The Role of the Transcription Factor Rbpj in the Development of Dorsal
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Hu et al. Neural Development 2011, 6:14 http://www.neuraldevelopment.com/content/6/1/14 RESEARCHARTICLE Open Access The role of the transcription factor Rbpj in the development of dorsal root ganglia Ze-Lan Hu1*†, Ming Shi2†, Ying Huang1, Min-Hua Zheng3, Zhe Pei1, Jia-Yin Chen1, Hua Han3 and Yu-Qiang Ding1* Abstract Background: The dorsal root ganglion (DRG) is composed of well-characterized populations of sensory neurons and glia derived from a common pool of neural crest stem cells (NCCs), and is a good system to study the mechanisms of neurogenesis and gliogenesis. Notch signaling is known to play important roles in DRG development, but the full scope of Notch functions in mammalian DRG development remains poorly understood. Results: In the present study, we used Wnt1-Cre to conditionally inactivate the transcription factor Rbpj, a critical integrator of activation signals from all Notch receptors, in NCCs and their derived cells. Deletion of Rbpj caused the up-regulation of NeuroD1 and precocious neurogenesis in DRG early development but led to an eventual deficit of sensory neurons at later stages, due to reduced cell proliferation and abnormal cell death. In addition, gliogenesis was delayed initially, but a near-complete loss of glia was observed finally in Rbpj-deficient DRG. Furthermore, we found P75 and Sox10, which are normally expressed exclusively in neuronal and glial progenitors of the DRG after the NCCs have completed their migration, were co-expressed in many cells of the DRG of Rbpj conditional knock-out mice. Conclusions: Our data indicate that Rbpj-mediated canonical Notch signaling inhibits DRG neuronal differentiation, possibly by regulating NeuroD1 expression, and is required for DRG gliogenesis in vivo. Background between the neural tube and the dermamyotome form The nervous system is made up of a wide variety of the DRG [3]. Between E9.25 and E13.5 in the mouse, neuronal and glial cell types. How these different cell NCCs first give rise to large neurons that express neuro- types are generated from multipotent progenitors during trophic tyrosine receptor kinase (Trk)C, and then to development is a fundamental and largely unanswered medium-sized TrkB+ and small-sized TrkA+ sensory question in neuroscience. The dorsal root ganglion neurons [4,5]. NCC-derived glial cells are also generated (DRG), which consists of several well-characterized during this period, including satellite cells and Schwann types of sensory neurons and glial cells, is an attractive cells, although these begin to differentiate about 1.5 model system to investigate the molecular processes days after sensory neurons [4,6]. underlying cellular differentiation in the nervous system Many genes are involved in the generation of sensory [1]. Sensory neurons and glial cells in the DRG derive neurons and glia from multipotent NCCs. Among the from the neural crest, a tissue that exists transiently various cell-surface proteins known to be expressed by during mammalian embryogenesis at the border NCCs, the low affinity neurotrophin receptor P75 has between the ectoderm and the neural plate [2]. Between been widely used to identify and isolate NCCs [7]. In embryonic day (E)8.5 and E10.0 in the mouse, neural addition, P75 interacts with TrkC, the high-affinity recep- crest stem cells (NCCs) begin to exit the neural tube, tor for Neurotrophin-3, to promote neuronal differentia- and those that migrate along the ventral pathway tion of NCCs in vitro [8]. The high-mobility group transcription factor SRY (sex determining region Y) box 10 (Sox10) is expressed in pre-migratory and migratory * Correspondence: [email protected]; [email protected] † Contributed equally NCCs and plays a role in maintaining the multipotency 1Department of Anatomy and Neurobiology, Tongji University School of of NCCs [9]. Expression of Sox10 turns off in daughter Medicine, 1239 Siping Road, Shanghai 200092, China cells committed to neuronal fates, but persists in Full list of author information is available at the end of the article © 2011 Hu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hu et al. Neural Development 2011, 6:14 Page 2 of 14 http://www.neuraldevelopment.com/content/6/1/14 glia-restricted progenitors and differentiated glia [9,10]. [26] conditionally knocked out Rbpj expression in NCCs The specification of DRG sensory neuron lineages is also and NCC-derived cells and observed a modest reduction controlled by several transcription factors. For example, in sensory neurons and profound defects in gliogenesis the basic helix-loop-helix (bHLH) transcription factors in the DRG of Wnt1-Cre;Rbpjflox/flox (Rbpj conditional Neurogenin-1 (Ngn1) and Neurogenin-2 (Ngn2) promote knock-out (CKO)) mice. However, the whole range of sensory fates, as opposed to autonomic ones [1,5,11,12], DRG phenotypes, especially the defects of neurogenesis, and are required for the initiation of neurogenesis [5]. in Rbpj CKO mice has not yet been described, and the Neurogenic differentiation 1 (NeuroD1) is thought to act mechanisms underlying the phenotype remain unclear. downstream of the neurogenins in the regulation of neu- In the present study, we focused on early developmen- ronal differentiation [13,14]. As terminal differentiation tal events in the DRG of Rbpj CKO mice. We found progresses, sensory neuron subtypes with distinct modal- that Ngn1 and Ngn2 expression was unchanged in the ities acquire specific patterns of Trk expression, uniquely absence of Rbpj,butNeuroD1 was up-regulated and expressing either TrkA, TrkB, or TrkC [1,15]. precocious neurogenesis occurred in the DRG. The ele- The function of Notch signaling in DRG development, vated rate of neuronal differentiation at early time as revealed by in vitro studies and in vivo chick studies, points was followed by a reduction in cell proliferation is consistent with its two fundamental roles in the devel- and abnormal cell death. In addition, the initiation of opment of the nervous system: maintaining undifferen- BFABP expression in glial progenitors was delayed, and tiated progenitors by inhibiting neuronal differentiation, this expression was lost at later stages. In wild-type and promoting glial determination [16,17]. Studies of mice, P75 and Sox10 are co-expressed in NCCs during chick development show that Notch signaling is essen- early DRG development, but are subsequently expressed tial for the maintenance of NCCs and prevents neuronal in two distinct populations as development progresses differentiation in the DRG and sympathetic ganglion [27]. In contrast, this separation did not occur in Rbpj- [18]. Furthermore, transient activation of Notch signal- deficient DRG, as evidenced by the presence of P75/ ing in NCCs in vitro prevents neuronal differentiation Sox10 co-expressing cells at later developmental stages, and promotes glial differentiation [19,20]. On the other suggesting that the multipotency of NCCs was abnor- hand, results from mutant mice with Notch signaling mally maintained, thus arresting their development. pathway deletions are not consistent. Delta-like-1 (a These data provide further insights into the physiologi- Notch ligand) null mice had aberrantly fused and/or cal functions of Notch signaling in the development of reduced DRG and sympathetic ganglia, suggesting that DRG. Notch signaling is also essential for the proper migra- tion of NCCs [21]. Hes1 and Hes5 (Notch signaling Results effectors) double-null mutant mice maintain the expres- Normal induction and early migration of NCCs in Rbpj sion of the specific marker for glial cell precursors, CKO embryos brain fatty acid binding protein (BFABP), in the DRG, In order to determine at what time point Rbpj becomes suggesting that Notch signaling is not involved in the inactivated in NCCs and their derivatives in Rbpj CKO generation of glial cell precursors from NCCs [22,23]. mice, we performed X-gal staining of Wnt1-Cre;Rosa26 By over-expressing or inactivating Notch signaling speci- reporter (R) embryos that express b-galactosidase (b-gal) fically in Schwann cell precursors in vivo,Notchsignal- in the Cre expression domain. The X-gal product did not ing has been shown to drive the differentiation of appear in the roof plate of the neural tube or in the pre- immature Schwann cells [23]. Thus, manipulating indivi- sumptive DRG until E9.5 (data not shown). At this stage, dual components of Notch signaling in the mouse yields nearly all P75+ NCCs were also positively labeled with b- varying results and the full scope of Notch functions in gal antibody in both Rbpj CKO (Wnt1-Cre;Rbpjflox/flox; mammalian DRG development remains to be elucidated. Rosa26R) and control (Wnt1-Cre;Rosa26R)embryos The presence of four Notch receptors and at least five (Figure 1A-F), demonstrating that Rbpj could be deleted Notch ligands, all of which are partially functionally in NCCs and their derivatives from E9.5 on. P75 and redundant, has made it difficult to investigate the phy- Sox10 are co-expressed in pre-migratory and migrating siological function of Notch signaling in mammals [16]. NCCs [27]. The double staining of P75 and Sox10 Recombination signal binding protein for immunoglobu- showed there was no obvious difference in the distribu- lin kappa J region (Rbpj) can interact with the intracel- tion and number of P75/Sox10-double labeled cells lular domains of all four Notch receptors and is between CKO and wild-type embryos at E9.5 (Figure 1G- required to mediate their transcriptional effects [24,25]. L). This result is consistent with a previous report [26] Therefore, deletion of Rbpj would be expected to com- and suggests that deleting Rbpj from E9.5 does not affect pletely abolish canonical Notch signaling. Taylor et al. the induction and initial migration of NCCs.