. (B,D) In TtrapMO and Smad3bOE embryos, DFCs are more spread out. Occasionally, DFCs are also ectopically located around and just underneath the blastoderm margin (black asterisks). (E-H) cdh1 is absent in DFCs and anterior axial hypoblast of TtrapMO and Smad3bOE embryos, 70% epiboly. General cdh1 expression in the epiblast remains unaltered in all embryos; the embryos are deliberately overstained. (E,G) cdh1 in DFCs (black arrowheads) and anterior axial hypoblast (white arrowheads) visible in controlMO and βgalOE embryos and missing in (F) TtrapMO and (H) Smad3bOE embryos. In Smad3bOE embryos, stronger staining in the prechordal plate (white asterisk) was also observed and may be a consequence of a thickening of this region because of hyperdorsalization. Dorsal views, anterior at top.

View larger version (49K): [in this window] [in a new window]   Fig. 10. Knockdown of Ttrap induces ectopic snai1a in the shield and DFCs. (A-D) WISH for snai1a, 60% epiboly. (A,B) snai1a transcripts are found in the margin and paraxial mesoderm in TtrapMO embryos, but are also present ectopically in the presumptive axial mesodermal region within the shield (red arrowheads and arrows). (C,D) Rescued Ttrap and Smad3b MO double-knockdown embryos display normal snai1a domain, particularly, the exclusion of snai1a from the shield (black arrowhead and arrows). Dorsal views, anterior at top. (E) Derepression of snai1a in axial mesoderm of TtrapMO embryos is mediated by Smad3. Graph depicts partial rescue of TtrapMO-induced snai1a phenotype in Ttrap and Smad3 double knockdowns. y-axis represents percentage of embryos exhibiting either snai1a exclusion from axial mesoderm (purple bars) or ectopic expression in axial mesoderm (blue bars), at 60% epiboly. x-axis represents types of MO treatment. 81% of TtrapMO embryos display abnormal snai1a domain, whereas simultaneous knockdown of Ttrap and Smad3 reverts up to 58% of these embryos back to the wild-type domain (purple bars).