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Dev Biol
2001 May 15;2332:495-512. doi: 10.1006/dbio.2001.0230.
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Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus.
Pozzoli O
,
Bosetti A
,
Croci L
,
Consalez GG
,
Vetter ML
.
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During primary neurogenesis in Xenopus, a cascade of helix--loop--helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.
FIG. 2. Xebf3 is expressed in the developing Xenopus nervous system. Whole-mount in situ hybridization was performed on Xenopus embryos by using a digoxigenin-labeled Xebf3 probe. Shown are embryos at stages 15.5 (A), 17 (B), 24 (C), 28 (D), and 32 (E). Xebf3 was detected at early neural plate stage in the trigeminal placode (st. 13.5) and turned on in the three stripes of primary neurons at stage 15.5 (A: m, medial; i, intermediate; l, lateral). The embryo in (A) is oriented with anterior to the top. (B) Posterior transverse section of a stage 17 embryo, showing Xebf3 expression in the three stripes of primary neurons (m, i, l). At this stage, the neural plate is invaginating to form the neural tube. In tailbud stage embryos (C), Xebf3 was expressed in some branchial arches, olfactory placode, neural tube (mesencephalon, rhombencephalon, and spinal cord), retina, and otic vesicle. Embryos in (C) were cleared using a 2:1 mixture of benzyl benzoate/benzyl alcohol. Abbreviations: ba, branchial arches; ms, mesencephalon; op, olfactory placode; ov, otic vesicle; r, retina; rh, rhombencephalon; sc, spinal cord.
Fig.3. XEBF3 binds the EBF/OLF-1 binding site and can activate transcription in vitro. The DNA binding and transactivation activities of XEBF3 and XEBF2/XCOE2 were compared to the mouse EBF proteins. (A, B) Gel mobility shift assay showing specific affinity of the EBF/XEBF factors for the EBF/OLF-1 palindromic binding site. Blank, no lysate; RRL, rabbit reticulocyte lysate only; other lanes, in vitro-synthesized, unlabeled EBF/XEBF proteins. All of the reactions were incubated with [gamma-32P] end-labeled double-stranded oligonucleotide containing either a wild-type or mutated EBF/ OLF-1 binding site derived from the OMP (Olfactory Marker Protein) promoter and then run on a nondenaturating polyacrylamide gel. A shifted band was observed after the incubation of all of the EBF/XEBF proteins with the wild-type binding site (A), but not with a mutated site (B). (b) Indicates the shifted bands and (f) the free probe. (C) Luciferase assay revealing the different levels of transactivation activity of the EBF family members. Luciferase activity was measured in luciferase units and was assayed from lysates of NIH3T3 cells. The cells were transfected with 5 ug of either the pcDNA3 expression plasmid or pcDNA3 containing coding sequence for the following EBF factors: mouse EBF2, XEBF2, XEBF3. A total of 10 ug of the luciferase reporter plasmid pT81 either containing (mb1) or lacking (pT81) the mb1 promoter sequence was cotransfected into the cells to monitor transactiva- tion activity of the expressed proteins. In all of the samples, 300 ng of pRL-TK was transfected as an internal control. The graph is labeled so that, for each bar, the expression plasmid is listed first and the reporter plasmid is listed last (e.g., EBF2-mb1). Values indicate the mean of the results from three independent experiments and all of the columns indicated by an asterisk (*) are statistically different from control luciferase values (*, P < 0.0002; **, P < 0.001 by Student t-test).
FIG. 4. Xebf3 and Xebf2/Xcoe2 overexpression promotes ectopic expression of neural markers in vivo and in animal caps. (A) RNA was injected into one cell of two-cell stage Xenopus embryos, and then embryos were collected at neural plate stages for in situ hybridization analysis, using the probes indicated at the top. Shown are dorsal views of representative embryos with anterior to the bottom and the injected side, revealed by X-gal staining, to the right. (A, B) Control embryos injected with RNA for Beta-galactosidase. N-tubulin is expressed in developing primary neurons, while XNF-M is normally expressed later, starting at stage 19. (C) Overexpression of Xebf3 (C, D) or Xebf2/Xcoe2 (E, F) promoted ectopic neurogenesis, as detected by whole-mount in situ hybridization using an N-tubulin probe or a XNF-M probe. Embryos injected with either Xebf3 or Xebf2/Xcoe2 showed a scattered pattern of N-tubulin-positive neurons restricted to the posteriorectoderm (C, E) compared with the more extensive and stronger distribution of ectopic XNF-M-positive neurons (D, F). (G) Stage 28 Xebf3-injected embryo immunostained with the anti-N-CAM antibody 6F11 (enlargement of the ventral side). Arrowheads indicate ectopic process-bearing neurons. (H) Xebf3-overexpressing cells show characteristics of sensory neurons. Dorsal view (anterior to the left) of a stage 26 Xebf3 overexpressing embryo with the injected side indicated by X-gal staining. Overexpression of Xebf3 caused ectopic activation of XHox11L2, a late marker of cranial sensory ganglia and Rohoneard neurons. (I) RT-PCR performed on animal caps isolated from embryos at stage 8 and cultured to neural plate stage. Overexpression of either Xebf3 or Xebf2/Xcoe2 (lanes 2 and 3) promoted ectopic expression of N-CAM, a marker of undifferentiated neural tissue, N-tubulin and XNF-M, markers of differentiating neurons, in the absence of mesodermal factors (muscle actin). Caps isolated from Beta-gal- and XNeuroD-injected embryos served as negative and positive controls, respectively (lanes 1 and 4). An additional positive control is represented by RNA extracted from total embryos at neural plate stage (TE, lane 5). Epidermal keratin was used as a marker for epidermis, and Elongation factor1-alpha(EF1-alpha) served as a loading control.
FIG. 5. Xebf2/Xcoe2, XNeuroD, and Xebf3 function in a unidirectional cascade during primary neurogenesis. Embryos were injected with RNA for XNeuroD (A, B), Xebf3 (C), and Xebf2 (F) at the two-cell stage and then assayed by whole-mount in situ hybridization at neural plate stages. Embryos are shown in a dorsal view with anterior to the bottom and injected side to the right (marked by X-gal staining). Overexpression of XNeuroD promoted ectopic expression of Xebf3 (A), while it suppressed the expression of Xebf2/Xcoe2 (B). Overexpression of Xebf3 did not affect Xngnr-1, XNeuroD, and Xebf2/Xcoe2 expression patterns (C), while Xebf2/Xcoe2-injected embryos showed ectopic expression of Xngnr-1, XNeuroD, and Xebf3 (F-H). (I) RT-PCR performed on animal caps isolated from XNeuroD (I), Xebf3 (L), or Xebf2/Xcoe2 (M) -injected embryos. The overexpression of XNeuroD caused a strong ectopic activation of Xebf3 (I, lane 2). Note that, in these caps, XNeuroD induced a very weak expression of Xebf2/Xcoe2 (I, lane 2). Overexpression of Xebf3 did not induce the expression of Xebf2 or XNeuroD (L, lane 2). In contrast, Xebf2-injected caps showed the activation of XNeuroD as well as Xebf3 (M, lane 2). Caps isolated from ﰁgal-injected embryos and RNA extracted from total embryos (TE) served as negative and positive controls (lanes 1 and 3). Muscle actin was used as a mesodermal marker, and EF1-alpha was used to normalize the samples.
FIG. 6. Xebf3 expression in animal caps can be activated by hGR-XNeuroD in the absence of protein synthesis. RT-PCR on animal caps isolated from hGR-XNeuroD-injected embryos, treated with or without dexamethasone (DEX) and/or cyclohexi- mide (CHX), an inhibitor of protein synthesis. Hormone-dependent activation of injected hGR-XNeuroD promoted ectopic expression of Xebf3, both in the absence and presence of protein synthesis (lanes 1 and 2), while activation of XNF-M expression was only observed in the presence (lane 2) but not the absence of protein synthesis (lane 1). Xebf3 and XNF-M expression were not induced in the absence of dexamethasone (lanes 3 and 4). TE (lane 5) indicates total embryo at stage 14, and B (lane 6) is a blank in which template was excluded from the PCR. Muscle actin is a mesoder- mal marker, and EF1-alpha was used as a loading control.
FIG. 7. Xebf3 is insensitive to Delta/Notch-mediated lateral inhibition mechanism. Embryos were injected with RNA at the two-cell stage then collected at neural plate stages and assayed by in situ hybridization for expression of the neural markers N-tubulin and XNF-M, and of Xebf3 and Xebf2/Xcoe2, as indicated at the top. Injected embryos are oriented with their anterior side down (dorsal view, injected side revealed by X-gal staining). Overexpression of an activated form of the Notch receptor (Notch-ICD) caused a suppression of N-tubulin expression (C) and no change in XNF-M expression (D) on the injected side. XNF-M is normally turned on at stage 19. Embryos coinjected with Notch-ICD and Xebf3 (E, F) gave a similar pattern of ectopic N-tubulin and XNF-M expression as with Xebf3 alone (see Figs. 4C and 4D), suggesting that Xebf3 function was not inhibited by activated Notch. In contrast, embryos coinjected with Notch-ICD and Xebf2/Xcoe2 showed an inhibition of neurogenesis (G, H), as compared with embryos injected with Xebf2/Xcoe2 alone (Figs. 4E and 4F). The block of the Delta/Notch pathway induced by injecting X-Dl1stu did not influence Xebf3 expression (I) but caused an increase in the Xebf2/Xcoe2-positive neurons within the three stripes of primary neurons (L). When lateral inhibition was similarly blocked in XneuroD-injected embryos, overexpression of XNeuroD promoted ectopic expression of both Xebf3 and Xebf2/Xcoe2 (M, N).
