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The basic helix-loop-helix (bHLH) factor Xath5 promotes retinal ganglion cell differentiation when overexpressed and may do so by regulating the expression of factors involved in the differentiation of these cells. Potential candidates include the Brn3 POU-homeodomain transcription factors, which have been implicated in retinal ganglion cell development. Here we have identified a new member of the Brn3 gene subfamily in Xenopus, XBrn3d. In situ hybridization analysis shows XBrn3d expression in developing sensory neurons and developing ganglion cells of the retina. Using a hormone-inducible Xath5 fusion protein, we have shown that in animal caps Xath5 can directly regulate the expression of XBrn3d. Since XBrn3d is also expressed in sensory populations where Xath5 is not expressed, we examined the regulation of XBrn3d expression by the bHLH factor XNeuroD. A XNeuroD-hGR fusion protein is similarly able to directly induce the expression of XBrn3d in animal caps. In addition, overexpression of XBrn3d by RNA injection promotes the expression of ectopic sensory neuronal markers in the lateralectoderm, suggesting a role in regulating neuronal development. Therefore, Xath5 and XNeuroD can directly regulate the expression of a neuronal subtype-specific factor, providing a link between neuronal differentiation and cell fate specification.
FIG. 2. Developmental expression pattern of XBrn3d. Whole-mount in situ hybridization was performed on Xenopus embryos using a
DIG-labeled XBrn3d probe. (A) Expression commences at neurula stages in the trigeminal placodes (arrowhead) and a stripe of primary
neurons along the midline. (B) At stage 26–27, XBrn3d is first detected in the neural retina (arrow). Expression is also seen in the olfactory
placode (right of the retina) and cranial ganglia (left of the retina). (C) At stage 29, XBrn3d is expressed in the optic tectum (arrowhead) and
the spinal cord. (D) At stage 40, cross sections in paraffin-embedded embryos reveal strong retinal staining restricted to the retinal ganglion
cell layer and ciliary marginal zone (arrow) as well as the entire dorsal half of the neural tube at the level of the optic tectum (top, middle).
The pigment epithelial layer surrounds the neural retina (arrowhead) and is not labeled. (E, F) At stage 35, staining is strong in a region of
the developing optic vesicle and dorsal neural tube at the level of the hindbrain and spinal cord, respectively.
FIG. 3. Expression of XBrn3d and Xath5 overlaps in the ciliary marginal zone of the Xenopus retina. Double in situ hybridization was perform ed on stage 41 em bryos using an antisense DIG probe (XBrn3d) and an antisense fluorescein probe (Xath5). (A) Schem atic showing a cross section of the Xenopus retina. The retinal pigment epithelium is shown as a thick line. The retinal ganglion cell layer (RGC) is closest to the lens (L), while the photoreceptor layer (ON L) borders the pigm ent epithelium and the inner nuclear layer (IN L) is between. The ciliary marginal zone (CMZ) is the region of continued proliferation, in which the central CMZ is the portion closest to the differentiated retinal layers. (B) XBrn3d expression is restricted to the retinal ganglion cell layer and the central CMZ. (C) The composite im age of both the Xath5 and the XBrn3d staining shows double-labeled cells. (D) Xath5 is expressed in the CMZ. Double-labeled cells are indicated with brackets (B, D).
FIG. 4. Xath5 can directly activate expression of XBrn3d. (A) The hormone-binding domain of the human glucocorticoid receptor was
fused to the C-terminal end of Xath5 as indicated. (B) Neural plate stage embryos analyzed by whole-mount in situ hybridization for
expression of N-tubulin following injection with Xath5hGR and b-gal mRNAs at the two-cell stage and incubation in the absence (2) or
presence (1) of dexamethasone (DEX). Dorsal is at the top and injected sides are on the right in each of the embryos. Arrowheads indicate
the three stripes of primary neurons formed in each half of the embryos. Xath5hGR had no effect on the initial pattern of neurogenesis in
the absence of the dexamethasone hormone (left), but produced ectopic neurogenesis in the presence of DEX (right), indicating that
transcriptional activity of this fusion protein is tightly regulated by the presence of DEX. (C) RT-PCR of animal caps injected with
Xath5hGR. 1/2 indicates the presence or absence of dexamethasone and cycloheximide (CHX) during incubation of animal caps. DEX
activates the hGR fusion protein. CHX was added 30 min prior to DEX to inhibit protein synthesis, limiting transcription to direct targets
of Xath5. Stage 35 cDNA from a whole embryo was used as a positive control for both XBrn3d and EF1a. EF1a primers were used as a
positive control for cDNA quality and to normalize across cDNAs. Xath5 can directly induce the expression of XBrn3d as indicated by the
amplification of a XBrn3d band in the 1DEX 1CHX lanes.
FIG. 5. XNeuroD can directly activate expression of XBrn3d. (A) The hormone-binding domain of the human glucocorticoid receptor was
fused to the N-terminus of XNeuroD as indicated. (B) Neural plate stage embryos analyzed by whole-mount in situ hybridization for
expression of N-tubulin following injection with XNeuroDhGR and b-gal mRNAs at the two-cell stage and incubation in the absence (2)
or presence (1) of dexamethasone (DEX). Dorsal is at the top and injected sides are on the right in each of the embryos. Arrowheads indicate
the three stripes of primary neurons formed in each half of the embryos. XNeuroDhGR had no effect on the initial pattern of neurogenesis
in the absence of the dexamethasone hormone (left), but produced ectopic neurogenesis (right) in the presence of DEX, indicating that
transcriptional activity of this fusion protein is tightly regulated by the presence of DEX. (C) RT-PCR of animal caps injected with
XNeuroDhGR. 1/2 indicates the presence or absence of dexamethasone and cycloheximide (CHX) during incubation of animal caps. DEX
activates the hGR fusion protein. CHX inhibits protein synthesis, limiting transcription to direct targets of XNeuroD. Stage 35 cDNA from
a whole embryo was used as a positive control for both XBrn3d and EF1a. XNeuroD can directly induce the expression of XBrn3d as
indicated by the amplification of a XBrn3d band in the 1DEX 1CHX lanes.
FIG. 6. Overexpression analysis of XBrn3d. Embryos were injected at the two-cell stage with mRNA for XBrn3d, then analyzed at stage 16 7 or stage 28 2 by whole-m ount in situ hybridization. ﰁ-Gal was coinjected to m ark the injected side of the em bryo. (A) Overexpression of XBrn3d causes loss of N-tubulin expression on the injected side (area under left arrowhead). (B) Expression of XamL, which marks Rohoneard cells, was also reduced at stage 17, indicating a loss of primary sensory neurons (area under left arrowhead). (C, D) At tailbud stages, ectopic N -tubulin positive neurons are seen on the injected side. (E, F) In situ hybridization with the sensory ganglia marker Xhox11L2 at stage 32. Ectopic neurons are seen on the injected side of the embryo, indicating that at least some of these ectopic neurons are sensory in nature. Staining in cranial ganglia is also disrupted. The sky blue staining in E and F is nonspecific background.
