Journal: The International journal of developmental biology

Article Title: Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

doi: 10.1387/ijdb.092922lh

Figure Lengend Snippet: Fig. 1. IGF-2 morpholinos efficiently and specifically knockdown IGF-2 in vivo. (A) Phenotype of (a) control injected and (b) IGF-2(a+b) morphant embryo at 24 hpf. IGF-2(a+b) morphant embryos are ventralised with a shorter body, disrupted brain structures (double arrows), reduced eyes, disrupted somites and an expanded intermediate cell mass (arrow). (B) Western blot showing IGF-2 and alpha-tubulin at 24 hpf in (1) uninjected control embryos, (2) embryos injected with control morpholinos or (3) embryos injected with IGF-2(a+b) morpholinos. (C) Igf-2a and igf-2b mediate their effects on development in a synergistic fashion. (D) Co-injection of igf-2a or igf-2b RNA with the corresponding morpholino rescues the IGF-2 morphant phenotype. Rescued embryos display normal body size, eyes and brain structures at 24 hpf. Results shown are a summary of three independent experiments (n ≥ 80). Error bars indicate the standard deviation of the mean.

Article Snippet: The membrane was incubated with primary antibodies rabbit anti-IGF-2 (1:1000, Gropep PAAA1) or mouse anti-α-tubulin (1:2000, Sigma) and then with the secondary antibodies rabbit peroxidase-conjugated (1:140,000, Sigma) or mouse peroxidase-conjugated (1:8000, Sigma).

Techniques: Knockdown, In Vivo, Control, Injection, Western Blot, Standard Deviation

Journal: The International journal of developmental biology

Article Title: Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

doi: 10.1387/ijdb.092922lh

Figure Lengend Snippet: Fig. 3. IGF-2 regulates the expression of genes involved in dorsal-ventral patterning. (A,C) Embryos injected with control morpholinos show normal chordin and goosecoid expression. (B,D) Expression of chordin and goosecoid is reduced in IGF-2(a+b) morphant embryos (arrows). (E,G,I) Embryos injected with control morpholinos show normal bmp2b, bmp4 and gata2 expression. (F,H,J) Expression of bmp2b, bmp4 and gata2 is expanded towards the dorsal side in IGF-2(a+b) morphant embryos (asterisks). Frequency of embryos displaying this staining pattern; B, 13/26; D, 17/31; F, 16/25; H, 18/32; J, 26/34. All embryos are shown in a lateral view.

Article Snippet: The membrane was incubated with primary antibodies rabbit anti-IGF-2 (1:1000, Gropep PAAA1) or mouse anti-α-tubulin (1:2000, Sigma) and then with the secondary antibodies rabbit peroxidase-conjugated (1:140,000, Sigma) or mouse peroxidase-conjugated (1:8000, Sigma).

Techniques: Expressing, Injection, Control, Staining

Journal: The International journal of developmental biology

Article Title: Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

doi: 10.1387/ijdb.092922lh

Figure Lengend Snippet: Fig. 2. IGF-2 is required for the development of anterior neural structures during gastrulation and plays an anti-apoptotic role during segmentation. (A,C) Control morpholino injected embryos show normal pax6.2 and rx3 expression. (B,D) IGF-2(a+b) morphant embryos show reductions in pax6.2 and rx3 expression. (E) Embryos injected with control morpholinos show low levels of apoptosis during zebrafish segmentation. (F) Knockdown of igf-2a and igf-2b results in an increase in apoptosis in the anterior region of the embryo and in the developing spinal cord. Frequency of embryos displaying this staining pattern; B, 14/ 32; D, 22/40; F, 17/28. (A-D) are views of the future anterior region; (E,F) are lateral views.

Article Snippet: The membrane was incubated with primary antibodies rabbit anti-IGF-2 (1:1000, Gropep PAAA1) or mouse anti-α-tubulin (1:2000, Sigma) and then with the secondary antibodies rabbit peroxidase-conjugated (1:140,000, Sigma) or mouse peroxidase-conjugated (1:8000, Sigma).

Techniques: Control, Injection, Expressing, Knockdown, Staining

Journal: The International journal of developmental biology

Article Title: Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

doi: 10.1387/ijdb.092922lh

Figure Lengend Snippet: Fig. 4. Blood circulation is disrupted when IGF signalling is reduced. (A) Control morpholino injected embryos stained with O-dianisidine at 72 hpf show normal blood circulation. (B) IGF-2(a+b) morphant embryo with reduced circulating blood. Grey arrows indicate blood in the heart region, red arrows blood circulation in the intersomitic vesicles and grey arrowheads the intermediate cell mass. (C,F) Embryos injected with control morpholinos showing normal scl and gata1 expression at 26 hpf. (D,G) Knockdown of igf-2a and igf-2b results in an increase in scl and gata1 expression and an expansion of expression outside the intermediate cell mass. (E,H) DN-IGF-1R injected embryos show an increase in scl and gata1 expression. Arrowhead indicates position of intermediate cell mass and arrows indicate extent of expression along the embryo. Frequency of embryos displaying this staining pattern; B, 73/86; D, 37/48; E, 30/30; G, 48/58; H, 26/29. All embryos are shown in lateral view.

Article Snippet: The membrane was incubated with primary antibodies rabbit anti-IGF-2 (1:1000, Gropep PAAA1) or mouse anti-α-tubulin (1:2000, Sigma) and then with the secondary antibodies rabbit peroxidase-conjugated (1:140,000, Sigma) or mouse peroxidase-conjugated (1:8000, Sigma).

Techniques: Control, Injection, Staining, Expressing, Knockdown

Journal: The International journal of developmental biology

Article Title: Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

doi: 10.1387/ijdb.092922lh

Figure Lengend Snippet: Fig. 5. Angiogenesis is compromised when IGF signalling is reduced. (A- D) fli1:EGFP transgenic em- bryos injected with control morpholinos showing normal vascular development. (E-H) Class I IGF-2 morphant em- bryo. The intensity of GFP expressing cells in the head and eyes is reduced while the basic pattern of vasculature remains intact. The intermediate cell mass is mildly expanded and the intersomitic vessels are reduced. By 72 hpf, the parachordal vessel is incompletely formed. (I-L) Class II IGF-2 morphant embryo. The sprouting of vessels in the head and eyes are reduced. Intersomitic vessel sprouting is irregular and reduced, the intermediate cell mass is expanded and the parachordal vessel is disrupted. (M,N) Control embryo at 26 hpf showing normal expression of flk1 in the vasculature. (O,P) Angiogenesis is disrupted in DN-IGF-1R injected embryos as shown by flk1 expression (n=14/29). White arrows indicate the vasculature in the head and eyes. White arrowheads point to the intersomitic vessels, red arrow points to the intermediate cell mass and the grey arrow indicates the parachordal vessel. All embryos are shown in a lateral view.

Article Snippet: The membrane was incubated with primary antibodies rabbit anti-IGF-2 (1:1000, Gropep PAAA1) or mouse anti-α-tubulin (1:2000, Sigma) and then with the secondary antibodies rabbit peroxidase-conjugated (1:140,000, Sigma) or mouse peroxidase-conjugated (1:8000, Sigma).

Techniques: Transgenic Assay, Injection, Control, Expressing