EGF-CFC proteins are essential coreceptors for the TGF-beta signals Vg1 and GDF1 - PubMed (original) (raw)

Figure 1

Dependence of Vg1 and GDF1 signaling on Oep but not on Nodal signals. (A–F) Live zebrafish embryos at 24 hpf, anterior to the left, dorsal up. (G–U) ntl mRNA expression in zebrafish embryos at shield stage, animal pole view; dorsal to the right in H, I, J, K, L, N, O, and Q. (A,G) LacZ control-injected wild type. (B,H) LacZ control-injected MZ_oep_. (A,C,E) Wild-type embryos. (G,J,M,P,S) Wild-type heterozygous siblings (cycm294/+;sqcz35/+ from a cycm294/+; sqtcz35/+ intercross). (B,D,F,H,K,N,Q,T) MZ_oep_ embryos. (I,L,O,R,U) cyc;sqt double mutant embryos. Embryos were injected with 250 pg of LacZ mRNA (A,B,G,H,I); 100 pg (C,J,K,L) or 250 pg (D) of aVg1 mRNA; 100 pg (E) or 250 pg (F,M,N,O) of bGDF1 mRNA; 20 pg of squint mRNA (P,Q,R); and 20 pg (T) or 100 pg (S,U) of activin mRNA. (G) Note the wild-type expression of ntl in marginal cells. (H) In MZ_oep_ mutants, ntl expression is absent dorsally at the margin. Wild-type embryos are severely dorsalized or have ectopic ntl expression when injected with aVg1 (C,J), bGDF1 (E,M), squint (P), and activin (S) mRNA. In contrast, MZ_oep_ embryos injected with aVg1 (D,K), bGDF1 (F,N), or squint (Q) mRNA are indistinguishable from the LacZ control-injected embryos. Similar to wild-type embryos, expression of ntl is expanded in cyc;sqt double mutants injected with aVg1 (L), bGDF1 (O), squint (R), or activin (U) mRNA.

Figure 2

Vg1 and GDF1 signaling is restored in MZ_oep_ embryos by divergent EGF-CFC proteins. (A–T) gsc mRNA expression in zebrafish embryos at shield stage, animal pole view; dorsal to the right in A, C, D, E, F, P, S, and T). (A,F,K,P) Wild-type embryos. (B–-E,G–-J,L–-O,Q–-T) MZ_oep_ embryos. (A) Uninjected wild type. (B) Uninjected MZ_oep_. Note the wild-type expression of gsc in the dorsal organizer. In MZ_oep_ mutants, gsc expression is absent. Wild-type embryos (F,K,P) and MZ_oep_ mutants (G,L,Q) injected with 100 pg of aVg1 (F,G), 250 pg of bGDF1 (K,L), and 100 pg of squint (P,Q) mRNA. gsc is ectopically expressed when aVg1 (F), bGDF1 (K), or squint (P) mRNA are injected in wild-type embryos, but not in MZ_oep_ embryos (G,L,Q, respectively). MZ_oep_ embryos injected with 50 pg each of cryptic (C), cripto (D), or oep (E) mRNA. Note the rescue of the dorsal organizer expression of gsc in injected MZ_oep_. One-hundred picograms of aVg1 mRNA was coinjected into MZ_oep_ with 50 pg each of cryptic (H), cripto (I), or oep (J) mRNA. Two-hundred-fifty picograms of bGDF1 mRNA was coinjected into MZ_oep_ with 50 pg each of cryptic (M), cripto (N), or oep (O) mRNA. One-hundred picograms of squint mRNA was coinjected into MZ_oep_ with 50 pg each of cryptic (R), cripto (S), or oep (T) mRNA. Coinjection of either cryptic, cripto, or oep mRNA with aVg1 (H,I,J, respectively), bGDF1 (M,N,O, respectively), or squint (R,S,J, respectively) mRNA results in ectopic gsc expression in MZ_oep_.

Figure 3

Vg1 and GDF1 signal through the Activin receptors, ActRIIB and Alk4, and Cripto. Synthetic mRNAs encoding aVg1 (50 pg), bGDF1 (50 pg), Alk4/3C (12.5 pg), _x_ActRIIB (12.5 pg), and Cripto (50 pg) were injected into Xenopus embryos. Activation of the chimeric receptor was measured by a BMP response element luciferase reporter, BRE(-243/-191)x4-luc. Results are shown from one experiment and are representative of three independent experiments. Note that Alk4/3C and xActRIIB only are not responsive to aVg1 and bGDF1 ligands. In the presence of Cripto, aVg1 and bGDF1 signaling through ActRIIB and Alk4/3C is greatly enhanced. Low levels of activation can sometimes be seen when coexpressing the ligand and receptors only. This is presumably due to the endogenous expression of EGF-CFC proteins, such as FRL1, in these embryos.

Figure 4

Vg1 and GDF1 binding to the ActRIIB and Alk4 receptor complex requires EGF-CFC proteins. aVg1 (A) and bGDF1 (B) binding to receptor complex. RNAs encoding aVg1/HA (100 pg), xActRIIB/Myc (500 pg), xAlk4 (500 pg), Cripto/Flag (1 ng), bGDF1/HA (2 ng), mActRIIB (KR)/Myc (1 ng), hAlk4(KR)/Flag (1 ng), and Cryptic/Flag (1 ng) were injected into Xenopus embryos. After chemical cross-linking with DTSSP, lysates were immunoprecipitated for either xActRIIB/Myc (A) or mActRIIB(KR)/Myc (B) with α-Myc antibody. Note that Cripto and Cryptic form a complex with the type II and type I receptors and the mature ligands Vg1 and GDF1, respectively. (C) Vg1 and GDF1 interact with EGF-CFC proteins. Synthetic mRNAs encoding Cripto/Flag (1 ng), Cryptic/Flag (1 ng), aVg1/HA (1 ng), and aGDF1/HA (1 ng) were injected into Xenopus embryos. After DTSSP cross-linking, lysates were immunoprecipitated for either aVg1/HA or aGDF1/HA with α-HA antibody. Proteins in the coimmunoprecipitates and total extracts were probed in Western blot analysis with the indicated antibodies: xActRIIB/Myc (∼120 kD; α-Myc), mActRIIB(KR)/Myc (∼120 kD, α-Myc), aVg1/HA (∼22 kD; α-HA), bGDF1/HA (∼22 kD, α-HA), aGDF1/HA (∼22 kD, α-HA), hAlk4(KR)/Flag (∼70 kD; α-Flag), Cripto/Flag (∼30 kD; α-Flag), and Cryptic/Flag (∼25 kD; α-Flag).