Proc Natl Acad Sci U S A
December 21, 1999;
X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina.
encodes a Xenopus neuronal-specific basic helix-loop-helix transcription factor related to the Drosophila proneural factor atonal. We show here that Xath3
acts downstream of X-ngnr-1
during neuronal differentiation in the neural plate
and that its expression and activity are modulated by Notch
by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm
have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina
overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1
regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.
Proc Natl Acad Sci U S A
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Figure 1 Sequential expression of X-ngnr-1, Xath3, and NeuroD during primary and retinal neurogenesis. (Upper) Whole-mount in situ hybridizations on stage-11.5, -12, -13, and -14 embryos with probes as indicated on the top of each column. (Lower) Double in situ hybridizations on sections of stage-40 Xenopus retina with X-ngnr-1 (blue) and Xath3 (red) probes. The three panels show the same section. Arrows indicate the positions where X-ngnr-1 and Xath3 expressions appear in the CMZ. (Scale bar = 20 μm.)
Figure 2 Xath3 participates in a network of bHLH genes. (A) Embryos were injected with the indicated RNA (top of each panel). Dorsal views of stage-14 embryos probed by in situ hybridization as indicated (bottom of each panel) are shown. The injected side is indicated (inj) or visualized by light blue staining for LacZ expression. (B) Embryos were injected with RNA encoding a X-ngnr-1-GR fusion protein at the two-cell stage. Blastula animal caps were isolated from injected embryos and were left untreated (lanes 1–3), or were incubated in the presence of CHX (lanes 4–6). After 1 hr of culture, dex was added for 2 (lanes 2 and 5) or 3 (lanes 3 and 6) hr. RNA was prepared and analyzed by RNase protection. RNAs isolated from caps derived from uninjected embryos plus or minus dex are shown in lanes 7 and 8. The addition of CHX did not block the activation of NeuroD expression, but, in contrast, abolished Xath3 expression induced by X-ngnr-1-GR (compare lanes 2 and 3 with 5 and 6). rFGF, fibroblast growth factor receptor.
Figure 3 Relationships between Xath3 and lateral inhibition. (A) Embryos were injected with the indicated RNA (top of each panel). Dorsal views of stage-14 embryos probed by in situ hybridization as indicated (bottom of each panel) are shown. (B) X-MyT1 can function with Xath3 to stimulate N-tubulin expression in the presence of Notch signaling. Animal caps were derived from embryos injected with RNA as indicated and analyzed for N-tubulin expression by RNase protection. rFGF, fibroblast growth factor receptor. Numbers below lanes indicate rFGF-normalized N-tubulin level with maximum level detected set to 10.
Figure 4 Misexpression of Xath3 causes ectopic development of distinct types of neurons. Embryos were injected with the indicated RNA (top of each panel). Embryos were analyzed by in situ hybridization with several different markers, as indicated (bottom of each panel), or by immunostaining with an anti-neurofilament marker (F). The injected side is indicated (inj) or visualized by light blue staining for LacZ expression. (A–E) and (L–N) Dorsal views of neurula-stage embryos. (F, O, and Q) Lateral views (F and O, posterior region; Q, anterior region) of tailbud embryos. (G–J, P, and R) Transverse sections of tailbud embryos (stage 30). (K) Double-labeling Xaml (black) and N-tubulin (red) on a section in the posterior part of a stage-15 embryo. Arrows indicate stained ectopic fibers in F, the endogenous signal on the injected side in H, J, and O, and the expanded signal in P.
Fig. 5. Xath3 and X-ngnr-1 lipofections lead to different effects on retinal
cell fate decisions. Percentage of retinal cell types generated by GFP alone, or
GFP plus Xath3, or GFP plus X-ngnr-1 lipofection. The percentage of each cell
type was calculated as a weighted average. The statistical analysis was performed
using Student’s t tests, except for the Mu¨ ller cell, for which we used a
x2 test because of the very low number of Mu¨ ller cells in each sample. *, P ,
Bellefroid, Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. 1998, Pubmed