Kato Y and He X (1999), Neuralization of the Xenopus embryo by inhibiti...

XB-ART-12097

J Neurosci November 1, 1999; 19 (21): 9364-73.

Neuralization of the Xenopus embryo by inhibition of p300/ CREB-binding protein function.

Kato Y , Shi Y , He X .

Abstract
p300/ CREB-binding protein (CBP) is a transcriptional coactivator for a plethora of transcription factors and plays critical roles in signal transduction pathways. We report that the inhibition of p300/CBP function in the Xenopus embryo abolishes non-neural tissue formation and, strikingly, initiates neural induction and primary neurogenesis in the entire embryo. The observed neuralization is achieved in the absence of anterior or posterior gene expression, suggesting that neural fate activation and anterior patterning may represent distinct molecular events. We further demonstrate that the neuralizing and anteriorizing activities of chordin and noggin are separable properties of these neural inducers. This study reveals that all embryonic cells possess intrinsic neuralizing capability and that p300/CBP function is essential for embryonic germ layer formation and neural fate suppression during vertebrate embryogenesis.

PubMed ID: 10531441
Article link: J Neurosci
Grant support: [+]
Genes referenced: a2m acvr1 bmp4 celf3 chrd.1 creb1 crebbp ctnnb1 egr2 ep300 gli1 gsc hoxb9 irx2 irx3 krt12.4 mix1 mixer mst1 msx1 ncam1 neurog1 nodal3.1 nodal3.2 nog otx2 sia1 sox17a sox3 tbx2 tbxt tubb2b ventx1.1 wnt8a zic1 zic2 zic3

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	Figure 1. Inhibition of p300/CBP by E1A, E1A–RG2, and p300N interferes with mesoderm and endoderm development. A, Phenotypes of embryos injected dorsally at four-cell stage with E1A, RG2, DL120– 140 (DL), and p300N RNAs, shown at stage 25. E1A, DL120–140, and p300N-injected embryos cleaved and developed normally up through the late blastula stage (stage 9) but began to exhibit severe gastrulation abnormality from the early gastrula stage (stage 10.5). A failure of blastopore closure was observed in many of the injected embryos (stage 13). Most of injected embryos displayed a lack of any axial development at stage 25. The RG2-injected embryo is phenotypically identical to normal controls. RNA injected per embryo: 0.1 ng for E1A, RG2, and DL120–140; 2 ng for p300N. Note that because p300N mRNA and protein sizes are each approximately seven times of those for E1A, the efficiency of synthesis and the local concentration of p300N protein are likely to be much smaller than those of E1A in the embryo if the same amount of RNA were to be injected. B, Sagittal sections of a RG2 (top) and an E1A-injected embryo (bottom) at stage 25. Note that the RG2-injected embryo exhibits normal development, whereas the E1A-injected one lacks any axial tissues, such as the notochord and muscles. Axial tissues are absent in all sections (data not shown). C, Inhibition of Xbra expression examined at stage 10.5 by in situ hybridization. Note that the control (CON) and RG2-injected embryo exhibit a uniform circumferential expression pattern, whereas expression is abolished on the injected side (either dorsal or ventral) in E1A, DL120–140, or p300N-injected embryos. Dorsal is to the right. RNA injected per embryo: 20 pg for E1A, RG2, and DL120–140; 500 pg for p300N. D, Lack of inhibition of Xnr3 expression by in situ hybridization analysis, examined at stage 10.5. Note that the endogenous Xnr3 expression is not blocked by dorsally injected E1A, and an ectopic induction of Xnr3 by ventrally injected b-catenin (0.5 ng RNA per embryo) is not inhibited by coinjected E1A or p300N. Ectopic activation of goosecoid expression by b-catenin was inhibited significantly by E1A, as expected (data not shown), because goosecoid expression depends on mesoderm-inducing signals (Watabe et al., 1995). Dorsal is to the right. RNA amount injected is the same as in C. E, Inhibition of Xsox-17a and Mixer expression assayed by quantitative RT-PCR. In this and all of the following RT-PCR results, EF-1a is used as a control for the template amount. The RNA amount injected is the same as in A. F, Immunoblot of stage 9 embryo extracts to examine protein levels of E1A, E1A–RG2 (RG2), and E1A–DL120–140 (DL) from injected synthetic RNAs. CON, Uninjected control extract.
	Figure 2. Inhibition of mesoderm, endoderm, and epidermis formation in animal pole explants. A, Inhibition of mesoderm markers induced by FGF examined at stage 10.5. WE is the positive control for RT-PCR, using the cDNA from whole embryos at the equivalent stage. RNA injected per embryo: 0.1 ng for E1A, RG, and DL; 3 ng for p300N. bFGF concentration is 50 ng/ml. B, Inhibition of mesoderm and endoderm markers induced by activin examined at stage 10.5. mix1 is a homeobox gene expressed in both mesoderm and endoderm. Endodermin induction by activin was examined at stage 35. The RNA amount that was injected is the same as in A. Activin concentration: 5 ng/ml (for mesoderm induction) and 50 ng/ml (for endoderm induction). C, Inhibition of early BMP-4 target genes induced by an activated ALK2, a BMP-4 receptor. RNA injected per embryo: 0.05 ng for E1A, RG, and DL; 3 ng for p300N; 0.2 ng for ALK2. D, A lack of inhibition of Xwnt-8 or b-catenin signaling. RNA injected per embryo is the same as in A, except 10 pg for Xwnt-8 and 0.1 ng for b-catenin. E, E1A or p300N inhibition of mesodermal and endodermal gene expression was rescued by p300(dl10), which has wildtype p300 function but is refractory to E1A inhibition (Eckner et al., 1994; Lee et al., 1996). RNA injected per embryo is the same as in A, except 1 ng for p300(dl10).
	Figure 3. Neuralization without anteroposterior patterning in animal pole explants. A, Induction of neural markers was examined at stage 20 by RT-PCR. Note the concomitant suppression of cytokeratin, an epidermal marker. RNA injected per embryo: 5 pg for E1A, RG, and DL; 3 ng for p300N; 1 ng for chordin (chd). Note that neurogenin was not induced by chordin at this stage, as expected from published results (see Discussion; Lamb et al., 1993; Papalopulu and Kintner, 1996). Similar results were obtained when noggin was used in place of chordin (data not shown). B, E1A or p300N induction of neural markers and suppression of cytokeratin could be rescued by p300(dl10), coinjected at 1 ng of RNA per embryo.
	Figure 4. Rescue of early neural development in UV-ventralized embryos. RNA injected per embryo: 20 pg for E1A and RG, 3 ng for p300N, and 150 pg for chordin. In situ hybridization and RT-PCR were performed on embryos at stage 14. A, Rescue of zic-r1 expression by E1A and chordin, but not by RG2 or b-galactosidase (used as a lineage-tracing marker). Note that the expression of zic-r1 overlaps with X-gal staining in E1A or chordin (chd)-injected embryos. B, Rescue of neural markers examined by RT-PCR.
	Figure 5. Neural gene induction in ventral marginal explants (prospective ventral mesoderm) and vegetal pole explants (prospective endoderm). RNA injected per embryo: 20 pg for E1A, RG, and DL; 3 ng for p300N. A, Induction of neural markers and suppression of a mesoderm marker in ventral marginal explants examined at stage 20 by RT-PCR. a-Globin is a hematopoietic (ventral mesoderm) marker. B, Induction of neural markers in vegetal explants examined at stage 20 by RT-PCR. Note the concomitant suppression of endoderm markers (see Fig. 1E).