Mammalian Achaete Homologs: Eye, olfactory and ear development

Mash1, a mammalian homolog of Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. Whereas Mash1 is expressed during retinal development, no apparent abnormalities are found both during embryogenesis as well as at birth in Mash1-null retina, suggesting that early differentiating cells such as ganglion, amacrine and cone cells develop normally. Because Mash1-null mice die soon after birth, their postnatal development cannot be examined in vivo. Thus, it remains to be determined whether or not Mash1 functions in postnatal development of retina. Here, the roles of Mash1 in postnatal development of the retina were examined by using a retinal explant that develops like in vivo retina. Without Mash1, differentiation of late appearing cells such as rod, horizontal, and bipolar cells is delayed and the final number of bipolar cells is significantly reduced. In contrast, vimentin-positive cells (probably Muller glial cells) are increased in Mash1-null retina. These results provide evidence that Mash1 promotes neuronal differentiation during retinal development and is essential for proper ratios of retinal cell types (Tomita, 1996).

Whereas vertebrate achaete-scute complex (as-c) and atonal (ato) homologs are required for neurogenesis, their neuronal determination activities in the central nervous system are not yet supported by loss-of-function studies, probably because of genetic redundancy. To address this problem, mice double mutant for the as-c homolog Mash1 and the ato homolog Math3 were generated. Whereas neurogenesis is only weakly affected in Mash1 or Math3 single mutants, in the double mutants, tectal neurons, two longitudinal columns of hindbrain neurons, and retinal bipolar cells are missing and, instead, those cells that normally differentiate into neurons adopt the glial fate. These results indicate that Mash1 and Math3 direct neuronal versus glial fate determination in the CNS and raise the possibility that downregulation of these bHLH genes is one of the mechanisms to initiate gliogenesis (Tomita, 2000).

To examine the possible fate switch in the double mutants, the retina was examined. The retina is an ideal system to analyze the cell fate because it has only six types of neuron and one type of glial cells, which can be clearly identified by position, cell morphology and specific markers. The mature retina consists of three cellular layers: the ganglion cell layer (GCL); the inner nuclear layer (INL), and the outer nuclear layer (ONL). The INL contains three types of interneuron (amacrine, bipolar and horizontal cells) and one type of glia (Müller glial cells). It has been shown that both Math3 and Mash1 are expressed by differentiating bipolar cells. Because most retinal cells including glial cells differentiate postnatally, the retinal explant culture, which mimics in vivo development well, was used. Retinal explants were prepared from E15.5 embryos and cultured for 2 weeks, during which period the majority of retinal cells finish differentiation. By this method, it was possible to monitor the later stage of cell differentiation well after the mutant hosts died. After 2 weeks of culture, the wild-type and mutant retina consisted of three cellular layers. Hematoxylin-eosin (HE) staining indicated that the cell number of each layer was normal in Math3(-/-), Mash1(-/-) and Math3(-/-)-Mash1(-/-) retina. However, whereas bipolar cells (PKC+, mGluR6+, L7+) are normally present in Math3(-/-), they are reduced in number in Mash1(-/-) retina and completely missing in the Math3(-/-)-Mash1(-/-) retina. Instead, Müller glial cells (vimentin+, glutamine synthetase+) are slightly increased in Mash1(-/-) and significantly increased in the double-mutant retina. The other cell types such as rods (rhodopsin+) and amacrine cells (HPC-1+) are not affected in the double-mutant retina. To determine whether any changes of birth date, which is important for cell type specification, are involved in the defects, the retinal explants were examined at days 5, 7 and 10 of culture. At day 5, no glial cells were detectable whereas at days 7 and 10 there were differentiating glial cells in both the wild type and the double mutants, indicating that the time course of gliogenesis is not affected in the double mutants. In addition, there were already more glial cells in the double mutants at day 7, suggesting that more cells initially adopt the glial fate. In contrast, bipolar cells were not detectable in the double mutants at any time points, excluding the possibility that once born, bipolar cells die in the double mutants. Furthermore, there was no significant difference in the Ki-67 staining and TUNEL assay between the wild type and double mutants. These results indicate that cell proliferation and apoptosis are not involved in the loss of bipolar cells and concomitant increase of Müller glial cells, supporting the idea that there is a fate switch from neurons to glial cells in the absence of Math3 and Mash1 (Tomita, 2000).

Thus, retinal explant assays demonstrate that Math3 and Mash1 are essential for bipolar cell development. It has been shown that the homeobox gene Chx10 (Drosophila homologs CG15782 and CG4136) is also essential for bipolar cell development: Chx10-null mice lack bipolar cells. Thus, at least three genes, Math3, Mash1 and Chx10, are involved in bipolar cell development although it is not clear at which stage Chx10 functions. Interestingly, in contrast to Math3(-/-)-Mash1(-/-) double-mutant retina, Müller glial cells are not increased and therefore a fate switch does not occur in Chx10-null mice, suggesting that the cells that normally differentiate into bipolar cells are likely to result in apoptosis in the absence of Chx10 rather than differentiate into glial cells. These results strongly suggest that the bHLH genes and Chx10 have distinct functions in bipolar cell development. Chx10 may not only confer the cell type-specific identities, as suggested by other homeobox genes, but may also regulate cell survival. In contrast, the main function of Math3 and Mash1 is determination of the neuronal fate but not cell survival. It remains to be analyzed whether the two bHLH genes can also directly confer some of the bipolar cell-specific identities. Further characterization of bHLH and homeobox genes will be necessary to decipher the roles of each factor in specification of particular neuronal subtypes (Tomita, 2000 and references therein).

The role that EGF-mediated signaling plays in vertebrate retinal development has been examined. During late retinal neurogenesis EGF delays rod photoreceptor differentiation: this EGF effect involves the modulation of expression of a homolog of Drosophila proneural gene, Mash1. EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retina in vitro. The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. This study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors it is proposed that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina (Ahmad, 1998).

The lineage of olfactory neurons has been relatively well characterized at the cellular level, but the genes that regulate the proliferation and differentiation of their progenitors are currently unknown. Murine Math4C/neurogenin1 is distantly related to the Drosophila proneural gene atonal. Math4C/neurogenin1 and the basic helix-loop-helix gene Mash1 are expressed in the olfactory epithelium by different dividing progenitor populations, while another basic helix-loop-helix gene, NeuroD, is expressed at the onset of neuronal differentiation. These expression patterns suggest that each gene marks a distinct stage of olfactory neuron progenitor development in the following sequence: Mash1>Math4C/neurogenin1>NeuroD. Inactivation of Mash1 function leads to a severe reduction in the number of olfactory neurons. Most cells in the olfactory epithelium of Mash1 mutant embryos fail to express Math4C/neurogenin1 or NeuroD. Strikingly, a subset of progenitor cells in a ventrocaudal domain of Mash1 mutant olfactory epithelium still express Math4C/neurogenin1 and NeuroD and differentiate into neurons. Cells in this domain also express Math4A/neurogenin2, another member of the Math4/neurogenin gene family, and not Mash1. These results demonstrate that Mash1 is required at an early stage in the olfactory neuron lineage to initiate a differentiation program involving Math4C/neurogenin1 and NeuroD. Another gene activates a similar program in a separate population of olfactory neuron progenitors (Cau, 1997).

The functions of the bHLH transcriptional repressors HES1 and HES5 in neurogenesis have been characterized, using the development of the olfactory placodes in mouse embryos as a model. Hes1 and Hes5 are expressed with distinct patterns in the olfactory placodes and are subject to different regulatory mechanisms. Hes1 is expressed in a broad placodal domain, which is maintained in the absence of the neural determination gene Mash1. In contrast, expression of Hes5 is restricted to clusters of neural progenitor cells and requires Mash1 function. Mutations in Hes1 and Hes5 also have distinct consequences on olfactory placode neurogenesis. Loss of Hes1 function leads both to expression of Mash1 outside of the normal domain of neurogenesis and to increased density of MASH1-positive progenitors within this domain, and results in an excess of neurons after a delay. A mutation in Hes5 does not produce any apparent defect. However, olfactory placodes that are double mutant for Hes1 and Hes5 upregulate Ngn1, a neural bHLH gene activated downstream of Mash1, and show a strong and rapid increase in neuronal density. Together, these results suggest that Hes1 regulates Mash1 transcription in the olfactory placode in two different contexts, initially as a prepattern gene defining the placodal domain undergoing neurogenesis and, subsequently, as a neurogenic gene controlling the density of neural progenitors in this domain. Hes5 synergises with Hes1 and regulates neurogenesis at the level of Ngn11 expression. Therefore, the olfactory sensory neuron lineage is regulated at several steps by negative signals acting through different Hes genes and targeting the expression of different proneural gene homologs (Cau, 2000).

The genes of the Hes family encode bHLH transcription factors that are most closely related to two groups of negative regulators of neurogenesis in Drosophila: hairy and the products of the Enhancer of Split complex (Espl). Both hairy and Espl products have been shown to directly repress transcription of the proneural gene achaete, but their activity is required in different contexts. hairy is a prepattern gene. It is required in large areas of the wing and leg imaginal discs to prevent ectopic expression of the proneural gene achaete and the formation of ectopic bristles. The genes of the Espl complex are neurogenic genes that are activated by Notch signaling in a process of lateral inhibition during embryonic and adult neurogenesis. Activation of the Espl genes blocks the accumulation of high amounts of proneural protein in most cells of the proneural clusters, thereby preventing them from adopting a neural fate. Hes1 regulation and mutant phenotype in the developping OE suggest that Hes1 has a dual role, acting as a prepattern (hairy-like) gene at the onset of neurogenesis in the olfactory placode and subsequently as a neurogenic (Espl-like) gene regulating Mash1 expression in olfactory sensory epithelium (OE) progenitors. In addition, the regulation of Hes5 expression in the olfactory placode and the placodal phenotype of Hes1;Hes5 double mutants support a role for Hes5 as a neurogenic gene acting at a later step in the olfactory sensory neuron (OSN) lineage (Cau, 2000).

Distinct stages of OSN progenitor maturation have been defined by the sequential expression of the three neural bHLH genes Mash1, Ngn1 and NeuroD. Mash1 is involved in the generation of basal OSN progenitors, whereas Ngn1 is required for their differentiation, and the function of NeuroD in this lineage has not yet been characterized. Analysis of the Hes1 mutant phenotype demonstrates that Hes1 regulates OSN development at the level of Mash1 expression. Despite the enlargement of the Mash1-positive cell population in Hes1 mutant olfactory placodes, there is only a relatively small increase in expression of Ngn1, suggesting that the step of Ngn1 expression is also subject to negative regulation. Indeed, the phenotype of Hes1;Hes5 double mutant placodes shows a further increase in the number of Ngn1-positive cells and of SCG10-positive neurons (SCG10 is a panneural marker that functions to depolymerize microtubules: there is no Drosophila homolog, but Drosophila CG5981 contains several domains that are partially homologous to SCG10) without change in the expression of Mash1, indicating that Hes5 is likely to regulate OSN development at the level of Ngn1 expression. Altogether, these data suggest that Notch signaling, acting through different Hes genes, regulates the production of OSNs by targeting the expression of two bHLH genes that control the development of the lineage at two distinct steps at least. This complex negative regulation of the OSN lineage at two levels, the determination and the differentiation of basal progenitors, allows for a fine tuning of the rate of neuronal production in the OE (Cau, 2000).

bHLH transcription factors are expressed sequentially during the development of neural lineages, suggesting that they operate in genetic cascades. In the olfactory epithelium (OE), the proneural genes Mash1 and neurogenin1 are expressed at distinct steps in the same olfactory sensory neuron (OSN) lineage. Loss-of-function analysis shows that both genes are required for the generation of OSNs. However, their mutant phenotypes are strikingly different, indicating that they have divergent functions. In Mash1 null mutant mice, olfactory progenitors are not produced and the Notch signaling pathway is not activated, establishing Mash1 as a determination gene for olfactory sensory neurons. In neurogenin1 null mutant mice, olfactory progenitors are generated but they express only a subset of their normal repertoire of regulatory molecules and their differentiation is blocked. Thus neurogenin1 is required for the activation of one of several parallel genetic programs functioning downstream of Mash1 in the differentiation of OSNs. These results illustrate the versatility of neural bHLH genes which adopt either a determination or a differentiation function, depending primarily on the timing of their expression in neural progenitors (Cau, 2002).

The analysis of the Ngn1 mutant phenotype in the OE has revealed the expression in the OE of two groups of regulatory genes distinct in their mode of regulation in basal progenitors. Genes of the first group, which include NeuroD and paired-homeobox gene Phd1, require Ngn1 function for their expression. Genes of the second group, which include the Lim-homeobox gene Lhx2 and the HLH gene Ebf1, are activated in basal progenitors independently of Ngn1 activity. None of these genes are expressed in Mash1 mutant OE, indicating that they belong to distinct regulatory pathways that are activated downstream of Mash1 in basal progenitors. It has been proposed that in neural crest-derived progenitors, Mash1 couples two parallel differentiation programs controlling the expression of neuronal subtype genes (e.g. the homeobox gene Phox2a and genes encoding the neurotransmitter synthesizing enzymes TH and BDH) and pan-neuronal genes (e.g., genes encoding peripherin and NF160). The idea that neuronal differentiation entails the activation of distinct regulatory programs implementing the different aspects of the neuronal phenotype has been amply supported experimentally. The results presented here indicate that Mash1 is likely to similarly couple the various components of the OSN phenotype. Among the regulatory genes expressed in basal progenitors and missing in Mash1 mutant OE, NeuroD and Ebf1 are likely to promote generic neuronal differentiation of OSNs. Both genes are broadly expressed in neurons in the embryonic nervous system and have the capacity to induce ectopic neurons when forcibly expressed in Xenopus embryos. In contrast, other basal OE progenitor genes, e.g., Phd1 and Lhx2, are likely to be involved in the specification of OSN identity. These genes belong to the paired-homeodomain and Lim-homeodomain families of transcription factors, respectively, which have been implicated in specification of various aspects of the neuronal phenotype. It is interesting to note that the Ngn1-dependent and -independent groups of regulators defined in this study do not segregate into 'pan-neuronal' (NeuroD and Ebf1) and 'neuronal subtype' (Phd1 and Lhx2) categories, but are on the contrary distributed in both. This suggests that the regulatory programs supporting OSN differentiation are not specialized in the acquisition of either generic or OSN-specific traits, but may instead control the acquisition of different combinations of both types of traits. The logic behind this complex regulation of the OSN phenotype remains to be elucidated (Cau, 2002).

Mice lacking both Mash1 and Ngn1 present a complete depletion of OSNs at all stages examined, indicating that together, Mash1 and Ngn1 are required for the progression of neurogenesis throughout the OE. Analysis of the olfactory placodes of Mash1;Ngn1 double mutant embryos shows that the two genes have redundant functions in the determination of OSN progenitors at this early stage. In particular, a lack of Dll1 and Hes5 expression reveals that Notch signaling is not activated and that OSN progenitors are likely missing from the olfactory placodes in the absence of both Mash1 and Ngn1 (Cau, 2002).

The redundancy of Mash1 and Ngn1 function in the determination of placodal progenitors at E10.0 is in marked contrast to the distinct and sequential roles of the two genes in the OE at E12.5. Even at E12.5, the Mash1/Ngn1 cascade does not operate in all OSN progenitors, since Ngn1 is expressed and required in a subset of Mash1 mutant OE progenitors at this stage. The fact that Mash1-independent progenitors are found at reproducible locations in different Mash1 mutant embryos argues for the existence of a distinct progenitor population in which a neurogenesis program including Ngn1 expression can be activated without Mash1 function. Whether Ngn1 is required for the determination of Mash1-independent progenitors at E12.5 as is the case for progenitors of olfactory placodes, or for their differentiation as is the case for other E12.5 OE progenitors, is difficult to address given the rarity of these cells. In any case, the epistatic relationship between Mash1 and Ngn1 observed in most OSN progenitors at E12.5 is not a general feature of the neurogenesis program in the OE, but is instead restricted both temporally (to the E12.5 OE and not the E10.0 placode) and spatially (to the rostral OE and less so to the caudal OE) (Cau, 2002).

How can these findings be reconciled with the observation that in regulatory cascades underlying cell lineage development, distinct subfamilies of bHLH factors are usually used as either early expressed determination factors or later expressed differentiation genes? These results support the idea that determination factors also have the necessary properties to participate to neuronal differentiation programs, and that the specific determination or differentiation function of genes like Ngn1 or Mash1 depends primarily on the timing of their expression and on the context of their activity. In contrast, other neural bHLH genes such as NeuroD and related genes, are consistently expressed in late precursors and post-mitotic neurons and have not been implicated in the process of progenitor selection during normal development, although NeuroD shares with Ngns the property to induce neurons and ectopically activate Notch signaling when overexpressed in Xenopus embryos, and NeuroD has been shown to participate to the choice between neuronal and glial fates in the retina. The fact that differentiation genes such as NeuroD are normally not involved in cell determination suggests that they may lack some of the necessary properties. Indeed, the myogenic determination factors MyoD and Myf5 are more efficient than the differentiation factor myogenin at remodelling chromatin and activate transcription at previously silent loci, an activity which is very likely relevant to their determination function. Possibly as a consequence of these divergent activities, myogenin cannot fully substitute for Myf5 when expressed from the Myf5 locus. It will be interesting to test the prediction that neural determination genes (Mash1 and Ngns) can efficiently substitute for differentiation genes (NeuroD and related genes) but not the reverse, in similar gene swapping experiments (Cau, 2002).

The basic helix-loop-helix (bHLH) gene Hes6, a novel member of the family of mammalian homologs of Drosophila hairy and Enhancer of split has been isolated. Hes6 is expressed by both undifferentiated and differentiated cells, unlike Hes1, which is expressed only by the former cells. Hes6 alone does not bind to the DNA but suppresses Hes1 from repressing transcription. In addition, Hes6 suppresses Hes1 from inhibiting the Mash1-E47 heterodimer and thereby enables Mash1 and E47 to upregulate transcription in the presence of Hes1. Furthermore, misexpression of Hes6 with retrovirus in the developing retina promotes rod photoreceptor differentiation, like Mash1, in sharp contrast to Hes1, which inhibits cell differentiation. These results suggest that Hes6 is an inhibitor of Hes1, supports Mash1 activity and promotes cell differentiation. Mutation analysis reveals that Hes1- and Hes6-specific functions are, at least in part, interchangeable by alteration of the loop region, suggesting that the loop is not simply a nonfunctional spacer but plays an important role in the specific functions (Bae, 2000).

Neurons and glial cells differentiate from common precursors. Whereas the gene glial cells missing determines the glial fate in Drosophila, current data about the expression patterns suggest that, in mammals, gcm homologs are unlikely to regulate gliogenesis. In mouse retina, the bHLH gene Hes5 is specifically expressed by differentiating Müller glial cells and misexpression of Hes5 with recombinant retrovirus significantly increases the population of glial cells at the expense of neurons. Conversely, Hes5-deficient retina show 30%-40% decrease of Müller glial cell number without affecting cell survival. These results indicate that Hes5 modulates glial cell fate specification in mouse retina (Hojo, 2000).

It remains to be determined how Hes5 specifies the glial fate in the retina. One possible mechanism is that Hes5, a DNA-binding repressor, may repress expression of neuronal bHLH genes such as Mash1, NeuroD and Math3 and lead to the glial fate. Supporting this idea, in the retina of Mash1- or NeuroD-deficient mice, Müller glial cells increase in number. The notion that Hes5 may repress Mash1 expression is also supported by the finding that Mash1 expression is prematurely upregulated in the regions where Hes5 expression disappears in RBP-J or Notch1 mutant mice. Thus, the antagonistic regulation between Hes5 and the neuronal bHLH genes such as Mash1 may determine the ratio of neuronal to glial cell numbers. However, in the retina of Hes5-deficient mice, many Müller glial cells still are differentiated, suggesting that Hes5 may be redundant in gliogenesis (Hojo, 2000 are references therein).

The inner ear (vestibular and cochlear) efferent neurons are a group of atypical motor-like hindbrain neurons that innervate inner ear hair cells and their sensory afferents. They are born in the fourth rhombomere, in close association with facial branchial motor neurons, from which they subsequently part through a specific migration route. The inner ear efferents depend on Phox2b for their differentiation, behaving in that respect like hindbrain visceral and branchial motor neurons. The vestibular efferent nucleus is no longer present at its usual site in mice inactivated for the bHLH transcription factor Mash 1. The concomitant appearance of an ectopic branchial-like nucleus at the location where both inner ear efferents and facial branchial motor neurons are born suggests that Mash1 is required for the migration of a subpopulation of rhombomere 4-derived efferents (Tiveron, 2003).

The inner ear (vestibular and cochlear) efferent neurons are a group of atypical motor-like hindbrain neurons that innervate inner ear hair cells and their sensory afferents. They are born in the fourth rhombomere, in close association with facial branchial motor neurons, from which they subsequently part through a specific migration route. The inner ear efferents depend on Phox2b for their differentiation, behaving in that respect like hindbrain visceral and branchial motor neurons. The vestibular efferent nucleus is no longer present at its usual site in mice inactivated for the bHLH transcription factor Mash 1. The concomitant appearance of an ectopic branchial-like nucleus at the location where both inner ear efferents and facial branchial motor neurons are born suggests that Mash1 is required for the migration of a subpopulation of rhombomere 4-derived efferents (Tiveron, 2003).

Ascl1 expression defines a subpopulation of lineage-restricted progenitors in the mammalian retina

The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To elucidate further the mechanisms of fate diversification, the expression was examined of three transcription factors made by retinal progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. It was observed that progenitors were heterogeneous, expressing every possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different cell fate outcomes, Ascl1- and Ngn2-inducible expression fate mapping was conducted using the Cre recombinase fused to the tamoxifen inducable CreER™/LoxP system. It was found that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. It was next determined whether Ascl1 prevented RGC development. Ascl1-null mice had normal numbers of RGCs and, interestingly, it was observed that a subset of Ascl1+ cells could give rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. These results link progenitor heterogeneity to different fate outcomes. It was shown that Ascl1 expression defines a competence-restricted progenitor lineage in the retina, providing a new mechanism to explain fate diversification (Brzezinski, 2011).

Ascl1+ progenitors did not significantly generate RGCs at any time point. Although it cannot be formally rule out that a biologically relevant, rare RGC subtype(s) derives from the Ascl1 lineage, the data strongly argue against this possibility. First, Ascl1-GFP and pan-Brn3 co-expression data suggests that this putative subtype would have to be exceedingly rare during development (one cell or fewer per retina). Second, whereas Brn3a/b/c expression might not label all ganglion cell subtypes, retrograde dextran uptake labels all RGCs; nonetheless, a significant number of Brn3+ or dextran-labeled RGCs in the Ascl1 lineage is not observed (Brzezinski, 2011).

Retroviral lineage-tracing studies have shown that all seven retinal cell types derive from a common progenitor population. However, throughout most of retinal development, several cell types are being formed concurrently. This implies that retinal progenitor cells form a heterogeneous population that expresses different intrinsic factors and responds differentially to extrinsic cues. Previous studies have demonstrated that Ascl1 is expressed in a subset of retinal progenitors, and proposed that Ascl1 expression defines a particular stage in the progenitor cells. The Ascl1 lineage-tracing experiment confirmed this proposal, as Ascl1+ progenitors significantly generated all retinal cell types except for RGCs. This is the first molecularly defined progenitor population that has competence to form all but one cell type in the retina. Analogously, Ascl1+ cells give rise to a competence-restricted lineage in the spinal cord, forebrain and other regions of the CNS (Brzezinski, 2011).

Although lineage-restricted progenitors have not been previously shown to exist in the mouse retina in vivo, it has been shown that committed precursors exist in the fish retina. Many of the rod photoreceptors in the teleost retina are generated through a committed rod precursor. Also, immature horizontal cells in the inner nuclear layer of the zebrafish retina have been shown to undergo a mitotic division to generate two horizontal cells. In Ath5-GFP transgenic zebrafish, GFP is made by cells in their terminal division such that one daughter becomes a ganglion cell and the other adopts a different neural fate. This cell is somewhat different from the committed horizontal and rod precursors, but nevertheless suggests that, in fish, there is something unique about progenitor cells in their last cell division. The results suggest that Ngn2 expression might mark mouse progenitors in their final mitotic division since nearly all of the traced cells were found in one- to two-cell clumps and because their cell fates were strongly biased towards those born at the time of tamoxifen treatment. However, this study did not find any clear pattern to the types of progeny generated in two-cell clumps in the Ngn2 or Ascl1 lineages. This appears to be true in vitro as well, where single isolated progenitors that undergo their final mitotic division in culture do not show a bias towards generating cells of the same identity (Brzezinski, 2011).

This study demonstrates that the E12.5/E13.5 Ascl1 and Ngn2 lineages are quite distinct from each other and from retroviral lineages traced at the same time points. However, these lineage-tracing techniques are quite different from each other. First, only progenitors can be infected and labeled by retroviruses, but it is possible for Ascl1- and Ngn2-Cre to persist transiently and catalyze recombination in newly postmitotic cells. Thus, the average clump size in the Ascl1 and Ngn2 lineages should be lower than that in the retroviral lineages, but the maximum clump size should be similar. Second, although clumps seen in the E12.5/E13.5 Ascl1 and Ngn2 lineage traces were sparsely distributed, it cannot be certain they were clones. Thus, the number of progeny that Ascl1+ and Ngn2+ cells generate might be overestimated (Brzezinski, 2011).

A major difference between these three lineage traces was the clump/clone size distribution. Whereas some retroviral clones were larger than 50 cells, no clumps were observed in the Ascl1 lineage that contained more than 15 cells and no clumps in the Ngn2 lineage that contained more than nine cells. Most of the clumps in the Ascl1 and Ngn2 lineages contained one or two cells (82.4% and 98.5%, respectively), but only a small fraction of retroviral clones (~33%) contained one or two cells. These differences in the clump/clone size distributions strongly suggest the following model. Retinal progenitors that do not express Ascl1 or Ngn2 can undergo a large number of mitotic divisions, whereas progenitors that express Ascl1 undergo few mitotic divisions and those that express Ngn2 are in their last cell cycle. This model is consistent with observations in other regions of the CNS, where it has been proposed that Ngn2-expressing progenitors in the spinal cord are in their last cell division and that Ascl1 overexpression can inhibit proliferation in certain contexts (Brzezinski, 2011).

The Ascl1, Ngn2 and retroviral lineages have distinct distributions of other cell fates. These distinctions, with the exception of RGCs, can be attributed to differences in proliferation. Ascl1+ cells at E12.5/E13.5 primarily adopted early neural fates, but some of the recombined cells proliferate until at least P0 and generate later fates, such as rods, bipolars and Müller glia. By contrast, the Ngn2 lineage fate distribution is consistent with near total cell cycle exit at the time of tamoxifen administration. The retroviral lineages from these same ages contained large clones with, predominantly, cells born later in development, such as rods, bipolars and amacrine cells. Small retroviral clones (<16 cells) had more early-born fates, but did not match the fate distribution of the Ascl1 or Ngn2 lineages. Although the Ngn2 lineage cannot be an obligate subset of the Ascl1 lineage, the data suggest that these two lineages overlap such that co-expressing cells are in their final cell division (Brzezinski, 2011).

It was observed that Ngn2+ progenitors generated few RGCs. Because RGCs and horizontal cells are similar in number and their birthdates overlap extensively these cell types should be similarly represented in the E12.5/E13.5 Ngn2 lineage trace. However, RGC frequency was considerably lower (~12-fold) than horizontal cells, indicating that only a subset of RGCs come from Ngn2+ progenitors. This suggests that Ngn2 lineage is largely RGC-competence restricted, probably a result of considerable overlap with the Ascl1 lineage. It is unclear whether Olig2+ progenitors are also competence restricted. An initial examination of the E14.5 Olig2 lineage revealed RGCs, amacrines and horizontal cells, suggesting that Olig2+ progenitors might be more similar to Ngn2+ than to Ascl1+ progenitors in the retina (Brzezinski, 2011).

Mammalian Achaete Homologs: Development of the peripheral nervous system

Analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 (Drosophila homologs CG15782 and CG4136) are essential for the generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. In Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression does not disappear but remains in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induces generation of the inner nuclear layer cells, including Müller glia, but few of them are mature bipolar cells. Misexpression of Mash1 or Math3 alone does not promote bipolar cell genesis either, but inhibits Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increases the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promote the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice (Hatakeyama, 2001).

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. The activities of the two genes have been systematically compared during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, it has been demonstrated that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity (Parras, 2002).

The molecular control mechanisms and regulatory molecules involved in nerve repair are not yet well known. Schwann cells have been attributed an important role in peripheral nerve regeneration; therefore, attention has been drawn to regulatory factors expressed by these glial cells. Mash2, a basic helix-loop-helix (bHLH) transcription factor previously shown to be crucial for placenta development, is expressed by Schwann cells of adult peripheral nerves. It has been observed that this gene is downregulated after nerve lesion and, using cDNA array hybridization technology, it was demonstrated that Mash2 is a regulator of Krox24, Mob-1, and CXCR4 expression in cultured Schwann cells. In addition, evidence is provided that Mash2 is a negative regulator of Schwann cell proliferation. Mash2 represents a first candidate for the missing class B bHLH proteins in peripheral nerves (Küry, 2002).

Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord

The neural basic helix-loop-helix transcription factor Ascl1 (previously Mash1) is present in ventricular zone cells in restricted domains throughout the developing nervous system. This study uses genetic fate mapping to define the stage and neural lineages in the developing spinal cord that are derived from Ascl1-expressing cells. Ascl1 is present in progenitors to both neurons and oligodendrocytes, but not astrocytes. Temporal control of the fate-mapping paradigm reveals rapid cell-cycle exit and differentiation of Ascl1-expressing cells. At embryonic day 11, Ascl1 identifies neuronal-restricted precursor cells that become dorsal horn neurons in the superficial laminae. By contrast, at embryonic day 16, Ascl1 identifies oligodendrocyte-restricted precursor cells that distribute throughout the spinal cord. These data demonstrate that sequentially generated Ascl1-expressing progenitors give rise first to dorsal horn interneurons and subsequently to late-born oligodendrocytes. Furthermore, Ascl1-null cells in the spinal cord have a diminished capacity to undergo neuronal differentiation, with a subset of these cells retaining characteristics of immature glial cells (Battiste, 2007).

Ascl1 is required for oligodendrocyte development in the spinal cord

Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitors (OLPs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. How these distinct steps are controlled remains unclear. The helix-loop-helix (HLH) transcription factor Ascl1 plays an important role in early generation of OLPs in the developing spinal cord. Ascl1 is also involved in terminal differentiation of oligodendrocytes late in development. Ascl1-/- mutant mice showed a deficiency in differentiation of myelin-expressing oligodendrocytes at birth. In vitro culture studies demonstrate that the induction and maintenance of co-expression of Olig2 and Nkx2-2 in OLPs, and thyroid hormone-responsive induction of myelin proteins are impaired in Ascl1-/-. Gain-of-function studies further showed that Ascl1 collaborates with Olig2 and Nkx2-2 in promoting differentiation of OLPs into oligodendrocytes in vitro. Overexpression of Ascl1, Olig2 and Nkx2-2 alone stimulates the specification of OLPs, but the combinatorial action of Ascl1 and Olig2 or Nkx2-2 is required for further promoting their differentiation into oligodendrocytes. Thus, Ascl1 regulates multiple aspects of oligodendrocyte development in the spinal cord (Sugimori, 2008).

Compensational regulation of bHLH transcription factors in the postnatal development of BETA2/NeuroD1-null retina

The bHLH transcriptional factor BETA2/NeuroD1 is essential for the survival of photoreceptor cells in the retina. Although this gene is expressed throughout the retina, BETA2/NeuroD1 knockout mice show photoreceptor cell degeneration only in the outer nuclear layer of the retina; other retinal neurons are not affected. Previous studies on retina explants lacking three bHLH genes revealed that retinal neurons in the inner nuclear layer require multiple bHLH genes for their differentiation and survival. However, single- or double-gene mutations show no or a lesser degree of abnormalities during eye development, likely because of compensation or cooperative regulation among those genes. Because not all null mice survive until the retina is fully organized, no direct evidence of this concept has been reported. To understand the regulatory mechanisms between bHLH factors in retinal development, a detailed analysis of BETA2/NeuroD1 knockout mice was performed. BETA2/NeuroD1 was expressed in all 3 layers of the mouse retina, including all major types of neurons. In addition, a null mutation of BETA2/NeuroD1 resulted in up-regulation of other bHLH genes, Mash1, Neurogenin2, and Math3, in the inner nuclear layer. These data suggest that compensatory and cross regulatory mechanisms exist among the bHLH factors during retinal development (Cho, 2007).

During postnatal development, BETA2/NeuroD1 expression is observed mainly in photoreceptor cells in the outer nuclear layer (ONL). However, moderate levels of expression remain in the outer half and innermost layer of the inner nuclear layer (INL) of the retina as well as in a certain population of cells in the ganglion cell layer (GCL). Therefore, the possibility that BETA2/NeuroD1 has functions in cell type specification in the INL together with other bHLH genes could not be ruled out. Both gain-of-function and loss-of-function studies have demonstrated that BETA2/NeuroD1 participates in the neuron/glia cell fate decisions, similar to other bHLH genes, including Mash1, Math3, and Math5 in retinal explants. Thus, attempts were made to identify differences in the population of major cell types in BETA2/NeuroD1-null retina compared with wild-type littermate retina. However, no differences were found. This result may be due to compensational regulation by other bHLH genes, such as Mash1, Math3, Neurogenin2, and Math5 (Cho, 2007).

Mash1, Math3, and Neurogenin2 are known to be expressed in the developing retina and act as positive regulators. Together with homeodomain factors such as Pax6 and Crx, these factors play important roles in cell type specification during early development. For example, Mash1 and Math3 are expressed predominantly in bipolar cells, and double knockouts of these genes decreases the bipolar cell population while increasing the Müller glial cell population. Neurogenin2 is also transiently expressed in all major neuron types in the mouse retina, and its expression is required for photoreceptor cells, horizontal cells, and bipolar cells. In contrast, BETA2/NeuroD1 is transiently expressed in differentiating amacrine cells. Although BETA2/NeuroD1-null mutation shows delayed amacrine cell development at earlier stages, the number of amacrine cells eventually is the same as that found in wild-type retinas. However, in double-knockout mutations with BETA2/NeuroD1 and Math3, the number of amacrine cells is decreased and that of retinal ganglionic cells is increased. Interestingly, amacrine cells adopt the ganglion cells' fate in this BETA2/NeuroD1;Math3 double-knockout mutant. In addition, the triple bHLH knockouts Mash1;Neurogenin2;Math3 and Math3;Neurogenin2; BETA2/NeuroD1 have been shown to have fewer horizontal cells, but any combination of double mutations of Mash1 or Math3 or BETA2/NeuroD1 with Neurogenin2 display abnormalities in retina development. Furthermore, single-knockout mutations of the genes barely affect the neuronal cell population in the INL and show no retinal abnormalities. Taken together, these results suggest that the bHLH factors cross regulate each others' expression and can specify neuronal subtypes cooperatively during late retinogenesis, especially in progenitor cells in the INL, to generate various subtypes of retinal neurons. Although the precise mechanism for retina cell type specification remains to be determined, these results provide further support for cooperative and compensational regulatory specification during postnatal retinogenesis (Cho, 2007).

Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord

During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Evidence is presented that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. Each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. Genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. A molecular code model is proposed whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord (Sugimori, 2007).

This study demonstrates the combinatorial actions of two classes of transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing ventral spinal cord. In vitro data have shown that the proneural HLH factors Ngns and Mash1 intrinsically possess the activity to induce neurons and oligodendrocytes, respectively, whereas the inhibitory HLH factors Id1 and Hes1 stimulate astrogenesis. Yet, the timing of differentiation of neurons and glia in vivo is not determined a priori by the expression of these HLH factors. The data have shown that they do so in collaboration with Pax6, Olig2 and Nkx2.2, the primary function of which has been thought to be to specify the positional identity of progenitors (Sugimori, 2007).

These patterning factors participate in controlling both the timing of differentiation and cell fate by two mechanisms. First, they act to maintain progenitors undifferentiated by suppressing otherwise strong neurogenic and astrogenic activities of Ngns and Id1 and/or Hes1. The suppression of the neurogenic activity of Ngn2 by Olig2 is in accordance with the fact that the Olig2+ domain markedly expands while producing a large number of motoneurons. Such an activity, however, is not limited to Olig2, but common among three patterning factors. Second, three patterning factors differentially modulate the activity of Mash1. Mash1 itself promotes differentiation of both neurons and oligodendrocytes. Pax6, however, converts Mash1 to become selectively neurogenic, whereas Olig2 selectively enhances Mash1-dependent oligodendrogenesis. Thus, it is proposed that these two classes of transcription factors comprise a molecular code for the coordinated spatiotemporal control of neuro/gliogenesis. According to this model, the relative expression levels of patterning and HLH factors at the single cell level are crucial to determine the fate of multipotent progenitors. How the timing and expression level of individual factors are precisely controlled remains to be further investigated. How these two classes of transcription factors coordinately regulate genetic programs for differentiation of neurons and glia also needs to be examined in the future studies (Sugimori, 2007).

Mammalian Achaete Homologs: Development of the central nervous system

Mice mutant for the gene Mash1 display severe neuronal losses in the olfactory epithelium and ganglia of the autonomic nervous system, demonstrating a role for Mash1 in development of neuronal lineages in the peripheral nervous system. Mash1 function in the central nervous system, has been analyzed, focusing on the ventral telencephalon where it is expressed at high levels during neurogenesis. Mash1 mutant mice present a severe loss of progenitors, particularly of neuronal precursors in the subventricular zone of the medial ganglionic eminence. Discrete neuronal populations of the basal ganglia and cerebral cortex are subsequently missing. An analysis of candidate effectors of Mash1 function reveals that the Notch ligands Dll1 and Dll3, and the target of Notch signaling Hes5, fail to be expressed in Mash1 mutant ventral telencephalon. In the lateral ganglionic eminence, loss of Notch signaling activity correlates with premature expression of a number of subventricular zone markers by ventricular zone (VZ) cells. In mutant embryos, GAD67 is ectopically expressed by Z cells of the ventral telencephalon at all developmental stages examined. Similarly, Dlx-5 is ectopically expressed in the VZ in mutant embryos. Dlx-1 transcripts are also found in virtually all cells of the mutant VZ, an expression normally observed only in the subventricular zone. Expression of Lhx2 expression is strongly reduced in the VZ of the mutant median ganglionic eminence, but remains at significant levels in the lateral ganglionic eminence. This result suggests that, although mutant VZ cells have prematurely acquired subventricular zone characteristics, they have only partially changed phenotype in the lateral ganglionic eminence, since they maintain expression of a ventricular zone marker. Therefore, Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production. Negative regulation of Dlx-1/2 by Mash1 is probably due to a process of lateral inhibition, rather than to a cell-intrinsic mechanism. Mice mutant for Dlx-1 and Dlx-2 present a striatal defect, interpreted as a block in differentiation specifically affecting late-born matrix neurons (Casarosa, 1999).

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, evidence is presented that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1, related to Drosophila Prospero, are involved in this process. In the developing rat forebrain and E13.5, the Mash-1+ domain covers the ventral thalamus, hypothalamus and ganglionic eminence. Strong expression is also seen in the dorsal midbrain, where neurogenesis proceeds earlier than in the forebrain. In contrast, the dorsal thalamus and the primordia of the cerebral cortex are devoid of expression of Mash-1 at this stage. Prox-1 follows the discrete patterns of Mash-1, which also demarcates sharp boundaries. These characteristic expression patterns are reminiscent of the two brain-specific homeobox genes, Dlx-1 and Pax-6. In vivo, Mash-1- and Prox-1-expressing cells are defined as a transient proliferating population that is molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, induction of Mash-1 and Prox-1 has been shown to coincide with an initial step of stem cell differentiation. Furthermore, forced expression of Mash-1 leads to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, specific defects are found in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, it is proposed that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS (Torri, 1999).

The pathfinding of thalamocortical axons (TCAs), which relay sensory information from the dorsal thalamus to the neocortex has been analyzed in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. This projection originates from neurons of the dorsal thalamus, which in mice are generated between embryonic days (E) 10 and 13. By E14, the first TCAs have reached their main target, the neocortex. Enroute to the neocortex TCAs extend ventrally in the diencephalon, make a sharp turn to cross the diencephalic-telencephalic border, and then extend dorsolaterally through the ventral telencephalon toward the neocortex. While the resultant V-shaped projection, the nascent internal capsule, passes through several forebrain domains, it appears to encounter but avoid others. For example, in their ventral course through the diencephalon, TCAs of normal mice turn laterally into the ventral telencephalon, rather than maintaining a straight ventral trajectory and thereby extending along the hypothalamic surface. In wild-type mice, four cell domains (two in the ventral thalamus, one in the hypothalamus and one in the telencephalon) have been identified that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, Mash-1 mutant mice were studied to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTPdelta domain in ventral thalamus (RPTPdelta codes for a transmembrane receptor-type protein tyrosine phosphatase) is correspondingly reduced and the normally graded expression of RPTPdelta in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and instead form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTPdelta expression. A small proportion of TCAs extend around the RPTPdelta domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon (Tuttle, 1999).

An intracellular timer in oligodendrocyte precursor cells is thought to help control the timing of their differentiation. The expression of the Hes5 and Mash1 genes, which encode neural-specific bHLH proteins, decrease and increase, respectively, in these cells with a time course expected if the proteins are part of the timer. Enforced expression of Hes5 in purified precursor cells strongly inhibits the normal increase in the thyroid hormone receptor protein TRbeta1, which is thought to be part of the timing mechanism; it also strongly inhibits the differentiation induced by either mitogen withdrawal or thyroid hormone treatment. Enforced expression of Mash1, by contrast, somewhat accelerates the increase in TRbeta1 protein. These findings suggest that Hes5 and Mash1 may be part of the cell-intrinsic timer in the precursor cells (Kondo, 2000).

The role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells has been addressed. Mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture shows that a large fraction of Ngn2;Mash1 double-mutant progenitors fail to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate (Nieto, 2001).

The analysis of bHLH gene function in progenitor cultures led to three important observations: (1) by studying separately the properties of Ngn2+ and Ngn2- progenitors, it has been demonstrated that Ngn2 expression is restricted to committed neuronal and astrocytic progenitors and is absent from pluripotent progenitors; (2) both neuronal and astrocytic lineages of the cortex are heterogenous for Ngn2 expression and Mash1 has an essential function in the Ngn2- subpopulation of cortical progenitors; (3) in vitro studies show that Ngn2 and Mash1 mutations result primarily in defects in fate commitment of cortical progenitors, which remain pluripotent or adopt a glial fate rather than becoming restricted to neuronal differentiation. Together, the data obtained in vivo and in culture support the conclusion that Ngn2 and Mash1 act in different populations of cortical progenitors to promote the neuronal fate and inhibit the astrocytic fate (Nieto, 2001).

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. Pax6 was used as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. The genomic binding locations were identified of Pax6 in neocortical stem cells during normal development, and the functional significance of genes were ascertained that were found to be regulated by Pax6. Pax6 was found to positively and directly regulate cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, a core network regulating neocortical stem cell decision-making was identified in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. It was found that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal (Sansom, 2009).

A transcriptional mechanism integrating inputs from extracellular signals to activate hippocampal stem cells

The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. This study shows that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 (Drosophila homolog: Achaete) and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate of hippocampal stem cells, and inactivating Ascl1 blocks quiescence exit completely, rendering them unresponsive to activating stimuli. Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating the expression of cell-cycle regulatory genes. Ascl1 is similarly required for stem cell activation in the adult subventricular zone. These results support a model whereby Ascl1 integrates inputs from both stimulatory and inhibitory signals and converts them into a transcriptional program activating adult neural stem cells (Andersen, 2014).

Dlx1 and Dlx2 control neuronal versus oligodendroglial cell fate acquisition in the developing forebrain: Mash1 promotes OPC formation by restricting the number of Dlx+ progenitors

Progenitors within the ventral telencephalon can generate GABAergic neurons and oligodendrocytes, but regulation of the neuron-glial switch is poorly understood. This study investigated the combinatorial expression and function of Dlx1&2, Olig2, and Mash1 transcription factors in the ventral telencephalon. Dlx homeobox transcription factors, required for GABAergic interneuron production, repress oligodendrocyte precursor cell (OPC) formation by acting on a common progenitor to determine neuronal versus oligodendroglial cell fate acquisition. Dlx1&2 negatively regulate Olig2-dependant OPC formation and Mash1 promotes OPC formation by restricting the number of Dlx+ progenitors. Progenitors transplanted from Dlx1&2 mutant ventral telencephalon into newborn wild-type mice do not produce neurons but differentiate into myelinating oligodendrocytes that survive into adulthood. These results identify another role for Dlx genes as modulators of neuron versus oligodendrocyte development in the ventral embryonic forebrain (Petryniak, 2007).

This analysis suggests a genetic model of GABAergic neuron versus oligodendrocyte specification in the ventral telencephalon. From as early as E10.5, OLIG2 is expressed in nearly all VZ progenitors in ventral telencephalon. By E12.5, two distinct populations of cells are observed in the VZ: OLIG2+/DLX2+/MASH1+ and OLIG2-only. The number of MASH1+/DLX2+ cells in the VZ is thought to be regulated non-cell-autonomously via Notch/Delta-mediated lateral inhibition. Thus, the increase in DLX2 expression from the VZ to SVZ1 is consistent with decreased Notch activity and progressive proneural functions of Mash1. Indeed, the proneural function of MASH1 might cell-autonomously, and positively, regulate Dlx expression as cells differentiate into GABAergic neurons. Reduced Notch/Delta-mediated lateral inhibition in Mash1 or Delta1 mutants results in an expansion of Dlx expression in nearly all ganglionic eminence progenitors in the VZ. Based on this model, expansion of DLX expression is predicted to lead to increased repression of OLIG2, and thereby to decreased OPC formation seen in the Mash1 mutants. Removal of Dlx1&2 function in the Dlx1&2;Mash1 triple mutants restores OLIG2 expression and OPC production. These findings suggest that MASH1 promotes oligodendrogenesis by nonautonomously inhibiting Dlx1&2 and maintaining the pool of OLIG2+/DLX2− progenitors (Petryniak, 2007).

After DLX expression is initiated in OLIG2+ cells within the VZ, there is a temporal delay between accumulation of DLX2 protein and suppression of OLIG2 expression. Progressive reduction in OLIG2 expression as DLX2 expression increases in the SVZ is consistent with DLX2+ cells downregulating OLIG2 via Dlx-mediated repression. In SVZ2, cells segregate into two pools expressing either DLX2 or OLIG2 that, in general, continue to differentiate along either the neuronal or oligodendroglial lineage, respectively. In this model, OPCs are primarily generated from cells that remain OLIG2+ from the VZ to SVZ, whereas GABAergic neurons arise from a DLX2+/OLIG2+ cell in which OLIG2 expression is downregulated. Consistent with this model, in vivo lineage analysis using inducible Olig2-Cre shows that Olig2+ progenitor cells in the forebrain give rise to GABAergic neurons, followed by oligodendrocytes. While the vast majority of Dlx-expressing cells repress Olig2 to become GABAergic neurons, a pathway may also exist whereby OLIG2+/DLX2+ progenitors downregulate Dlx expression to produce oligodendrocytes. Consistent with this hypothesis, a small number of OPCs arise from Dlx2/tauLacZ+ cells, and a few PDGFRα+ cells are generated from the Dlx2-Cre lineage. Thus, DLX2 expression does not represent an irreversible state of neuronal commitment, and in rare DLX2+/OLIG2+ cells, OLIG2 expression may predominate the driving of OPC development (Petryniak, 2007).

In conclusion, these findings show a genetic mechanism for GABAergic neuron and oligodendrocyte specification regulated by DLX2, MASH1, and OLIG2. It is proposed that DLX1&2 regulate a transcriptional hierarchy to control neuron versus oligodendroglial cell fate within a common bi-potent progenitor, based on the following lines of evidence: (1) GABAergic neuron formation is defective, while oligodendrogenesis is substantially increased in Dlx1&2 mutants; (2) transplants of wild-type medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) progenitors generate both neurons and oligodendrocytes, whereas those from Dlx1&2 mutants produce only oligodendrocytes; (3) loss of Dlx1&2 does not result in either increased proliferation or earlier onset of oligodendrogenesis; (4) Dlx1&2 are sufficient to autonomously repress OLIG2 expression, which is necessary for OPC production in Dlx1&2 mutants; and (5) Olig2-Cre and Dlx2-Cre lineages give rise to both GABAergic interneurons and oligodendrocytes. Thus, these data support that the transient DLX2+/OLIG2+ cells represent a common progenitor capable of generating GABAergic neurons and oligodendrocytes (Petryniak, 2007).

The SVZ of the lateral wall of the lateral ventricles is a site of neurogenesis in the adult mammal. Neurogenic astrocytes (type B cells) give rise to transit amplifying type C cells that produce type A neuroblasts. Intriguingly, type C cells express MASH1 and DLX2, and generate type A neuroblasts that develop into GABAergic interneurons in the olfactory bulb. Recent studies have found that OLIG2 is expressed in a small, heterogeneous population of type C cells that give rise to oligodendrocytes that populate the corpus callosum, striatum, and fimbria. The striking similarity in the transcription factors expressed within MGE progenitors and type C cells suggests that parallel mechanisms control GABAergic interneuron formation and oligodendrocyte production in the adult SVZ. In light of the current findings, it is speculated that DLX proteins suppress OLIG2 expression in the majority of type C cells to produce DLX2+/MASH1+ type A neuroblasts. However, a minority of type C cells may downregulate DLX expression to enable OLIG2 expression and produce OLIG2+/PDGFRα+ OPCs. It remains to be determined whether interactions between DLX, MASH1, and OLIG2 that occur in the embryonic forebrain play a similar role in regulating GABAergic neuron and oligodendrocyte production within the adult SVZ (Petryniak, 2007).

In the mouse, the first OPCs are generated in the MGE and AEP and produce oligodendrocytes that populate all regions of the forebrain by the time of birth. Lineage-mapping experiments using Nkx2.1-Cre have shown that these early-born oligodendrocytes are replaced postnatally by oligodendrocytes derived from more dorsal regions, including the LGE, caudal ganglionic eminence, and cortex. Thus, Nkx2.1-lineage oligodendrocytes were not detected at P30 in the corpus callosum or cortex. The data show that OPCs transplanted from E15.5 MGE and AEP into newborn mice can survive into adulthood. These transplanted cells incorporate into white matter tracts, especially the corpus callosum and fimbria, and express markers of mature oligodendrocytes. These contrasting results could be explained if only a subset of OPCs from the MGE and AEP were labeled using Nkx2.1-Cre recombination, or if the contribution of labeled cells was diluted below detection by OPCs from different regions. These transplants include the entire VZ and SVZ of the MGE and AEP and represent the potential of progenitors from the ventral telencephalic oligodendrocyte precursor region to produce oligodendrocytes that are maintained in the adult. It is possible that a heterotopic and heterochronic transplant introduces OPCs into an environment that may enable their long-term survival. Nevertheless, the results show that embryonic OPCs are not intrinsically programmed to be eliminated during the early postnatal period, raising the possibility that embryonically derived neural stem cells could be used as a source of oligodendrocytes in neurological disorders involving white matter loss, such as periventricular leukomalacia, a cause of cerebral palsy, and multiple sclerosis (Petryniak, 2007).

Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. This study revealed a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an 'on-target' pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myelin transcription factor 1 (Myt1l) zinc finger transcription factor do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, this study identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types (Wapinski, 2013).

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achaete: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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