Park BY et al. (2012), Xaml1/Runx1 is required for the specification o...

XB-ART-44588

Dev Biol 2012 Feb 01;3621:65-75. doi: 10.1016/j.ydbio.2011.11.016.

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Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus.

Park BY , Hong CS , Weaver JR , Rosocha EM , Saint-Jeannet JP .

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Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons.

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Species referenced: Xenopus laevis
Genes referenced: astl2c astl3a.1 ccndx cdkn1a egr2 fgf8 isl1 kcna1 neurog2 notch1 npb nsg1 pak1 pak3 pax3 runx1 snai2 tbx2 tub tubb2b zic1
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	Fig. 1. Expression of Runx1/Xaml1 in Rohon-Beard sensory neurons by whole-mount in situ hybridization. (A–F) Runx1 expression in stage 15 embryos. (A) Runx1 is detected at the posterior portion of the NPB. (B) Double in situ hybridization for Runx1 and the HG marker Xhe. Runx1 positive cells are located posterior to HG cells (arrow). (C) Double in situ hybridization for Runx1 and the neural crest marker Snail2. Runx1 expression (purple) is distinct from and posterior to the neural crest forming region (Snail2, green staining). (D) Double in situ hybridization for Runx1 and the hindbrain marker Krox20 (arrow). Panels (A–D), dorsal view, anterior to top. (E) Transverse section, dorsal to top, showing that Runx1 is restricted to two discrete domains at the neural plate border. (F) Higher magnification of the neural plate region of the embryo shown in (E). (G) At stage 22 as the neural tube closes, Runx1 expression is restricted to the dorsal aspect of the spinal cord. Dorsal view, anterior to top. (H–I) Transverse section through a stage 23 embryo, dorsal to top. Runx1 is confined to the dorso-lateral region of the spinal cord. Runx1 is also detected ventrally in the lateral plate mesoderm, precursor of the hematopoietic lineage (arrow). (I) Higher magnification of the neural tube region of the embryo shown in (H). (J) Runx1 expression in a stage 28 embryo. Runx1 is detected in the olfactory epithelium (yellow arrow), periotic mesenchyme (black arrow), and blood precursors (white arrow). Lateral view, anterior to left. (K) Transverse section through the spinal cord of a stage 45 embryo shows Runx1 expression in RB sensory neurons in the dorsal spinal cord. Dorsal to top.
	Fig. 2. Comparative expression of Runx1, N-Tub and Ngnr1 in primary neurons. (A) Developmental expression of Runx1, N-Tub, and Ngnr1 at the neural plate border in stage-matched embryos. The arrows indicate the position of the row of RB sensory neurons. Dorsal views, anterior to top. (B–C) In situ hybridization on adjacent transverse sections of stage 15 and stage 19 embryos, dorsal to top. (B) At stage 15 Runx1 is co-expressed with N-Tub and Ngnr1, as indicated by the red overlay in the lower panels. (C) At stage 19 while Runx1 and N-Tub are still co-expressed, Ngnr1 expression is lost in the RB neurons population, as indicated by the red overlay in the lower panels. The dotted lines demarcate the position of the spinal cord.
	Fig. 3. Wnt, Fgf and Notch signaling pathways regulate Runx1. Overexpression of Fgf8a by injection of Fgf8a mRNA in one blastomere at the 2-cell stage results in an expansion of the Runx1 expression domain. The expression domain of N-Tub and Ngnr1 is also expanded in these embryos. Knockdown of Fgf8a (Fgf8aMO) causes a severe reduction of all three genes. Similarly, interference with canonical Wnt signaling, by injection of Wnt8MO or β-catMO, reduces Runx1, N-Tub and Ngnr1 expression in all three populations of primary neurons. Expression of an activated form of Notch (Notch-ICD) also represses Runx1 expression. In all panels embryos are viewed from the dorsal side, anterior to top. The injected side is on the right.
	Fig. 4. Runx1 expression in RB sensory neurons depends on Pax3 and Zic1. (A) Comparative expression of Pax3, Zic1 and Runx1 in stage-matched embryos indicates that the Runx1 expression domain overlaps with that of these two NPB specifiers. (B) Embryos injected with Pax3MO (50 ng) or Zic1MO (45 ng) in one blastomere at the 2-cell stage exhibit a strong reduction of Runx1 as well as N-Tub and Ngnr1 expression in RB progenitors. The injected side is on the right. In all panels embryos are viewed from the dorsal side, anterior to top.
	Fig. 5. Runx1-deficient tadpoles lack Rohon-Beard sensory neurons and lose response to touch. (A) Two-cell stage embryos received a bilateral injection of control (CoMO), Runx1 (Runx1MO) or Aml1 (Aml1MO) morpholino antisense oligonucleotides. At stage 28 the corresponding embryos were sectioned in the trunk region (red line) and analyzed by in situ hybridization. (B) Expression of Rohon-Beard (Kv1.1) and motor neuron (Ccndx) marker genes in the spinal cord of morphant embryos. Runx1MO and Aml1MO show a loss of Kv1.1 expression, while the ventral motor neurons are largely unaffected. (C–D) At stage 32 Runx1MO and Aml1MO injected embryos have a severely reduced response to touch (pokes and strokes) as compared to control uninjected or control morpholino (CoMO) injected embryos. (C) Pokes: Control (uninjected), 10.00 ± 0.00 (n = 30); CoMO (60 ng), 9.83 ± 0.53 (n = 30); Runx1MO (60 ng), 3.14 ± 1.45 (n = 28); Aml1MO (30 ng), 3.03 ± 1.82 (n = 20); Aml1MO (40 ng), 1.00 ± 1.19 (n = 15). (D) Strokes: Control (uninjected), 9.68 ± 0.56 (n = 30); CoMO (60 ng), 9.47 ± 0.63 (n = 30); Runx1 MO (60 ng), 1.30 ± 1.26 (n = 28); Aml1MO (30 ng), 1.95 ± 1.28 (n = 20); Aml1MO (40 ng), 0.33 ± 0.75 (n = 15). Statistical significance was determined using one-way ANOVA. The values are presented as mean SEM; * = P < 0.0001, versus Control and CoMO).
	Fig. 6. Runx1-deficient embryos downregulate N-Tub, Pak3 and Islet1. (A) Embryos were injected in one blastomere at the 2-cell stage with Runx1MO, Aml1MO or Runx1SMO and analyzed at stage 15 for the expression of Ngnr1, N-Tub, Pax3 and Islet1. The RB expression domain of N-Tub, Pak3 and Islet1 is reduced while Ngnr1 expression is expanded (arrows). In all panels the injected side is on the right. Dorsal view anterior to top. (B) Transverse sections of representative Runx1MO-injected embryos. The expression of N-Tub is lost in RB progenitors while N-Tub expression in primary motor neurons precursors is unaffected (arrow heads). Ngnr1 expression is expanded (arrows). The injected side is on the right. Dorsal to top. no, notochord; so, somites.
	Fig. 7. Ngnr1 expression is not sufficient to activate Runx1 expression. (A) Embryos at the 2-cell stage were injected in one blastomere with 0.5 ng of Ngnr1-GR mRNA. Embryos were subsequently incubated with dexamethasone at early (+ Dex 10.5) or late (+ Dex 12.5) gastrula stages, and fixed at stage 15 for detection of Runx1, N-Tub or Pak3 by whole mount in situ hybridization. N-Tub and Pak3 are dramatically upregulated while the Runx1 expression domain is only marginally affected. The injected side is on the right. Dorsal view anterior to top. (B) At the tailbud stage (stage 27) these embryos show ectopic Kv1.1 and Islet1 expression in the ectoderm, independently of any upregulation of Runx1. Lateral views dorsal to top. Control and injected sides of the same embryo are shown for comparison.
	Fig. 8. Model of the gene regulatory network regulating cell fate at the neural plate border. This model is an extension of the model proposed by [Meulemans and Bronner-Fraser, 2004] and [Litsiou et al., 2005] for NC and PE specification. Based on our current observations and other studies (Hong and Saint-Jeannet, 2007) this regulatory cascade has been expanded to include two additional NPB cell types, RB neurons and HG cells.

References [+] :

Ahrens, Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. 2005, Pubmed, Xenbase