Zheng X et al. (2015), cnrip1 is a regulator of eye and neural develop...

XB-ART-50168

Genes Cells 2015 Apr 01;204:324-39. doi: 10.1111/gtc.12225.

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cnrip1 is a regulator of eye and neural development in Xenopus laevis.

Zheng X , Suzuki T , Takahashi C , Nishida E , Kusakabe M .

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Cannabinoid receptor interacting protein 1 (CNRIP1), which has been originally identified as the binding partner of cannabinoid receptor 1 (CNR1), is evolutionarily conserved throughout vertebrates, but its physiological function has been unknown. Here, we identify a developmental role of CNRIP1 using Xenopus laevis embryos. During early embryogenesis, expression of Xenopus laevis cnrip1 is highly restricted to the animal region of gastrulae where neural and eye induction occur, and afterward it is seen in neural and other tissues with a temporally and spatially regulated pattern. Morpholino-mediated knockdown experiments indicate that cnrip1 has an essential role in early eye and neural development by regulating the onset of expression of key transcription factor genes, sox2, otx2, pax6 and rax. Also, over-expression experiments suggest that cnrip1 has a potential to expand sox2, otx2, pax6 and rax expression. These results suggest an instructive role of Xenopus laevis cnrip1 in early eye and neural development. Furthermore, Xenopus laevis cnr1 knockdown leads to eye defects, which are partly similar to, but milder than, those caused by cnrip1 knockdown, suggesting a possible functional similarity between CNRIP1 and CNR1. This study is the first characterization of an in vivo role of CNRIP1 in the context of whole organisms.

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Species referenced: Xenopus laevis
Genes referenced: cnr1 cnrip1 frzb2 myc nppa odc1 otx2 pax6 rax sox2 uqcc6
???displayArticle.morpholinos??? cnr1 MO1 cnr1 MO2 cnrip1 MO1

Phenotypes: Xla Wt + cnr1 MO (fig.7.c) [+]

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	Figure 1 Expression of cnrip1 in early Xenopus development. (A) qRT-PCR analysis of cnrip1 mRNA expression during Xenopus development. The expression level of cnrip1 was normalized to that of ornithine decarboxylase (odc). The bar graph indicates the average ratio of the normalized expression level in each stage to that in stage 33/34, from three independent experiments. The error bar represents SD. (B–Q) Whole-mount in situ hybridization for cnrip1 mRNA at stage 10.5 (B, C), 12 (D, E), 15 (F, G), 19 (H, I), 25 (J-M), 33/34 (N-Q). (B, D) Animal views. (C, E) Lateral views with blastopores downwards (arrowheads). (F, H, M, Q) Dorsal views with anterior upwards. (G, K) Anterior views with dorsal upwards. (I, J, N) Lateral views with dorsal upwards and anterior leftwards. (L) A transverse trunk section of the middle part of a stage 25 embryo. (O) A magnified view of the stage 33/ 34 embryo shown in N. (P) A transverse trunk section of the middle part of a stage 33/34 embryo. anp, anterior neural plate; np, neural plate; nt, neural tube; not, notochord; cg, cement gland; tp, trigeminal placode; op, olfactory placode; fb, forebrain; pg, pineal gland; mb, midbrain; hb, hindbrain; e, eye; ap, alar plate; bp, basal plate.
	Figure 2 cnrip1 knockdown disrupts eye and neural development. (A) Control MO or cnrip1 MO (40 ng each/embryo) was coinjected with N- or C-terminally myc-tagged cnrip1 mRNA (1 ng each/embryo) at the 2-cell stage. GFP mRNA (200 pg/ embryo) was also co-injected to check that the injection was successful and that the protein loading was controlled. Injected embryos were collected at stage 10.5 and extracted for immunoblotting analysis. (B, C) Control MO, cnrip1 MO or cnrip1-5mis MO (10 ng each/blastomere) was injected into the animal region of two dorsal blastomeres at the 4-cell stage. The representative results from three independent experiments are shown. (B) Stage 19, anterior views with dorsal upwards. Embryos injected with control MO developed normally (100%, n = 65). Embryos injected with cnrip1 MO displayed open neural tube defects (96%, n = 72). Embryos injected with cnrip1-5mis MO developed normally (100%, n = 70). (C) Stage 37/38, lateral views with dorsal upwards and anterior leftwards. Embryos injected with control MO developed normally (100%, n = 65). Embryos injected with cnrip1 MO displayed head defects, eye defects and shortened curved body axes (100%, n = 72). Embryos injected with cnrip1- 5mis MO developed normally (100%, n = 70).
	Figure 3 cnrip1 knockdown reduces expression of sox2, otx2, pax6 and rax. (A) qRT-PCR analysis for the pan-neural marker sox2 (stage 10.5 and 12.5), the anterior marker otx2 (stage 10.5 and 12.5), the eye and neural marker pax6 (stage 12.5 and 24) and the eye marker rax (stage 12.5 and 24) in embryos injected with control MO or cnrip1 MO (10 ng each/blastomere) into the animal region of two dorsal blastomeres at the 4-cell stage. The expression level of each gene was normalized to that of odc. Values are mean SD of three independent experiments; *P < 0.01; P < 0.05. (B) Whole-mount in situ hybridization was carried out on MO-injected embryos. Control MO or cnrip1 MO (10 ng each) was injected into the animal region of the left dorsal blastomere at the 4-cell stage. The expression of sox2, otx2, pax6 and rax was reduced in 100% of cnrip1 MO-injected embryos (sox2, stage 10.5, n = 20; sox2, stage 12.5, n = 24; otx2, stage 10.5, n = 20; otx2, stage 12.5, n = 24; pax6, stage 12.5, n = 30; pax6, stage 24, n = 24; rax, stage 12.5, n = 24; rax, stage 24, n = 24). Anterior views with dorsal upwards. The representative results from two (sox2, stage 10.5; otx2, stage 10.5) or three (sox2, stage 12.5; otx2 stage 12.5; pax6, stage 12.5 and 24; rax, stage 12.5 and 24) independent experiments are shown. The black brackets indicate MO-injected left sides. The dashed lines indicate midlines of embryos.
	Figure 4 Effects of cnrip1 over-expression on spatial expression patterns of sox2, otx2, pax6 and rax. Whole-mount in situ hybridization analysis was carried out on embryos uninjected or injected with 500 pg of cnrip1 mRNA into the left dorsoanimal blastomere at the 8-cell stage. The representative results from two (B, C) or three (A, D, E) independent experiments are shown. Anterior views with dorsal upwards. The black brackets indicate mRNA-injected left sides. The dashed lines indicate midlines of embryos. (A) sox2 expression at stage 12.5. Injected sides of embryos (n = 24) displayed expanded expression (29%, second and third left) or no obvious change (71%, fourth left to rightmost). (B) otx2 expression at stage 12.5. Injected sides of embryos (n = 20) displayed expanded expression (15%, second and third left) or no obvious change (85%, fourth left to rightmost). (C) otx2 expression at stage 24. Injected sides of embryos (n = 22) displayed expanded expression (23%, second and third left), expanded expression in the brain but reduced expression in the eye (14%, fourth left), or no obvious change (64%, rightmost and second right). (D) pax6 expression at stage 24. Injected sides of embryos (n = 32) displayed expanded expression (31%, second and third left), expanded expression in the brain but reduced expression in the eye (34%, fourth left), or no obvious change (34%, rightmost and second right). (E) rax expression at stage 24. Injected sides of embryos (n = 30) displayed expanded expression (47%, second and third left), reduced expression (10%, fourth left), or no obvious change (43%, rightmost and second right).
	Figure 5 Partial rescue of eye defects in cnrip1 knockdown embryos. Cnrip1 MO (5 ng) was co-injected with or without myccnrip1 mRNA (350 pg) into the left dorsoanimal blastomere of 8-cell stage embryos. Injected embryos were cultured until stage 37/38. (A) The respective morphologies are shown as lateral views with anterior leftwards and dorsal upwards. (B) Left bar, 64% of embryos injected with cnrip1 MO alone exhibited severe eye defects (absent or very small eyes). Seventy-one embryos were examined in three independent experiments. Right bar, 51% of embryos injected with cnrip1 MO plus myc-cnrip1 mRNA displayed severe eye defects. Ninety-six embryos were examined in three independent experiments. Values are mean SD of the percentages of embryos with severe eye defects from three independent experiments; *P < 0.05. ‘Very small eyes’ include small eyes with a dot-like, crescent, or gibbous shape, but not small eyes with a nearly round shape.
	Figure 6 Knockdown of cnrip1 in the D1.1 blastomere, but not in the D1.2 blastomere, causes eye and head defects. (A) Schematic diagram of a 16-cell stage Xenopus laevis embryo. (B–E) Cnrip1 MO (2.5 ng) was injected into the left D1.1 or D1.2 blastomere at the 16-cell stage, and injected embryos were cultured until stage 41. The uninjected right sides served as controls. The representative results from two independent experiments are shown. (B, D) Lateral views with anterior leftwards and dorsal upwards. (C, E) Dorsal views with anterior upwards. (B, C) Embryos injected with cnrip1 MO into the left D1.1 blastomere showed eye and head defects in the left sides (100%, n = 40). (D, E) Embryos injected with cnrip1 MO into the left D1.2 blastomere showed no significant defects in eyes and heads, but exhibited slight bending of axes to the left (90%, n = 40).
	Figure 7 cnr1 knockdown leads to eye defects. (A) qRT-PCR analysis of cnr1 mRNA expression during Xenopus laevis development. The expression level of cnr1 was normalized to that of odc. The bar graph indicates the ratio of the normalized expression level in each stage to that in stage 33/34, from each of two independent experiments. (B) Whole-mount in situ hybridization for cnr1 mRNA. The representative results from two independent experiments are shown. Anterior is upwards in the dorsal view and leftwards in the lateral views. Dorsal is upwards in lateral and anterior views. The lateral view of a stage 33/34 embryo is magnified in the lower middle panel. e, eye; br, brain. (C, D) Control MO, cnr1 MO1, cnr1-5mis MO or cnr1 MO2 (20 ng each/blastomere) was injected into the animal region of two dorsal blastomeres at the 4-cell stage. Injected embryos were cultured until stage 41. Lateral views with anterior leftwards and dorsal upwards. Representative results from three independent experiments are shown. (C) Embryos injected with either control MO or cnr1-5mis MO developed normally (control MO, 100%, n = 60; cnr1-5mis MO, 100%, n = 60), whereas those injected with cnr1 MO1 displayed eye defects (100%, n = 60). (D) Embryos injected with control MO developed normally (100%, n = 60), whereas those injected with cnr1 MO2 displayed eye defects (100%, n = 60).
	Figure 8 Effects of cnr1 knockdown on expression of sox2, otx2, pax6 and rax. (A) qRT-PCR analysis for marker gene expression in embryos injected with control MO or cnr1 MO1 (20 ng each/blastomere) into the animal region of two dorsal blastomeres at the 4-cell stage. The expression level of each gene at stage 12.5 or 24 was normalized to that of odc. Values are mean SD of three independent experiments; *P < 0.05. (B, C) Whole-mount in situ hybridization analysis was carried out on embryos injected with 20 ng of control MO or cnr1 MO1 into the animal region of the left dorsal blastomere at the 4-cell stage. The representative results from two independent experiments are shown. Anterior views with dorsal upwards. The black brackets indicate MOinjected left sides. The dashed lines indicate midlines of embryos. (B) A mild reduction of pax6 expression in the cnr1 MO1- injected side was observed at stage 12.5 (52%, n = 21) and stage 24 (39%, n = 18). (C) A mild reduction of rax expression in the cnr1 MO1-injected side was observed at stage 12.5 (44%, n = 18) and stage 24 (56%, n = 16).
	cnrip1 (cannabinoid receptor interacting protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal view, anterior up.
	cnrip1 (cannabinoid receptor interacting protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anterior left, dorsal up.
	cnrip1 (cannabinoid receptor interacting protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	cnrip1 (cannabinoid receptor interacting protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
	cnr1 (cannabinoid receptor 1 (brain)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.