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Figure 4. Expression patterns of selected retinoic acid receptor (RAR) target genes. Expression pattern of selected upregulated genes at stage 18 and stage 22 were determined by whole-mount in situ hybridization. Embryos were hybridized with probes made from indicated expressed sequence tag (EST) clones. The staining patterns in whole and sectioned embryos were summarized and used to categorize the genes broadly into four groups (group 1, 2, 3, and 4) by expression patterns. The signature staining of group 1 is present in the neural tube, neural crest, and optic anlagen; the staining patterns of group 2 are similar to those of group 1 but absent from the neural crest. Group 3 genes show staining in the neural tube but not in the optic anlagen. Group 4 genes have staining in the lateral plate mesoderm where RALDH2 is predominantly expressed. A, B, and C are in group 1; D, E, and F are in group 2; G, H, and I are in group 3; and J, K, and L are in group 4. A1-L1, A3-L3, M1-R1, and M2-R2: dorsal view; A2-I2 and A4-I4: anterior view; C6 and J2-L2: lateral view; C7: ventral view; C5: J4-L4 and M3-R3: sagittal section of stage 22 embryos. VLCS, very-long-chain acyl-CoA synthetase; Hypothetical, similar to ESTs from other organisms; Unidentified, no significant similarity to database sequences from any organism; Sm, somites; rp, roof plate of the neural tube; no, notochord; nt, the neural tube.
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Fig. 2. Northern blot analysis. Equal amounts of total RNA isolated from controls or embryos
treated with TTNPB or AGN 193109 were electrophoresed, blotted to nylon membranes, and
hybridized with probes derived from each clone. The name of each clone used for probe-templates
and its closest match found in public databases are given on the left of each panel. Fold and (PCR)
indicate change in microarray analysis and a validation result of quantitative real-time reverse
transcriptase-polymerase chain reaction (QRT-PCR). C, T, and 109 indicate control, TTNPB, and
AGN193109 treatment, respectively. The asterisks indicate known RA target and/or posterior
marker genes, and ISH indicates clones used as probes for whole-mount in situ hybridization. MK,
Midkine; VLCS, very-long-chain acyl-CoA synthetase; SDR1, short-chain dehydrogenase/reductase;
LCS, long-chain acyl-CoA synthetase.
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Fig. 5. The effects of modulated retinoid signaling on expression pattern of retinoic acid receptor
(RAR) target genes. The effects of AGN 193109, TTNPB, or vehicle control on expression pattern
of three expressed sequence tags (ESTs), XL016p20 (S1�S6), XL049b08 (T1�T6), and XL043c08
(U1�U6), were determined in stage 18 embryos by whole-mount in situ hybridization. AGN193109
treatment reduced posterior neural expression of all three genes compared with controls (S1 vs. S2,
T1 vs. T2, and U1 vs. U2) but increased medial staining of XL016p20 (S1 vs. S2), XL049b08 staining
in the eye anlagen (T4 vs. T5), and XL043C08 expression in the forebrain (U1 vs. U2). In contrast,
TTNPB treatment increased expression of XL016P20 throughout the embryo (S3, S6 vs. S2, S5)
and reduced Xl043C08 staining in the forebrain and presumptive trigeminal ganglion (U3, U6 vs.
U2, U5). S1�S3, T1�T3, and U1�U3: dorsal view; S4�S6, T4�T6, and U4�U6: anterior view; TTNPB,
TTNPB-treated embryos; Control, vehicle control embryo; AGN193109, AGN193109-treated
embryos.
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cyp11a1 (cytochrome P450, family 11, subfamily A, polypeptide 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
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nkain4 (Na+/K+ transporting ATPase interacting 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
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pkdcc.2 (protein kinase domain containing, cytoplasmic) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, dorsal up, anterior left.
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shd (Src homology 2 domain containing transforming protein D) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
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slc27a2 (solute carrier family 27 (fatty acid transporter), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
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tfcp2l1 (transcription factor CP2-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
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tmem38a (transmembrane protein 38A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
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tmem150c (transmembrane protein 150C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
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1a11 (71 kDa protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, dorsal up (J1 left), and lateral view (J2, right) anterior left.
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1a11 (71 kDa protein) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, dorsal up (J3 left), and lateral view (J4, right) anterior left.
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