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Figure 1.
Sequences of Rat and Xenopus NEUROGENIN
(A) Alignment of the entire predicted amino acid sequences of rat NEUROGENIN and a Xenopus NEUROGENIN-related protein, X-NGNR-1.a. The bHLH region is marked in bold type. Solid lines indicate amino acid identity; the dots, conservative substitutions. The initiator methionine was selected based on Kozak's rules (Kozak 1984) and identification of in-frame up-stream termination codons (data not shown).
(B) Alignment of the NEUROGENIN bHLH domain with other bHLH domains. Identity is shown by bold type. References for the compared sequences are as follows: NeuroD (Lee et al. 1995) /BETA2 (Naya et al. 1995), MATH-2/Nex-1 (5 and 43), MATH-1 (Akazawa et al. 1995), KW8 (Kume et al. 1996), Drosophila atonal (Jarman et al. 1993b), MASH1 (Johnson et al. 1990), AS-C T5 (Villares and Cabrera 1987). |
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Figure 2.
Sequential Expression of neurogenin and NeuroD in Rat Embryos
Adjacent transverse sections of E13.5 rat trunk spinal cord (A and B) and tangential sections of E14.5 forebrain (top part) (C and D). Arrows in (A) and (B) indicate dorsal root sensory ganglia; no signal was detected in sympathetic or other autonomic ganglia (data not shown). Note that neurogenin and NeuroD are expressed in similar regions (arrowheads, A and B), but that NeuroD is displaced lateral to the ventricular zone where NEUROGENIN is expressed (arrows, C and D). |
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Figure 3.
Sequential Expression of X-ngnr-1 and XNeuroD mRNAs in Xenopus
A series of embryos at St. 12 (A–C) and St. 13–13.5 (D and E) and St. 14 (F) are shown, hybridized with probes for X-ngnr-1 (A and D), XNeuroD (B and E) and N-tubulin (C and F). Note that X-ngnr-1 is expressed prior to both XNeuroD and N-tubulin, in patches that define the three prospective territories of primary neurogenesis (A, m, i, and l). Arrows in (A) and (D)–(F) indicate trigeminal placode (A) or ganglia (D–F). Note also that at St. 13.5, the domain of X-ngnr-1 expression within the m, i, and l regions of the neural plate is larger than the domain of either XNeuroD or N-tubulin expression. |
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Figure 4.
Induction of Endogenous XNeuroD Expression and Neurogenesis by Injection of X-ngnr-1 mRNA
Ectopic neurogenesis was visualized in St. 13.5 (neural plate stage) embryos by whole mount in situ hybridization with an N-tubulin probe (A and D). Embryos were injected on one side (inj) with either X-ngnr-1 (A and B) or XNeuroD (C and D) synthetic RNA. X-NGNR-1 induced ectopic neurogenesis (A) as well as expression of endogenous XNeuroD (B); XNeuroD also induced ectopic neurogenesis (D) but did not induce X-ngnr-1 (C). No ectopic expression of N-tubulin was observed in lacZ RNA-injected embryos (E). At the tail bud stage (F), ectopic neurogenesis is induced by X-NGNR-1 in the skin (arrow) and the entire anterior region (arrowhead). A similar phenotype is seen in XNeuroD-injected embryos at this stage ( Lee et al. 1995; and data not shown). |
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Figure 5.
X-NGNR-1 Induces Expression of Neural and Neuronal Markers in the Absence of Mesodermal Markers, in Animal Caps
Neither of two mesodermal markers, Xenopus brachyury (A) and muscle-specific actin (M.Actin) (B), is induced in animal caps cultured from embryos injected with X-ngnr-1 RNA (A and B, lanes 3), whereas both are induced in positive control caps cultured with activin (A and B, lanes 2). In contrast, X-NGNR-1 induces expression of both NCAM and N-tubulin (B, lane 3). For comparison, caps injected with noggin RNA express NCAM but not N-tubulin (B, lane 4). Elongation factor-1α (EF-1α) serves as a control for RNA loading ( Ferreiro et al. 1994). “Total embryo†indicates total RNA from embryos of the equivalent stage (St. 11 for [A], St. 15–16 for [B]); “tRNA†indicates carrier tRNA control, while “Control†animal caps represents RNA from animals caps cultured from uninjected embryos. |
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Figure 6.
Expression of X-ngnr-1 Preceeds and Induces That of X-Delta-1
(A–D) in situ hybridization of embryos at St. 10.5–11.0 (A and B) and St. 11.5 (C and D) showing that expression of X-ngnr-1 (A and C) precedes that of X-Delta-1 (B and D), in both the prospective neural plate (A, arrow) and trigeminal placode (C and D, arrows). At St. 11.5, the X-Delta-1-expressing cells within the medial, intermediate, and lateral (m, i, and l) regions of the neural plate (D) overlap the domains of X-ngnr-1-expressing cells (C).
(E) injection of X-ngnr-1 mRNA induces ectopic expression of endogenous X-Delta-1 mRNA on the injected (inj) side of a St. 13.5 embryo.
(F) control injection of lacZ mRNA alone has no effect on X-Delta-1 expression.
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Figure 7.
Expression and Function of X-ngnr-1 Are Restricted to Subsets of Cells by Lateral Inhibition Mediated by X-Notch and X-Delta-1
(A and B) opposite effects of injection of constitutively active X-NotchICD RNA (A) and dominant negative X-Delta-1Stu RNA (B) on expression of endogenous X-ngnr-1 mRNA. In both cases, only the lateral stripe of X-ngnr-1 expression is affected (arrowhead, A and B; compare to arrow indicating uninjected side), due to restricted distribution of the injected RNA as defined by the lacZ tracer (light blue staining). X-NotchICD RNA represses expression of X-ngnr-1 (A, arrowhead), while X-Delta-1Stu RNA increases the density of X-ngnr-1-expressing cells (B, arrowhead). A similar effect on N-tubulin expression is seen in animals expressing X-NotchICD (C) or X-Delta-1Stu RNA (D) in the lateral stripe (light blue stain, C). In other embryos exhibiting a different distribution of the injected RNAs, the development of the medial or lateral stripes was similarly affected (data not shown). (E and F) suppression of X-NGNR-1 neurogenic function by NotchICD. Blastomeres were coinjected either with X-ngnr-1 plus lacZ RNAs, (E) or in addition with X-NotchICD RNA (F), and hybridized with N-tubulin probes at St. 13.5. Note that ectopic neurogenesis is inhibited by NotchICD (F, arrowhead), in regions receiving the highest levels of injected RNAs (light blue staining), but that some ectopic neurogenesis is still seen in other parts of the injected side (F, arrow). By contrast ectopic neurogenesis on the injected side of embryos receiving X-NGNR-1 plus lacZ is relatively uniform (E, arrowhead). |
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Figure 8.
Model for the Role of X-NGNR-1 and XNeuroD in Lateral Inhibition and Neuronal Determination
The model draws heavily on analogies to Drosophila (Ghysen et al. 1993). X-NGNR-1 initially activates expression of X-Delta-1 and is inhibited by signaling through X-Notch (left side of diagram). The inhibition of X-ngnr-1 expression may be mediated by Suppressor of Hairless [Su(H)] and enhancer of split [E(spl)] proteins, by analogy to Drosophila ( Artavanis-Tsakonas et al. 1995). Expression of X-NGNR-1 above a certain threshhold, or in the presence of a hypothetical cofactor (see text), leads to expression of XNeuroD and neuronal differentiation in a manner insensitive to inhibition by Notch signaling (dashed blunt arrow) ( Chitnis and Kintner 1996). |
