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Fig. 4. Biological activity of XXBP-1 (I)Phenotypes of XXBP-1-injected embryos. (A) Examples of ventralized phenotypes after dorsal injection of 200 pg XXBP-1 mRNA at the four cell stage. (D, E) The ventralized embryos exhibited no anteroposterior or dorsoventral axes, after either dorsal injection of 400 pg XXBP-1 mRNA or radial injection of 800 pg XXBP-1 mRNA. (F) A transverse section through the embryo injected dorsally with 200 pg XXBP-1 mRNA shows a reduced notochord (nt) and an indiscernible spinal cord (sc) as compared with the control embryo (F). pn, pronephros; sm, somite. (II) Disruption of neural marker genes in vivo by XXBP-1. A single dorsal blastomere was coinjected with 200 pg XXBP-1 and 100 pg LacZ mRNA at the four cell stage. The embryos were fixed at the late neurula stages and stained for lacZ to identify the injected side, and whole-mount in situ hybridization with antisense probes for the cement gland marker, XAG2, and neural marker genes was carried out. (A) The expression pattern in control embryos is shown at the left, and expression after XXBP-1 overexpression on one side is shown at the right. (A) XAG2 expression was not changed by XXBP-1 overexpression at this dose. (C) XXBP-1 overexpression inhibited the expression of Otx2 (anterior neural marker), Rx2A (lens marker), En2 (marker for the mid/hindbrain boundary), Krox20 (marker for r3 and r5), Pax6 (neural marker), and Sox3 (pan-neural marker). (O, P) Embryos injected only with lacZ mRNA as a control showed no changes in En2 or Sox3 expression. (III) Overexpression of XXBP-1 downregulates the dorsal mesoderm markers, Chordin and goosecoid. Embryos were injected dorsally into the marginal zone with 0.1 ng/blastomere of XXBP-1 mRNA at the four cell stage and fixed at midgastrula for whole-mount in situ hybridization with Chd and gsc antisense RNA. (A, C) Expression in control embryos. (B, D) XXBP-1 overexpression reduced both Chd and gsc expression. (IV) Overexpression of XXBP-1 leads to ventralization of dorsal mesoderm. Dorsal and ventral marker genes were analyzed by using RT-PCR in whole embryos (st. 22 embryo), dorsal or ventral marginal zone explants (DMZ or VMZ, respectively) from uninjected embryos, or embryos radially injected with 0.1 or 0.4 ng XXBP-1 mRNA per blastomere at the four-cell stage. Marginal zones were explanted at the start of gastrulation and cultured until sibling embryos reached stage 22. ODC served as a loading control. RT−, control without reverse transcriptase. (V) XXBP-1 inhibits neural induction in dissociated ectodermal explants. Animal caps were dissected from uninjected embryos (AC) and embryos injected with either 0.4 ng XXBP-1 or 0.8 ng BMP-4 mRNA into all four blastomeres at the four cell stage. The ectodermal cells from the animal caps were dissociated then allowed to reassociate and cultured subsequently for 24 h. (A) RT-PCR was used to analyze the expression of the neural marker, NCAM; ectodermal marker, epidermal keratin (EK); and mesoderm markers, actin and Xbra. BMP-4 was injected as a positive control for inhibition of neural induction. ODC was employed as a loading control. (B) At 24 h after reaggregation, the ectodermal cells from XXBP-1 animal caps were not able to reaggregate as well as cells from control or BMP-4 animal caps. The majority of XXBP-1-overexpressing ectodermal cells did not reaggregate. Only reaggregated cells (indicated by arrow) were harvested for RT-PCR analysis. RT−, control without reverse transcriptase. Image published in: Zhao H et al. (2003) Copyright © 2003. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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