Xenbase Image ID: 86697

FIGURE 5. dhrs3 morphants exhibit shortened axis and reduced head structure. A, 25-base-long morpholino was designed to target the 5′-UTR region of dhrs3. The start codon ATG is underlined. B, Western blot shows that Dhrs3 MO inhibited in vitro translation of C-terminal FLAG-tagged full-length dhrs3 (Dhrs3-FLAG). Dhrs3-FLAG contained the targeting sequence of Dhrs3 MO. C, Dhrs3 MO decreased translation of Dhrs3-FLAG in embryos. Embryos were first injected with dhrs3 mRNA with or without Dhrs3 MO, followed by protein extraction at stage 10. Anti-FLAG antibody was used to detect Dhrs3-FLAG, and endogenous actin was employed as the loading control. D, stage 35 embryos injected with 30 ng of Dhrs3 MO at the two-cell stage or treated with 1 μM atRA during gastrulation showed a shortened AP axis and reduced head structure compared with uninjected embryos. Co-injection of 1 ng of dhrs3 mRNA lacking 5′-UTR rescued the morphant phenotypes. E, quantification of the relative head diameter of uninjected and Dhrs3 MO-injected embryos. F, expression of en2, krox20, and hoxb3 in Dhrs3 morphants. At the four-cell stage, one dorsal blastomere was injected with 12.5 ng of Dhrs3 MO together with 100 pg of lacZ mRNA as the lineage tracer. Injected side is on the right (asterisk). Whole mount in situ hybridization was performed at stage 19. Expression of en2, krox20, and hoxb3 was localized in the anterior neuroectoderm. Knockdown of dhrs3 suppressed the expression of these three genes on the injected side (asterisk). Percentages of embryos showing the phenotype are marked in the figure. G, real time PCR indicated that expression level of en2, krox20, and hoxb3 at stage 19 was down-regulated by dhrs3 knockdown. ***, analysis of variance, p < 0.0005. Image published in: Kam RK et al. (2013) Copyright © 2013. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Gene Synonyms Species Stage(s) Tissue en2.L En-2, en2-a, en2-b, eng2, engrailed 2, engrailed homolog 2, engrailed-2, maben, xen2 X. laevis Throughout NF stage 18 presumptive midbrain-hindbrain boundary egr2.L EGR-2, Krox-20, krox20, XKr20, XKrox-20 X. laevis Throughout NF stage 18 rhombomere rhombomere R3 rhombomere R5 hoxb3.S hox2.7, hoxb-3, LOC108700804, LOC108702517 X. laevis Throughout NF stage 18 rhombomere rhombomere R5 Image source: Published Larger Image Printer Friendly View

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