Analysis of cyp26d1 expression during zebrafish early development from cleavage to gastrulation using in situ hybridization and RT-PCR. (A–E) Animal pole top; (F–G) dorsal right, anterior top; (H) dorsal forward, anterior top. (A) No maternal cyp26d1 message during cleavage is detected in 3 hpf embryos by whole-mount in situ hybridization. (B) Weak expression of cyp26d1 is first found in inner layer of blastula at sphere stage (4 hpf). (C) The expression is found in all blastomeres at dome stage (4.3 hpf). (D–E) Expression of cyp26d1 is decreased at 30% epiboly (4.7 hpf) (D) and cannot be detected at 50% epiboly (5.3 hpf) (E). (F) cyp26d1 resumes its expression in dorsal region at 75% epiboly (8 hf). (G, H) Expression of cyp26d1 is restricted to two separate regions that would presumptively develop into hindbrain at 90% epiboly (9 hpf). Arrows point to the expression areas of cyp26d1 gene. (I) RT-PCR analysis shows the dynamic change of cyp26d1 expression from cleavage to gastrulation of zebrafish embryos. ct, Positive control lane using cyp26d1 cDNA as template; 3–9 h, lanes using total RNA isolated from 3 to 9 hpf as templates; PCR gives a 837 bp amplified fragment from cyp26d1 cDNA. Amplification of a 716 bp β-actin cDNA fragment is a control of RT-PCR sensitivity in the assay. The dynamic changes of cyp26d1 expression from cleavage to gastrulation as detected by RT-PCR is consistent with that detected by whole-mount in situ hybridization.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: 1k-cell to 90%-epiboly

Expression pattern of cyp26d1 mRNA during segmentation period of zebrafish. (A, J, Q) Lateral view, dorsal right, anterior top; (B–I, K) dorsal view, dorsal forward, anterior top; (L, P) flatmount of anterior part of embryo; anterior top; (M, O) lateral view; anterior left, dorsal top; (N) dorsal view, dorsal forward, anterior left. cyp26d1—blue; krox20—red. (A) Expression of cyp26d1 gene is in presumptive hindbrain region at bud stage (10 hpf). (B) Double in situ hybridized embryos at bud stage with krox20 and cyp26d1 shows that the two separate dorsal regions of cyp26d1 expression are in presumptive r2–r4. (C) cyp26d1 mRNA is present in two stripes that are limited to lateral expression and do not meet at the midline of the anterior neuroectoderm at 2-somite stage (10.5 hpf). (D) Double in situ hybridization shows that zebrafish cyp26d1 is expressed in presumptive r2 and r4 and part of r3 region at 2-somite stage. (E–G) The lateral expression stripes at presumptive r2 merge at the midline of embryos at 2–3-somite stages (10.7–11 hpf) while the expression stripes at r4 do not. At 3-somite stage, cyp26d1 gene is also expressed in the stripes that are presumptive r6 and in pharyngeal arch (pa) one in addition to expression at r2 and r4 (G). (H, I) At 6-somite stage (12 hpf), the expression of cyp26d1 is in continuous bands at r2–r6 and also in pa1 with broad expression at r2 and high expression at r6. (J–P) The expression pattern is maintained from 10-somite stage (J–L), 18-somite stage (M, N) through 21-somite stage (19.5 hpf) (O, P) except that the expression level of the gene is greatly reduced at r2 and hardly detectable in r4 (J–P). At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons (O). (Q) A schematic diagram summarizes expression domains of the three cyp26 genes (cyp26a1, cyp26b1, and cyp26d1) in embryos at 3-somite stage. Blue areas show the expression regions of cyp26a1, red ones mark the expression regions of cyp26d1 and the yellow area is the region where cyp26b1 and cyp26d1 are co-expressed. d, Diencephalon; fb, forebrain; mb, midbrain; pa, pharyngeal arch; r, rhombomere; t, telencephalon; tb, tail bud.

Expression pattern of cyp26d1 mRNA during pharyngula period of zebrafish. (A, C, E, G, I, K, M) lateral view, anterior left, dorsal top; (B, D, F, H, J, L, N) flatmount of anterior part of embryo, anterior top. (A, B) At 25 hpf, cyp26d1 gene is expressed in telencephalon, boundary between telencephalon and diencephalons, midbrain, dorsal part of hindbrain, pharyngeal arches (A) and otic vesicles (B). (C–F) The expression pattern of 25 hpf is maintained from 28 hpf through 31 hpf with high expression levels. Additionally, cyp26d1 is also expressed in the neural retina of eyes during 28–31 hpf (D, F). (G, H) At 35 hpf, the expression of cyp26d1 is mainly restricted to a dorsal part of telencephalon, otic vesicles, pharyngeal arches 2–7 and pectoral fins. At this stage, the expression in neural retina disappears except for a weak presence in lens. (I–L) With development, the expression level in telencephalon and otic vesicles is reduced and the expression at eyes, pharyngeal arches, and pectoral fins is hardly detectable from 42 hpf through 48 hpf. (M, N) Schematic diagrams summarizing the expression domains of the three cyp26 genes (cyp26a1, cyp26b1, and cyp26d1) in embryos at 24 hpf. Blue areas show the expression regions of cyp26a1, green ones mark the expression regions of cyp26b1, red ones show the expression regions of cyp26d1, yellow ones mark the regions where cyp26b1 and cyp26d1 are co-expressed and gray ones are the regions where cyp26a1, cyp26b1, and cyp26d1 are co-expressed. c, Cerebellum; d, diencephalon; ey, eye; h, hindbrain; m, midbrain; mh, midbrain–hindbrain boundary; nc, notochord; ot, otic vesicle; r, rhombomere; pa, pharyngeal arch; t, telencephalon; d, diencephalon.

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Reprinted from Gene expression patterns : GEP, 5(6), Gu, X., Xu, F., Wang, X., Gao, X., and Zhao, Q., Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development, 733-739, Copyright (2005) with permission from Elsevier. Full text @ Gene Expr. Patterns