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Neurogenesis or Neuronal Specification: Phosphorylation Strikes Again!

2008; Cell Press; Volume: 58; Issue: 1 Linguagem: Inglês

10.1016/j.neuron.2008.03.023

1097-4199

Helen C. Lai, Jane E. Johnson,

Genetics and Neurodevelopmental Disorders

How basic helix-loop-helix (bHLH) transcription factors control neurogenesis and neuronal subtype specification through transcriptional mechanisms mediated by cell signaling remains to be fully elucidated. In this issue of Neuron, Ma et al. discover that phosphorylation via GSK3 of the bHLH factor, Ngn2 (Neurog2), adds a neuronal subtype-specific program to its functional repertoire that is activated in the developing neural tube in vivo. How basic helix-loop-helix (bHLH) transcription factors control neurogenesis and neuronal subtype specification through transcriptional mechanisms mediated by cell signaling remains to be fully elucidated. In this issue of Neuron, Ma et al. discover that phosphorylation via GSK3 of the bHLH factor, Ngn2 (Neurog2), adds a neuronal subtype-specific program to its functional repertoire that is activated in the developing neural tube in vivo. The formation of nervous system complexity has fascinated researchers for over a century. In the last few decades, molecular mechanisms by which progenitor cells differentiate and acquire their cell identity are only just beginning to be fully understood. Neuronal differentiation involves two closely linked processes, neurogenesis and neuronal specification. Neurogenesis is the process whereby the neural progenitor cell exits the cell cycle and initiates panneuronal gene expression. In contrast, neuronal specification refers to the acquisition of a specific neuronal identity such as neurotransmitter or projection phenotype (i.e., an interneuron versus a motor neuron). The process of neuronal differentiation is regulated by two major classes of transcription factors. Basic helix-loop-helix (bHLH) factors have a primary function in neurogenesis, but in some contexts also influence neuronal specification (for review, see Bertrand et al., 2002Bertrand N. Castro D.S. Guillemot F. Nat. Rev. Neurosci. 2002; 3: 517-530Crossref PubMed Scopus (1115) Google Scholar). Whereas, the LIM-homeodomain (LIM-HD) factors, whose pattern of expression is regulated by signaling pathways such as sonic hedgehog (Shh) and bone morphogenic protein (Bmp), have a primary function in specifying neuronal identity (for reviews, see Jessell, 2000Jessell T.M. Nat. Rev. Genet. 2000; 1: 20-29Crossref PubMed Scopus (1569) Google Scholar, Lee and Jessell, 1999Lee K.J. Jessell T.M. Annu. Rev. Neurosci. 1999; 22: 261-294Crossref PubMed Scopus (416) Google Scholar). How members of the bHLH and LIM-HD classes of transcription factors interact mechanistically to generate the entire nervous system with the correct number and type of neurons remains a fundamental question in neurobiology. Mechanistic insight into how neural bHLH factors work in conjunction with LIM-HD factors to specify neuronal subtype was first provided in studies of the regulation of the motor neuron-specific gene, Hb9, in the ventral neural tube (Lee and Pfaff, 2003Lee S.K. Pfaff S.L. Neuron. 2003; 38: 731-745Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). This study demonstrated that the enhancer of Hb9 integrates the activity of the two classes of factors via transcription factor binding site organization. While the Lee and Pfaff, 2003Lee S.K. Pfaff S.L. Neuron. 2003; 38: 731-745Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar study addressed how bHLH and LIM-HD factors could coordinate neuronal subtype specification, it suggested that the bHLH merely enhanced the specification function of the LIM-HD factors and did not have a direct role in specification itself. This conclusion is consistent with the broad expression of bHLH factors in ventral neural progenitors and the observation that mice mutant for the bHLH factor Ngn2 have decreased expression of most classes of ventral interneurons and motor neurons (Scardigli et al., 2001Scardigli R. Schuurmans C. Gradwohl G. Guillemot F. Neuron. 2001; 31: 203-217Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This is in contrast to the role of bHLH factors demonstrated in the dorsal neural tube that show a clear function in neuronal subtype specification as well as neurogenesis (Bertrand et al., 2002Bertrand N. Castro D.S. Guillemot F. Nat. Rev. Neurosci. 2002; 3: 517-530Crossref PubMed Scopus (1115) Google Scholar, Gowan et al., 2001Gowan K. Helms A.W. Hunsaker T.L. Collisson T. Ebert P.J. Odom R. Johnson J.E. Neuron. 2001; 31: 219-232Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, Nakada et al., 2004Nakada Y. Hunsaker T.L. Henke R.M. Johnson J.E. Development. 2004; 131: 1319-1330Crossref PubMed Scopus (85) Google Scholar). In this issue, Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar lend further insight into the relationship between bHLH and LIM-HD factors by demonstrating that Ngn2 does function as a neuronal specification factor for motor neurons (Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Ngn2 has this function only in a specific phosphorylated state that allows for Ngn2 interaction with a LIM-HD transcription complex. Furthermore, the phosphorylation of Ngn2, at least at serine residues S231 and S234, has no apparent role in the function of Ngn2 as an inducer of neurogenesis. The work by Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar provides the first in vivo mechanistic insight into how cell signaling can alter the activity of a bHLH to produce differential functional outputs. The authors identify two serines in the C terminus of Ngn2 that are conserved across multiple vertebrate species. They demonstrate that these serines are phosphorylated in vivo in the ventral neural tube in progenitors positioned to give rise to motor neurons and ventral interneurons. To determine the in vivo function of Ngn2 phosphorylation, mice were generated by targeted mutagenesis to knock in (KI) a form of Ngn2 where S231 and S234 were mutated to alanine, thus abrogating phosphorylation at these sites. This definitive strategy revealed that phosphorylated Ngn2 has a discrete function in specifying motor neurons but is not required for neurogenesis in general (Figure 1). The latter conclusion comes from the observation that neuronal differentiation in the cranial ganglia and dorsal root ganglia, sites of Ngn2 expression, appears normal in the Ngn2S231A&S234A mice. Consistent with this, cultured cortical rat progenitors expressing Ngn2 or Ngn2 with the alanine mutations differentiate normally. The most striking phenotype detected in the Ngn2S231A&S234A mouse is the decrease in motor neuron number, with a concomitant increase in the neighboring V2 interneuron population. This phenotype is in contrast to the Ngn2 null that has a decrease in multiple ventral neurons including both the V2 and motor neuron populations (Scardigli et al., 2001Scardigli R. Schuurmans C. Gradwohl G. Guillemot F. Neuron. 2001; 31: 203-217Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), a phenotype that was used to conclude that Ngn2 is required for neurogenesis but plays only a permissive role in neuronal subtype specification. Thus, comparing the consequences of the different Ngn2 alleles in neural tube development, phosphorylated Ngn2 has a role in specifying motor neurons over V2 interneurons. A role for this modified form of Ngn2 for specifying other ventral neuronal subtypes or dorsal interneurons requires further testing. In a previous study, it was demonstrated that a transcription complex containing the LIM-HD factors Isl1 and Lhx3 with the adaptor NLI drives motor neuron-specific expression while Lhx3 with NLI in the absence of Isl1 drives V2 interneuron specific expression (Thaler et al., 2002Thaler J.P. Lee S.K. Jurata L.W. Gill G.N. Pfaff S.L. Cell. 2002; 110: 237-249Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). For these experiments, expression in chick neural tube of constructs fusing the dimerization domain of NLI with the homeodomains of Isl1 and Lhx3 (DD-Isl1-Lhx3) or Lhx3 only (DD-Lhx3) resulted in ectopic motor neurons or V2 interneurons, respectively. Later experiments showed that coexpression of the bHLH factors NeuroD4 (NeuroM) or Ngn2 with the DD-Isl1-Lhx3 complex enhanced the ectopic motor neuron phenotype and that this enhancement could be attributed to interactions of the transcription factor complexes on an Hb9 enhancer sequence (Lee and Pfaff, 2003Lee S.K. Pfaff S.L. Neuron. 2003; 38: 731-745Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). To build on these earlier mechanistic experiments, Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar demonstrate that Ngn2S231A&S234A does not enhance the ectopic motor neuron phenotype in the chick neural tube when coexpressed with DD-Isl1-Lhx3, a finding that supports the conclusions from the mouse KI experiments that phosphorylation at these sites is important for motor neuron specification. Biochemical support came from the cofractionation of Ngn2 with NLI, Isl1, and Lhx3, and coimmunoprecipitation of Ngn2 with NLI from endogenous neural tube tissue. The dependence on the phosphorylated form of Ngn2 for this interaction with NLI was shown in cultured cells overexpressing wild-type or mutant forms of Ngn2. Together, these findings support a mechanism by which phosphorylated Ngn2 gains a function in neuronal subtype specification by forming a transcription complex with NLI-Isl1-Lhx3 (Figure 1C). The other function of Ngn2, that of inducing neurogenesis, was not assessed in these overexpression experiments. Another major gap in our understanding of neural development is how known signaling pathways, such as Shh and Wnt, function in the neural tube to control the activity levels of essential transcription regulators like Ngn2. Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar attempt to close this gap by identifying a kinase responsible for the serine phosphorylation of Ngn2. The ubiquitously expressed glycogen synthase kinase 3 (GSK3) was identified as the responsible kinase since manipulation of GSK3 activity, or levels, in embryonic stem cells induced to differentiate to motor neurons alters Ngn2 phosphorylation. Unfortunately, the identification of GSK3 as a relevant kinase does little to tie the activation function of the Ngn2 to a signaling pathway since it is not known how GSK3 is involved in this context. However, in embryonic stem cells induced to differentiate to motor neurons, Shh, a known inducer of ventral neuron identity (Jessell, 2000Jessell T.M. Nat. Rev. Genet. 2000; 1: 20-29Crossref PubMed Scopus (1569) Google Scholar), was shown to induce Ngn2 levels without altering GSK3 activity. Thus, Shh and GSK3 may work independently in this process with Shh regulating levels of Ngn2 and GSK3 phosphorylating at least a subset of Ngn2 factors for its activity in the NLI-Isl1-Lhx3 transcription complex (Figure 1D). Future experiments to identify signaling pathways that modulate GSK3 activity in the ventral neural tube and thus indirectly alter levels of Ngn2 available for specifying motor neurons are still required to gain a full molecular understanding of how neuronal diversity is generated. In summary, Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar demonstrate that GSK3-phosphorylated Ngn2 works in concert with a specific LIM-HD transcription complex to specify motor neuron cell type through a protein-protein interaction with the adaptor protein NLI. Phosphorylation at S231 and S234 of Ngn2 adds a new function of neuronal subtype specification that is absent in Ngn2 not phosphorylated at these sites. This is not the first report for phosphorylation of Ngn2 revealing a new function as phosphorylation at a tyrosine (Y241) is required for Ngn2 function in radial migration and dendritic morphology in cortical pyramidal neurons (Hand et al., 2005Hand R. Bortone D. Mattar P. Nguyen L. Heng J.I. Guerrier S. Boutt E. Peters E. Barnes A.P. Parras C. et al.Neuron. 2005; 48: 45-62Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). However, it is the first report for this type of posttranslational modification altering Ngn2's transcriptional activity. Further investigation will be required to determine how widespread the binding of phosphorylated Ngn2 is to NLI containing LIM-HD complexes in the neural tube. Ngn2, while not ubiquitous in the developing nervous system, is broadly expressed in neural progenitor domains. NLI is also broadly expressed throughout the developing neural tube and is thought to be a common adaptor for LIM-HD transcription complexes (Jurata et al., 1998Jurata L.W. Pfaff S.L. Gill G.N. J. Biol. Chem. 1998; 273: 3152-3157Crossref PubMed Scopus (142) Google Scholar). This sets up a seeming conundrum since Ngn2 and NLI are coexpressed in several domains, but different neuronal subtypes are encoded. Clearly, there is some specificity since V2 interneurons do not require the modification of Ngn2, yet a NLI-LIM-HD complex is involved in specifying V2 as well (Thaler et al., 2002Thaler J.P. Lee S.K. Jurata L.W. Gill G.N. Pfaff S.L. Cell. 2002; 110: 237-249Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Further analysis of the Ngn2S231A&S234A mutant mice will shed more light on this issue. The broader implication for bHLH function raised from this study is whether posttranslational modification reflects a universal mechanism for modulating bHLH factor activities. This type of mechanism would allow inductive signals such as Shh, Wnt, Bmp, or FGF to influence differential phosphorylation of bHLH factors along the dorsal-ventral axis of the neural tube. Phosphorylation could alter protein-protein or protein-DNA interactions in different cellular contexts influencing the diversity of neuronal cell types in the nervous system. The C termini of other neural bHLH factors such as Neurog1 (Ngn1), NeuroD4, and Ascl1 (Mash1) that are patterned along the dorsal ventral axis of the neural tube appear rich in candidate residues for phosphorylation that are conserved between multiple species. Ma et al., 2008Ma Y.-C. Song M.-R. Park J.P. Ho H.-Y.H. Hu L. Kurtev M.V. Zieg J. Ma Q. Pfaff S.L. Greenberg M.E. Neuron. 2008; 58 (this issue): 65-77Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar provide us with an exciting glimpse into the next generation of research as we attempt to understand how cell signaling results in posttranslation modifications of transcription factors essential for regulating the complexities of neuronal differentiation throughout the developing nervous system. Regulation of Motor Neuron Specification by Phosphorylation of Neurogenin 2Ma et al.NeuronApril 10, 2008In BriefThe mechanisms by which proneural basic helix-loop-helix (bHLH) factors control neurogenesis have been characterized, but it is not known how they specify neuronal cell-type identity. Here, we provide evidence that two conserved serine residues on the bHLH factor neurogenin 2 (Ngn2), S231 and S234, are phosphorylated during motor neuron differentiation. In knockin mice in which S231 and S234 of Ngn2 were mutated to alanines, neurogenesis occurs normally, but motor neuron specification is impaired. Full-Text PDF Open Archive