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EVOLUTIONARY HOMOLOGS (part 2/3)

Mammalian Achaete Homologs: Expression patterns

Two mouse homologues of Achaete-Scute Complex genes, Mash-1 and Mash-2, have been isolated using the conservation of the basic-helix-loop-helix domain in this family. Mouse Mash-1 mRNA expression is restricted to cells of the developing central and peripheral nervous systems. There are three successive phases in the distribution of Mash-1 transcripts in the developing central nervous system. Initially, between embryonic day 8.5 and 10.5, Mash-1 transcripts are found in restricted domains in the neuroepithelium of the midbrain and ventral forebrain, as well as in the spinal cord. Between embryonic day 10.5 and 12.5, Mash-1 expression pattern changes from a restricted to a widespread one. Mash-1 transcripts are then found at variable levels in the ventricular zone in all regions of the brain. From embryonic day 12.5 to post-natal stages, Mash-1 is also expressed in cells outside of the ventricular zone throughout the brain. In addition, Mash-1 is expressed during development of the olfactory epithelium and neural retina. Overall, its expression pattern suggest that Mash-1 plays a role at early stages of development of specific neural lineages in most regions of the central nervous system and of several lineages in the peripheral nervous system. Mash-1 and mouse Notch show very similar expression patterns, both spatially and temporally, in the early developing brain and in the retina, suggesting that both genes may participate in the development of the same neural lineages. mash1 is not expressed during the determination of neural precursors and is not required for their specification. Instead, mash1 is present in terminally dividing neural progenitors to control in subsequent differentiation. (Guillemot, 1993).

The expression of murine Mash1 and neurogenin1 (ngn1) have been compared. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. In the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. There is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation (Ma, 1997).

Mammalian Achaete Homologs: Transcriptional regulation

Mash1 is a transcription factor required during embryogenesis for the development of multiple neural lineages. It is expressed in restricted domains at specific stages in the developing central and peripheral nervous systems and in the developing olfactory epithelium. The regulation of Mash1 has been investigated using transgenic mice containing Mash1/lacZ reporter constructs. Cis acting regulatory elements controlling Mash1 expression in the central nervous system are located within an 8-kb sequence of the Mash1 coding region. This 8-kb sequence does not contain elements directing expression to the peripheral nervous system, olfactory epithelium, or retina. Sequences outside this 8 kb region, but within 36 kb of the Mash1 locus, contain elements responsible for expression in the autonomic division of the peripheral nervous system. However, transgene expression in embryos containing the 36-kb sequence is never detected in the olfactory epithelium and the retina. Thus, regulatory elements driving expression in these lineages may be at even greater distances from the Mash1 coding region (Verma-Kurvari, 1997).

Mash1, a transcription factor of the basic helix-loop-helix class, is expressed during embryogenesis in restricted regions of the nervous system. An essential role for Mash1 in neural development has been previously demonstrated in mice carrying a targeted disruption of the Mash1 gene. Regulation of the precise temporal and spatial expression of Mash1 is thus likely to be important for proper neural development. Sequences that regulate Mash1 expression in the central nervous system have been characterized by assaying the expression of lacZ reporter genes in transgenic embryos. A 1158-bp enhancer localized approximately 7 kb upstream of the Mash1 coding region has been identified. Deletions within this enhancer region reveal the presence of both positive and negative cis-acting elements. Analysis of multiple sequences within the enhancer demonstrate that different elements preferentially function in different regions within the Mash1-specific CNS expression domain. Specific sequences confering expression in the hypothalamic region of the diencephalon and the dorsal mesencephalon have been identified, and a region having an essential role for the whole enhancer region has been localized. A role for sequences 3' downstream of the Mash1 coding region has been identified, providing evidence for posttranscriptional control of Mash1 expression in multiple CNS domains (Verma-Kurvari, 1998).

Mash1, a neural-specific bHLH transcription factor, is essential for the formation of multiple CNS and PNS neural lineages. Transcription from the Mash1 locus is elevated in mice null for Mash1, suggesting that MASH1 normally acts to repress its own transcription. This activity is contrary to the positive autoregulation of other proneural bHLH proteins. To investigate the mechanisms involved in this process, sequences flanking the Mash1 gene were tested for the ability to mediate negative autoregulation. A Mash1/lacZ transgene containing 36 kb of cis-regulatory sequence exhibits an increase in lacZ expression in the Mash1 mutant background, which phenocopies the observation of transcriptional autoregulation at the endogenous Mash1 locus. Using Mash1/lacZ lines with progressively less cis-acting sequence, autoregulatory responsive elements have been demonstrated to colocalize with a previously characterized 1.2-kb CNS enhancer. Mutations of E-box sites within this enhancer do not result in an apparent loss of autoregulation, suggesting that MASH1 does not directly repress its own transcription. Interestingly, these mutations do not indicate any underlying positive auto- or cross-regulation of Mash1. Furthermore, the loss of autoregulation in the Mash1 mutant background is reminiscent of a loss of lateral inhibitory signaling. However, mutations in Hairy/Enhancer-of-split-like (HES) consensus sites, the likely purveyors of Notch-mediated lateral inhibition, do not support a role for these sites in negative autoregulation. It is hypothesized that MASH1 normally inhibits its own expression indirectly, possibly through a HES-mediated repression of positive regulators or through novel HES binding sites (Meredith, 2000).

Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promote generic neuronal differentiation. The underlying genetic interactions have been examined. Phox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2b represses expression of the inhibitors of neurogenesis Hes5 and Id2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation (Dubreuil, 2002).

Patterning of the dorsal neural tube involves Bmp signaling, which results in activation of multiple pathways leading to the formation of neural crest, roof plate and dorsal interneuron cell types. Constitutive activation of Bmp signaling at early stages (HH10-12) of chick neural tube development induces roof-plate cell fate, accompanied by an increase of programmed cell death and a repression of neuronal differentiation. These activities are mimicked by the overexpression of the homeodomain transcription factor Msx1, a factor known to be induced by Bmp signaling. By contrast, the closely related factor, Msx3, does not have these activities. At later stages of neural tube development (HH14-16), dorsal progenitor cells lose their competence to generate roof-plate cells in response to Bmp signaling and instead generate dorsal interneurons. This aspect of Bmp signaling is phenocopied by the overexpression of Msx3 but not Msx1. Taken together, these results suggest that these two different Msx family members can mediate distinct aspects of Bmp signaling during neural tube development (Liu, 2004).

Two lines of evidence suggest that the neural bHLH genes that are crucial for neuronal differentiation might be direct transcriptional targets of Msx1. (1) In pursuit of factors that regulate the expression of the neural bHLH genes by yeast one-hybrid screening, Msx1 was identified to be potential regulator for both Math1 and Mash1 and several consensus sites for Msx1 binding are present in the enhancer regions of Math1/Cath1 and Mash1/Cash1. (2) Both Msx1 and Msx3 can bind to these consensus sites in vitro. However, because in vivo Msx1 represses the bHLH factor expression and Msx3 induces Cath1 expression, additional in vivo co-factors or chromatin properties that modulate these activities must be invoked (Liu, 2004).

Induction of the sympathetic nervous system (SNS) from its neural crest (NC) precursors is dependent on BMP signaling from the dorsal aorta. To determine the roles of BMP signaling and the pathways involved in SNS development, components of the BMP pathways were conditionally knocked out. To determine if BMP signaling is a cell-autonomous requirement of SNS development, the Alk3 (BMP receptor IA) was deleted in the NC lineage. The loss of Alk3 does not prevent NC cell migration, but the cells die immediately after reaching the dorsal aorta. The paired homeodomain factor Phox2b, known to be essential for survival of SNS precursors, is downregulated, suggesting that Phox2b is a target of BMP signaling. To determine if Alk3 signals through the canonical BMP pathway, Smad4 was deleted in the NC lineage. Loss of Smad4 does not affect neurogenesis and ganglia formation; however, proliferation and noradrenergic differentiation are reduced. Analysis of transcription factors regulating SNS development shows that the basic helix-loop-helix factor Ascl1 is downregulated by loss of Smad4 and that Ascl1 regulates SNS proliferation but not noradrenergic differentiation. To determine if the BMP-activated Tak1 (Map3k7) pathway plays a role in SNS development, Tak1 was deleted in the NC lineage. Tak1 was shown not to be involved in SNS development. Taken together, these results suggest multiple roles for BMP signaling during SNS development. The Smad4-independent pathway acts through the activation of Phox2b to regulate survival of SNS precursors, whereas the Smad4-dependent pathway controls noradrenergic differentiation and regulates proliferation by maintaining Ascl1 expression (Morikawa, 2009).

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. Two precursor classes were characterized in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, it was found that Sox2 dose and Meis1 -- independent of Pbx co-factors -- regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, proliferative characteristics and a dose-dependent transcriptional network were characterized that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells (Tucker, 2010).

Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. The proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. This study identifies, by in silico screening, a number of additional candidate target genes that are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. It is thus proposed that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis (Castro, 2006).

Delta1 is a common target of the proneural genes Mash1 and Neurogenin1/2 in mouse embryos. To determine whether Mash1 and Neurogenin1/2 directly transcribe Delta1, the regulatory sequences of this gene were analyzed. Two evolutionarily conserved enhancers active in different CNS regions have been identified in the Delta1 gene. To determine whether these enhancers mediate the regulation of Delta1 by proneural genes, transgenic mouse lines were generated. A transgene containing the full-length 4.3 kb mouse Delta1 promoter driving lacZ was expressed broadly in the embryonic brain and spinal cord at E11.5, in a pattern similar to that of endogenous Delta1. A transgene containing the proximal Delta1 neural enhancer (hereafter called DeltaM) and a minimal promoter driving lacZ was, in contrast, only expressed in parts of the Delta1 expression domain, including the dorsal spinal cord and ventral telencephalon, which also express Mash1. A transgene containing the distal Delta1 enhancer (hereafter called DeltaN) driving lacZ was expressed in a complementary manner in the neural tube, including the ventral spinal cord and dorsal telencephalon, which also express Ngn1 and Ngn2. To test whether these enhancers are regulated by proneural genes, the transgenic lines were bred with proneural null mutant mice. On a Mash1 null mutant background, the DeltaM-lacZ transgene was not expressed in the CNS at E11.5, demonstrating that the DeltaM enhancer requires Mash1 function for its activation. On a Ngn1;Ngn2 double mutant background, the DeltaN-lacZ transgene showed reduced expression in the spinal cord and was not expressed in the brain, except near the hindbrain border, showing that the DeltaN enhancer is activated by Neurogenins (Castro, 2006).

It was next asked whether proneural proteins directly interact with the Delta1 enhancers in the embryonic CNS by performing chromatin immune precipitation (ChIP) experiments. An antibody to Mash1 coprecipitated the DeltaM sequence in chromatin prepared from E12.5 wild-type telencephalon, but not from Mash1 mutant telencephalon, and the antibody did not precipitate the DeltaN sequence or the Delta1 coding sequence. Conversely, an antibody to Ngn2 coprecipitated the DeltaN sequence from wild-type, but not from Ngn2 null mutant telencephalon, and it did not precipitate the DeltaM or Delta1 coding sequences. Therefore, Mash1 and Ngn2 specifically bind in vivo to the DeltaM and DeltaN enhancers, respectively (Castro, 2006).

To further examine the interaction of Mash1 with the DeltaM enhancer, transcription assays were performed in P19 cells. It was first verified that Mash1 induces Delta1 transcription in these cells. After transfection of P19 cells with a Mash1 expression vector, the Mash1 transcript level increased in less than 4 hr, while the Delta1 transcript level increased less than 3 hr later, suggesting that Delta1 is directly transcribed by Mash1 in this system. By performing ChIP experiments with the Mash1 antibody on Mash1-transfected and mock-transfected P19 cells, it was shown that Mash1 specifically binds to the DeltaM sequence, indicating that Mash1 uses the same enhancer element to activate Delta1 in P19 cells and in the embryo. To examine the regulation of DeltaM by Mash1, the DeltaM sequence was inserted in a luciferase reporter vector and its transcriptional activity was tested in P19 cells. The DeltaM reporter was strongly activated when cotransfected with a Mash1 expression construct (Castro, 2006).

Two E-boxes (hereafter called E1-box and E2-box) were identified in the DeltaM sequence that are completely conserved in the human, mouse, chick, and zebrafish Delta1/DeltaD gene. To test whether these motifs mediate the direct binding of Mash1 to DeltaM, the two E-boxes were mutated either separately or together and examined the activity of the resulting DeltaM mutants. Mutation of each E-box separately or the two E-boxes together abolished activation of DeltaM by Mash1 and severely reduced the capacity of Mash1 to activate the full-length Delta1 promoter in P19 cells. A shorter version of DeltaM that mostly contains the 2 E-boxes and the 17 nucleotides in between (DeltaM short was activated by Mash1 as efficiently as DeltaM (Castro, 2006).

To determine whether activation of DeltaM by Mash1 involves motifs other than the two E-boxes, the DeltaM short element was mutated further. Interestingly, a perfect evolutionarily conserved consensus binding site for the POU family of homeodomain proteins, or octamer, is present in this element, one nucleotide 5′ from the E2-box. Point mutations in each half-site of the octamer motif, octT64G and octC68G, known to disrupt the interaction of the octamer with the homeodomain (POUH) and the POU-specific domain (POUS) of POU factors, respectively, abolished activation of DeltaM short by Mash1, and the same mutations introduced in the complete DeltaM element or the full-length Delta1 promoter also severely reduced their activation by Mash1. This raised the possibility that activation of Delta1 by Mash1 requires binding of both Mash1 and a POU protein to adjacent motifs in the DeltaM enhancer (Castro, 2006).

The proximity of the two sites suggested that binding of Mash1 to the E2-box might be influenced by binding of a putative POU protein to the adjacent octamer. To address this possibility, a luciferase reporter vector containing the multimerized E2 sequence and a minimal promoter (E26 construct) was generated, so that the interaction of Mash1 with E2 could be analyzed in P19 cells independently of the rest of the DeltaM element. It was asked whether the octamer sequence adjacent to E2 had an effect on the Mash1::E2 interaction by generating a reporter construct containing three copies of a sequence containing both the octamer and the E2-box in the same configuration as in DeltaM ([oct+E2]3). Mash1 activated this construct more efficiently. Moreover, a mutation in the octamer that interferes with POU protein binding (octT64G abolished activation of (octmut+E2)3 by Mash1, thus suggesting that binding of a POU protein to the octamer sequence increases the efficiency of the Mash1::E2 interaction (Castro, 2006).

This study shows that the activation of Delta1 expression by Mash1, a key aspect of its proneural function, involves a functional synergy between Mash1 and the POU genes Brn1 and Brn2. The synergistic activation of Delta1 by Mash1 and Brn1/2 likely reflects recruitment of Mash1 by a Brn protein to the DeltaM enhancer. Brn1/2 proteins on their own bind strongly to the consensus octamer sequence present in this enhancer, while Mash1 alone binds only poorly to the adjacent E2-box, but Mash1 efficiently forms a complex with Brn proteins on the octamer-E2 motif. The configuration of this binding motif plays an essential role in the recruitment process, since increasing the distance between the octamer and the E2-box by just one nucleotide is sufficient to abolish Mash1 recruitment and enhancer activity. The importance of keeping the two DNA-binding sites in close proximity strongly suggests that Mash1-E47 and Brn1/2 physically interact when bound to DNA (Castro, 2006).

The interaction of Mash1 and Brn proteins may also enhance the transcriptional activity of the complex. This is another well-documented mechanism of functional synergy, operating, for example, in the interaction between NeuroM, Isl1, and Lhx3 on the HB9 promoter. Although the primary mechanism underlying the functional synergy of Mash1 and Brn1/2 on the Delta1 promoter is cooperative binding to DNA, the role of Brn proteins does not appear to be restricted to Mash1 recruitment. Indeed, direct binding of Mash1 to the Mash1/Brn motif in the absence of Brn protein binding, e.g., when the low-affinity E2-box sequence in the Mash1/Brn motif is converted to a high-affinity one, is not sufficient to activate a Delta1 reporter construct. This suggests that Brn1/2 also potentiate the transcriptional activity of Mash1, perhaps by recruiting an essential coactivator or by initiating a conformational change that exposes the Mash1 activation domain (Castro, 2006).

An evolutionarily conserved Mash1/Brn-binding motif was found in the vicinity of 21 mouse genes. Six of them are components of the Notch pathway, which, together with the finding that a dominant-negative Brn construct blocks Notch activity in the chick neural tube, suggests that Mash1/Brn protein complexes play a major role in regulating Notch signaling in the CNS. Other genes associated with a Mash1/Brn motif also have important roles in neural development but act independently of Notch signaling. This is notably the case of Dcamkl1 or doublecortin-like kinase, a microtubule-associated protein that has recently been implicated in multiple aspects of development of the cerebral cortex, including cell cycle progression, neuronal commitment, neuronal migration, and axon growth (Castro, 2006).

Some of the other genes associated with a Mash1/Brn motif have not been previously studied in the developing nervous system, but studies in other systems suggest that they may also have varied functions during neurogenesis downstream of Mash1 and Brn1/2. The zinc finger transcription factor Insm1 is regulated by the bHLH gene Neurogenin3 in the pancreas, where it promotes neuroendocrine cell differentiation. Fbw7 is an ubiquitin ligase with an important role in promoting cell cycle arrest in G1/G0 through degradation of cyclin E, c-myc, and c-jun. Fbw7 has also been implicated in degradation of Notch1 (Castro, 2006).

These data thus support the idea that Mash1 acts in synergy with Brn proteins to activate a genetic program that controls multiple steps of neurogenesis, including precursor selection through Notch activation, cell cycle exit, neuronal differentiation, and migration. Analysis of Brn1/Brn2 double mutant mice has shown that these two genes regulate neuronal migration and the proliferation of subventricular zone precursors in the cerebral cortex, a region where neurogenesis is primarily regulated by the proneural gene Ngn2. Whether Brn1/Brn2 mutant mice also display neurogenesis defects in regions where Mash1 is the main proneural gene remains to be analyzed (Castro, 2006).

An important question raised by these results is whether Mash1 regulates aspects of neurogenesis independently of Brn proteins. In support of this notion, additional direct targets of Mash1 have been identified in the brain that are not associated with a conserved Mash1/Brn motif. Moreover, a study of Mash1 function in a neuroendocrine prostate cell line has revealed a number of putative direct targets in this tissue that are not associated with a conserved Mash1/Brn motif, and some of these genes are also regulated by Mash1 in the telencephalon in overexpression experiments. These different findings thus support a model whereby Mash1 interacts with different DNA-binding cofactors to activate different subprograms of neurogenesis, similar to the regulation of different subprograms of myogenesis by MyoD (Castro, 2006).

Direct conversion of fibroblasts to functional neurons by defined factors

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. It was hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, was found to suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine (Vierbuchen, 2010).

Activating the PARP-1 sensor component of the Groucho/ TLE1 corepressor complex mediates a CaMKinase II-dependent MASH1 activation pathway

Switching specific patterns of gene repression and activation in response to precise temporal/spatial signals is critical for normal development. This study reports a pathway in which induction of CaMKII triggers an unexpected switch in the function of the HES1 transcription factor from a TLE-dependent repressor to an activator required for neuronal differentiation. These events are based on activation of the poly(ADP-ribose) polymerase1 (PARP-1) sensor component of the groucho/TLE-corepressor complex mediating dismissal of the corepressor complex from HES1-regulated promoters. In parallel, CaMKII mediates a required phosphorylation of HES1 to permit neurogenic gene activation, revealing the ability of a specific signaling pathway to modulate both the derepression and the subsequent coactivator recruitment events required for transcriptional activation of a neurogenic program. The identification of PARP-1 as a regulated promoter-specific exchange factor required for activation of specific neurogenic gene programs is likely to be prototypic of similar molecular mechanisms (Ju, 2004).

A covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins mediated by the poly(ADP-ribose) polymerase1 (PARP-1) enzyme. PARP-1 catalyzes the transfer of ADP-ribose chains onto glutamic acid residues of acceptor proteins, including itself (automodification), histones, transcription factors, and DNA repair proteins using NAD+ as a substrate involved in chromatin decondensation, DNA replication, and DNA repair. Therefore, poly(ADP-ribosyl)ation by PARP-1 affects cellular processes such as apoptosis, necrosis, cellular differentiation, malignant transformation, and modulations activities of transcription factors. While it has been recently reported that PARP influences both the expression and silencing at diverse times during Drosophila development (Tulin, 2002), it has been demonstrated that high PARP enzymatic activity is observed in areas of high transcriptional activity and chromatin decondensation on the polytene chromatin (Tulin, 2003). Together these observations suggest that PARP-1 may exert its function in transcription through direct binding to the gene-regulating sequences and through modification of transcription factors by poly(ADP-ribosyl)ation (Ju, 2004).

This study finds that HES1-dependent repression of MASH1 is dependent upon the actions of the TLE1 corepressor complex. Not only are additional insights provided into the molecular mechanisms of TLE1-mediated repression but also the molecular mechanism of the switch to activation function has been uncovered. The composition of this TLE1 complex is distinct from those of other reported corepressor complexes such as N-CoR/SMRT and CtBP. Interestingly, roles for transcriptional regulation and chromatin remodeling activities have been described for most of the components of the TLE1 complex identified, but no component alone is indispensable for at least some level of TLE1-mediated repression (Ju, 2004).

Consistent with the observation that the enzymatic activity of PARP-1 is not required for HES1-mediated MASH1 repression, these data favors a model predicting that in the TLE1 holorepressor complex, the enzymatic activity of PARP-1 is inhibited. Exposure to a signal inducing neuronal differentiation causes activation of CaMKIIdelta, which is proven to be required for activation of neurogenic genes. Once sufficient levels of CaMKIIdelta are achieved (5-7 hr), it will, directly or indirectly, mediate phosphorylation and activation of PARP-1, which then catalyzes poly(ADP-ribosyl)ation of TLE1 and most of the other components to the corepressor complex. This is consistent with the observation that calcium signaling evoked by extrinsic and intrinsic cues can induce auto-poly(ADP-ribosyl)ation of PARP-1; however, CaMKIIdelta may also be activated in a calcium-independent fashion. This covalent modification is suggested to result in their dismissal from the biochemical complex and derepression of the MASH1 gene. The role of PARP-1 in derepression of MASH1 and its retention on the activated MASH1 promoter is quite consistent with reports that poly(ADP-ribosyl)ation of chromatin-associated proteins induce major changes in chromosomal architecture. However, in the case of MASH1, it was found that derepression alone is insufficient for induction. This is in accord with the findings for other regulated transcription factors. For example, the loss of the N-CoR corepressor is not alone sufficient to activate most AP-1-regulated genes, and only in a subset of RAR target genes does derepression result in a signal-independent 'default' activation of gene targets.

These data also indicate that, in addition to Ca2+-CaMKII-dependent dissociation of the TLE1 corepressor from HES1, a covalent modification of HES1 itself is required to permit activation of MASH1. Thus, activation of MASH1 is linked to sequential CaMKIIdelta-dependent activation of PARP-1 enzymatic activity, which was previously inhibited in the TLE1 holocorepressor complex, permitting dismissal of the TLE1 complex, derepression and phosphorylation of HES1, and recruitment of specific coactivators and thus causing and maintaining derepression of genes mediating neuronal differentiation. The actions of PARP-1 in the TLE1-mediated events are thus analogous to the effects of covalent modifications by phosphorylation and acetylation, as mediators of switches from repressor to activator function (Ju, 2004).

While HES1 is recognized to regulate tissue morphogenesis by maintaining undifferentiated cells and preventing differentiation, continued occupancy of the MASH1 promoter by HES1 in the differentiating neural stem cells has surprisingly proven to be required to initiate MASH1 activation events. Indeed, previous, seemingly contradictory reports of transient transfection assays in which HES1 can inhibit acid beta-glucosidase genes in HepG2 cells but cause activation in fibroblasts are likely to be explained by findings of signal-dependent HES1 switching events (Ju, 2004).

This cortical progenitor culture system has permitted identification of a regulatory pathway that may be, at least in part, partially compensated in vivo because many nestin-positive neural stem cells in the subventricular zone proliferate without losing the multipotentiality to differentiate into neurons in HES1 mutant or even HES1/HES5 double mutant mice. It is suggested that there may be additional HES1-like repressors or unidentified protein partners, including HERP and other E box binding proteins, that are also potentially involved in MASH1 gene activation. The identification of this unexpected mechanism of HES1 action in cortical progenitor cell cultures suggest that, in this system, the other molecules that could assume a similar function were either not expressed or required. The finding of a requirement of HES1 in activation of neurogenic genes is consistent with suggestions that HES1 might promote differentiation, in addition to its role in maintenance of the undifferentiated state, at multiple steps of neural stem cell development (Ju, 2004).

In summary, a pathway is suggested in which a PARP-1-containing TLE1 complex is recruited by the Notch-induced bHLH factor, HES1, initially mediating repression of MASH1 in the proliferating neural stem cells. The data suggest that signals that induce neuronal differentiation, such as PDGF in neural stem cells, act to induce the CaMKIIdelta isoform, which, in turn, is required for HES1-mediated MASH1 activation . The temporal aspects of CaMKIIdelta induction appear to account for the delay in derepression and activation of MASH1 expression following critical PDGF signaling. CaMKIIdelta-induced phosphorylation of a specific serine residue in the orange domain of HES1 permits it to recruit coactivators, including CBP, and has proven to be required for activation of the MASH1. The conserved relationship of the HES1 orange domain with the HLH domain raises the possibility of an additional role in protein interactions that include dimerization (Ju, 2004).

In a sense, the observation that a component of the TLE1-mediated repression complex, PARP-1, is also required for derepression events and maintenance of activation, parallels the requirement of TBL1/TBLR1 complex for ligand-dependent exchange of N-CoR corepressor complexes for coactivators in the switch of nuclear receptor function from repression to activation. TBLR1 is required for recruitment of the ubiquitylation/19S proteosome complex to prevent N-CoR/SMRT-dependent maintenance of a repression checkpoint. It is suggested that PARP-1 may achieve the same effects by a distinct modification strategy and serves as a regulated sensor of neuron-inducing signals based on the actions of CaMKIIdelta induced by the initial stimulus to neuronal differentiation. Therefore, it is tempting to speculate that PARP-1 and TBLR1 may be critical for the exchange events required to overcome the repression checkpoint for TLE- and N-CoR-regulated repressors, respectively (Ju, 2004).

The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway indicate that while the initial Notch signal causes repression of neurogenic genes by induction of HES1, it simultaneously arms the response to subsequent Ca2+-CaMKII signals that permit MASH1 gene activation events. The data illustrate how a single signaling pathway can mediate a sequential, two-step derepression/activation process required for development in gene activation. Induction of a Ca2+/CaMKII-dependent program initiates both PARP-1 activation, which is required for dismissal of the TLE1 corepressor complex, and a second event, covalent modification of HES1, which is required for target gene activation. The requirement for both a derepression and independently mediated activation event is likely to be prototypic of many similar functions of PARP-1 factors in development. The sequential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic gene expression to signals and to permit temporally precise patterns of cellular response (Ju, 2004).

MASH1 transcriptional targets

Establishment of neuronal networks is an extremely complex process involving the interaction of a diversity of neuronal cells. During mammalian development, these highly organized networks are formed through the differentiation of multipotent neuronal progenitors into multiple neuronal cell lineages. In the developing forebrain of mammals, the combined function of the Dlx1, Dlx2, Dlx5 and Dlx6 homeobox genes is necessary for the differentiation of the GABAergic interneurons born in the ventricular and subventricular zones of the ventral telencephalon, as well as for the migration of these neurons to the hippocampus, cerebral cortex and olfactory bulbs. The 437 bp I12b enhancer sequence in the intergenic region of the Dlx1/2 bigene cluster is involved in the forebrain regulation of Dlx1/2. Using DNase I footprinting, six regions of I12b potentially bound by transcription factors were identified. Mutagenesis of each binding site affected the expression of reporter constructs in transgenic mice. However, the effects of impairing protein-DNA interactions were not uniform across the forebrain Dlx1/2 expression domains, suggesting that distinct regulatory interactions are taking place in the different populations of neuronal precursors. Analyses of protein-DNA interactions provide evidence of a direct role for MASH1 in Dlx1/2 regulation in the forebrain. DLX proteins play a crucial role in the maintenance of their own expression, as shown by transgenic and co-transfection experiments. These studies suggest that the seemingly continuous domains of Dlx gene expression in the telencephalon and diencephalon are in fact the combination of distinct cell populations within which different genetic regulatory interactions take place (Poitras, 2007).

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. A genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures was carried out by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. Identified targets included Sox4, Dlx2, Ebf3, Gli3, and Nf1b. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation (Castro, 2011).

The most unexpected finding from this study is that Ascl1 activates a large number of positive cell cycle regulators in the embryonic telencephalon and NS cells. Ascl1 target genes include several canonical cell cycle regulators that are essential for G1/S transition (e.g., E2f1, Cdk1, Cdk2, and Skp2) or entry into mitosis (e.g., Cdk1 and Cdc25b). Other Ascl1 targets have additional roles besides cell cycle control. FoxM1 has an important role in progression of normal and tumor cells through M phase and also promotes angiogenesis through activation of vascular endothelial growth factor (VEGF) expression. Cyr61/Ccn1, a secreted heparin-binding protein, also promotes both cell proliferation and angiogenesis. Ascl1 therefore promotes cell cycle progression at both G1/S and G2/M transitions, and may also coordinate cell cycle regulation with other aspects of the biology of neural progenitors. Ascl1 shares some of its targets with c-Myc and N-Myc—including Birc5, Cdca7, Ccnd2, Foxm1, and the Notch ligand Dll3 - suggesting that its role in mitotic progenitors may involve cooperation with Myc proteins. In the absence of Ascl1, Myc proteins may be able to sustain the division of certain progenitors (e.g., in the telencephalic VZ) (Castro, 2011).

Direct targets of Ascl1 in the embryonic telencephalon also include genes involved in cell cycle arrest—including Fbxw7, Gadd45g, Ccng2, Hipk2, and Prmt2—thus demonstrating that Ascl1 regulates two sets of targets with opposite roles in cell cycle control. Interestingly, several of the cell cycle arrest targets have distinct spatial and/or temporal expression patterns from those of cell cycle progression targets in both the embryonic brain and NS5 cells, suggesting that they are regulated during a later phase of neurogenesis. Two findings support this assertion. First, cell cycle arrest targets are strongly up-regulated when NS5 cells are induced to differentiate by overexpression of Ascl1, and they are not affected by overexpression of DN-Ascl1. Second, their transcripts are found mostly outside of the VZ in the embryonic telencephalon. Gadd45g and Fbxw7 transcripts are localized to the SVZ and may be expressed by progenitor cells that are going through their last division or are already post-mitotic. Ccng2 and Prmt2 transcripts are present mostly in the MZ, where these genes may prevent cell cycle re-entry of post-mitotic neurons (Castro, 2011).

Together, these results suggest that Ascl1 has a role in both cell cycle promotion and cell cycle termination, and that the transition between these two functions involves the coordinated down-regulation and induction of multiple target genes. It is unusual that the same TF promotes in the same lineage both the expansion of progenitors and their subsequent division arrest and differentiation. Interestingly, Asense, the Ascl1 ortholog in Drosophila, has been shown recently to promote neuroblast self-renewal, while earlier studies had suggested a role of Asense in inhibiting cell divisions of neuroblast daughter cells, suggesting that this dual activity is an evolutionarily conserved feature of the Ascl1 gene family. A selective advantage may be that it allows for a rapid and efficient switch from a proliferating progenitor state to a post-mitotic neuronal state and ensures that the regulatory programs that underpin these two states are mutually exclusive (Castro, 2011).

The processes that select which set of target genes Ascl1 regulates in progenitors versus differentiating neurons are not currently known. A differential response of cell cycle progression genes and cell cycle arrest genes to different levels of Ascl1 is ruled out by the observation that Ascl1 protein levels do not increase substantially when NS cells differentiate. Other mechanisms, however, can be envisaged. The differential binding of Ascl1 to the Ccng2 and Foxm1 promoters in proliferating versus differentiating NS5 cells suggests that changes in Ascl1 structure (e.g., post-translational modifications such as phosphorylation) and/or Ascl1 regulation (e.g., a switch from oscillatory to stable expression) control Ascl1-promoter interactions and possibly other aspects of target gene regulation. Other pathways coregulating Ascl1 targets may also contribute to the change in the Ascl1 transcriptional program as neural progenitors stop proliferating and begin to differentiate. The enrichment of a consensus motif for CBF1/RBPj binding in promoters of cell cycle progression targets raises the intriguing possibility that coregulation by Ascl1 and the Notch pathway results in expression of these genes in proliferating progenitors where Notch is signaling and their repression in differentiating neurons where the Notch pathway is inactive. Elucidating the mechanisms involved in the selection of Ascl1 targets will shed light on the pathways that control the switch from proliferation to differentiation during neural development (Castro, 2011).

A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord

The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, their direct transcriptional targets were tested genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. Ascl1 and Ptf1a were shown to directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification (Borromeo, 2014).

Ascl1 controls the number and distribution of astrocytes and oligodendrocytes in the gray matter and white matter of the spinal cord

Glia constitute the majority of cells in the mammalian central nervous system and are crucial for neurological function. However, there is an incomplete understanding of the molecular control of glial cell development. This study found that the transcription factor Ascl1 (Mash1), which is best known for its role in neurogenesis, also functions in both astrocyte and oligodendrocyte lineages arising in the mouse spinal cord at late embryonic stages. Clonal fate mapping in vivo reveals heterogeneity in Ascl1-expressing glial progenitors and shows that Ascl1 defines cells that are restricted to either gray matter (GM) or white matter (WM) as astrocytes or oligodendrocytes. Conditional deletion of Ascl1 post-neurogenesis shows that Ascl1 is required during oligodendrogenesis for generating the correct numbers of WM but not GM oligodendrocyte precursor cells, whereas during astrocytogenesis Ascl1 functions in balancing the number of dorsal GM protoplasmic astrocytes with dorsal WM fibrous astrocytes. Thus, in addition to its function in neurogenesis, Ascl1 marks glial progenitors and controls the number and distribution of astrocytes and oligodendrocytes in the GM and WM of the spinal cord (Vue, 2014).

Mammalian bHLH proteins: Distinct domains within the bHLH motif have different activities

Many members of the basic helix-loop-helix (bHLH) family of transcription factors play pivotal roles in the development of a variety of tissues and organisms. Activities have been identified for the neural bHLH proteins Mash1 and Math1 in inducing neuronal differentiation, and in inducing the formation of distinct dorsal interneuron subtypes in the chick neural tube. Although both factors induce neuronal differentiation, each factor has a distinct activity in the type of dorsal interneuron that forms: overexpression of Math1 increases dI1 interneurons; Mash1 increases dI3 interneurons. Math1 and Mash1 function as transcriptional activators for both of these functions. Furthermore, discrete domains within the bHLH motif have been defined that are required for these different activities in neural development. Helix 1 of the Mash1 HLH domain is necessary for Mash1 to be able to promote neuronal differentiation, and is sufficient to confer this activity to the non-neural bHLH factor MyoD. In contrast, helix 2 of Math1, and both helix 1 and 2 of Mash1, are the domains required for the neuronal specification activities of these factors. The requirement for distinct domains within the HLH motif of Mash1 and Math1 for driving neuronal differentiation and cell-type specification probably reflects the importance of unique protein-protein interactions involved in these functions (Nakada, 2004).

Mash1 and Math1 are class II bHLH factors that characteristically have tissue-specific expression, and bind E-box DNA (CANNTG) as heterodimers with class I bHLH factors such as E12, E47, HEB and E2-2. Crystal structures of a non-neural class II bHLH factor MyoD, or a class I bHLH factor E47, demonstrate that the basic region interacts with DNA and the HLH forms an amphipathic helix that is involved in protein-protein interactions in the formation of homo- or heterodimers. Studies with other bHLH factors have demonstrated the importance of the basic region for specific functions. Studies of the Xenopus neural bHLH factors Xash1 (Mash1 homolog) and Xngnr1 (Ngn homolog) have identified the HLH domain as the region encoding information for induction of specific downstream targets. The current findings are similar to these latter experiments, demonstrating that the HLH domain, and not the basic region, encodes the necessary information for the specific functions in neurogenesis of the neural bHLH factors Mash1 and Math1 in the chick neural tube (Nakada, 2004).

Since the HLH domain functions in protein-protein interactions, it is reasonable to propose that specificity of function may be conferred by interactions with specific cofactors that vary between the different bHLH proteins. It is known that in vitro, DNA binding activity is most efficient with heterodimers of the neural bHLH with an E-protein such as E12. There are no reports that the individual E-proteins (E2a, HEB, E2-2) can confer specificity of function on the neural bHLH/E-protein heterodimer, and knockout studies with the E-protein genes suggest they have a high level of functional redundancy. Thus, it may be that context-dependent co-factors that form higher order complexes with bHLH heterodimers are important for specific functions. Further support for the importance of cellular context for bHLH factor function is provided by the chick electroporation experiments. For example, electroporation of pMiWIII-Math1 results in overexpression of Math1 along the extent of the dorsoventral axis, however, the increase in the dI1 interneurons is biased to the dorsal regions. This is in contrast to the neuronal differentiation phenotype that is seen throughout the dorsoventral axis. Invoking context-dependent protein-protein interactions to explain the specificity of bHLH function along the dorsoventral axis of the spinal neural tube is also helpful in explaining how bHLH factors are required for different types of neurons in different regions of the nervous system. Consistent with the involvement of specific co-factor interactions is the presence of conserved surface amino acids within each neural bHLH sub-family that are distinct between the different sub-families (Nakada, 2004).

Besides the E-proteins, a number of other co-factors have been described that form complexes with Class II bHLH factors. In Drosophila, Chip, a LIM homeodomain binding protein (homolog of Ldb factors in vertebrates), has been shown to form a complex with Achaete (Mash1 homolog) and Daughterless (E12 homolog), possibly as an adapter protein bringing in another transcription factor, Pannier, to form a higher order transcriptional complex. In studies of Tal1, a class II bHLH factor important in hematopoiesis, a LIM only factor (LMO) and Ldb factors were seen to form a complex with Tal1 and E12. In the neural tube, it has been shown that higher order complex formation of homeodomain factors and bHLH factors control ventral cell fates. In this case, the homeodomain factor Lhx3 alone is not sufficient to generate motor neurons, but in combination with Ldb it specifies V2 interneurons. When co-expressed with islet1, a higher order complex is formed, resulting in different DNA binding characteristics, and the specification of motor neurons rather than V2 interneurons. Furthermore, two bHLH factors, Ngn2 and NeuroM, transcriptionally synergize with the homeodomain complex to specify the motor neurons. This type of combinatorial interactions of transcription factors is an attractive hypothesis for the context-dependent functions seen with the bHLH factors studied here for dorsal neural tube development. The identity and role of co-factors in forming higher order transcriptional complexes with Mash1 and Math1 is yet to be determined (Nakada, 2004).

The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.

Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. This study demonstrates the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro (Ali, 2014).

Mammalian Achaete Homologs: Protein degradation

In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes, which leads to transcriptional repression of neural determination and differentiation genes. In addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, it has been shown that the initial appearance of Notch1 coincides with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone is capable of down-regulating hASH1 mRNA but can not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 is coinfected with Notch1, a dramatic and abrupt loss of the exogenous hASH1 protein is still observed, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreases the apparent half-life of the adenoviral hASH1 protein and increases the fraction of hASH1 that is polyubiquitinylated. The proteasome inhibitor MG132 reverses the Notch1-induced degradation. The Notch RAM domain is dispensable but a lack of the OPA and PEST domains inactivates this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 protects hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer (Sriuranpong, 2002).

Mammalian Achaete Homologs: Neural crest development

An investigated has been carried out of the genetic circuitry underlying the determination of neuronal identity, using mammalian peripheral autonomic neurons as a model system. Treatment of neural crest stem cells (NCSCs) with bone morphogenetic protein-2 (BMP-2) leads to an induction of MASH1 expression and consequent autonomic neuronal differentiation. BMP2 also induces expression of the paired homeodomain transcription factor Phox2a, and the GDNF/NTN signaling receptor tyrosine kinase c-RET. Constitutive expression of MASH1 in NCSCs from a retroviral vector, in the absence of exogenous BMP2, induces expression of both Phox2a and c-RET in a large fraction of infected colonies, and also promotes morphological neuronal differentiation and expression of pan-neuronal markers. In vivo, expression of Phox2a in autonomic ganglia is strongly reduced in Mash1 -/- embryos. These loss- and gain-of-function data suggest that MASH1 positively regulates expression of Phox2a, either directly or indirectly. Constitutive expression of Phox2a, in contrast to MASH1, fails to induce expression of neuronal markers or a neuronal morphology, but does induce expression of c-RET. These data suggest that MASH1 couples expression of pan-neuronal and subtype-specific components of autonomic neuronal identity, and support the general idea that identity is established by combining subprograms involving cascades of transcription factors, which specify distinct components of neuronal phenotype (Lo, 1998).

The sympathetic, parasympathetic and enteric ganglia are the main components of the peripheral autonomic nervous system, and are all derived from the neural crest. The factors needed for these structures to develop include the transcription factor Mash1, the glial-derived neurotrophic factor GNDF and its receptor subunits, and the neuregulin signaling system, each of which is essential for the differentiation and survival of subsets of autonomic neurons. All autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the anlagen of the enteric nervous system and the sympathetic ganglia, Phox2b is needed for the expression of the GDNF-receptor subunit Ret and for maintaining Mash1 expression. Mutant ganglionic anlagen also fail to switch on the genes that encode two enzymes needed for the biosynthesis of the neurotransmitter noradrenaline, dopamine-beta-hydroxylase and tyrosine hydroxylase, demonstrating that Phox2b regulates the noradrenergic phenotype in vertebrates (Pattyn, 1999).

The mechanisms that establish and maintain the multipotency of stem cells are poorly understood. In neural crest stem cells (NCSCs), the HMG-box factor SOX10, 32% identical to Drosophila Sox50E over the region of homology, preserves not only glial, but surprisingly, also neuronal potential from extinction by lineage commitment signals. The latter function is reflected in the requirement of SOX10 in vivo for induction of MASH1 and PHOX2B, two neurogenic transcription factors. Simultaneously, SOX10 inhibits or delays overt neuronal differentiation, both in vitro and in vivo. However, this activity requires a higher Sox10 gene dosage than does the maintenance of neurogenic potential. The opponent functions of SOX10 to maintain neural lineage potentials, while simultaneously serving to inhibit or delay neuronal differentiation, suggest that it functions in stem or progenitor cell maintenance, in addition to its established role in peripheral gliogenesis (Kim, 2002).

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. MASH1 function is shown to be necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1–/– mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1+/+ mice is maintained in Mash1–/– mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1–/– mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1–/– mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chromaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation (Huber, 2002).

Notch signaling is involved in neurogenesis, including that of the peripheral nervous system as derived from neural crest cells (NCCs). However, it remains unclear which step is regulated by this signaling. To address this question, advantage was taken of the Cre-loxP system to specifically eliminate the protein O-fucosyltransferase 1 (Pofut1: see Drosophila O-fucosyltransferase 1/neurotic) gene, which is a core component of Notch signaling, in NCCs. NCC-specific Pofut1-knockout mice died within 1 day of birth, accompanied by a defect of enteric nervous system (ENS) development. These embryos showed a reduction in enteric neural crest cells (ENCCs) resulting from premature neurogenesis. Sox10 expression, which is normally maintained in ENCC progenitors, was decreased in Pofut1-null ENCCs. By contrast, the number of ENCCs that expressed Mash1, a potent repressor of Sox10, was increased in the Pofut1-null mouse. Given that Mash1 is suppressed via the Notch signaling pathway, a model is proposed in which ENCCs have a cell-autonomous differentiating program for neurons as reflected in the expression of Mash1, and in which Notch signaling is required for the maintenance of ENS progenitors by attenuating this cell-autonomous program via the suppression of Mash1 (Okamura, 2008).

Mammalian Achaete Homologs: Roles in non-neural development

The Mash2 gene, which encodes a basic helix-loop-helix transcription factor, is one of the mammalian homologs of the Drosophila achaete-scute genes. It is strongly expressed in diploid trophoblast cells of the postimplantation mouse embryo. Targeted mutagenesis of Mash2 reveals that loss of function results in embryonic lethality at midgestation, due to placental failure associated with a lack of spongiotrophoblast and reduced labyrinthine trophoblast layers. For the further study of Mash2 function in development of the trophoblast cell lineage, chimeric analysis was performed combining Mash2 mutant and wild-type embryos. Two questions were asked: (1) Is the phenotype of the Mash2 mutant embryo, which affects all of the three trophoblast cell layers, caused by a cell autonomous or non-autonomous defect? (2) Is Mash2 required in both spongiotrophoblast and labyrinthine trophoblast development?

It was found that there is no contribution of Mash2 mutant cells to the spongiotrophoblast layer in chimeric placentae at 10.5 and 12.5 days postcoitum, suggesting that the product of the Mash2 gene is required cell autonomously during the development of the spongiotrophoblast. However, it seems that Mash2 is not required for development of labyrinthine trophoblast or giant cells, since high contributions of Mash2 mutant cells are observed in those trophoblast cell layers in the chimeric placentae analyzed. It can therefore be concluded that the primary and cell-autonomous function of Mash2 appears to be an involvement in the development of diploid trophoblast cells in the ectoplacental cone to form the spongiotrophoblast cell layer of the mature chorioallantoic placenta (Tanaka, 1997).

Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development, since overexpression of Mash-2 reduces giant cell formation. The role that the Notch signaling pathway may play in trophoblast development has been investigated. Mash-2 is a homolog of Drosophila achaete/scute complex genes. In the developing mouse placenta, all elements of the Notch pathway are expressed. In particular, the Notch-2, HES-2, and HES-3 genes are coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family are expressed in trophoblasts; TLE3 is expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 is limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development (Nakayama, 1997).

The basic helix-loop-helix transcription factor, Mash2, has been shown to be necessary for the development of the spongiotrophoblast of the mature chorioallantoic placenta of the mouse. Mash2 is transcribed during oogenesis and expressed throughout preimplantation development, only becoming restricted to the diploid trophoblast around the time of implantation. This expression raised the possibility that Mash2 has earlier functions in the trophoblast lineage that were not detectable in mutant embryos because of the persistence of oogenetically derived protein. This was tested by generating viable Mash2-/- females by tetraploid rescue of the extraembryonic defect. Mutant embryos derived from such females show no enhanced phenotype over embryos produced from heterozygous females, demonstrating unequivocally that neither maternal nor zygotic Mash2 is required for early trophoblast development. If Mash2 functions in other aspects of trophoblast development, it must act cooperatively with other factors (Rossant, 1998).

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble the neoplastic cells. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. The protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, Hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines that lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer (Chen, 1997a).

Malignancies with neuroendocrine (NE) features such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are prototypic neoplasms arising from peripheral endocrine cells. The mechanisms that regulate the NE phenotype in these tumors and their cellular precursors are not well understood. However, a basic helix-loop-helix transcription factor that is homologous to Drosophila neural fate determination proteins may have a central role. Human achaete-scute homolog-1 (hASH1), a human homolog of the Drosophila achaete-scute complex, is highly expressed in MTC, SCLC, and pheochromocytomas. To determine what mechanisms allow constitutive expression of hASH1 in NE tumors, human genomic DNA fragments containing the hASH1 gene were cloned and the gene's promoter region was sequenced. hASH1 expression is shown to be restricted to NE cell lines by a transcriptionally regulated mechanism. Dual promoters initiate hASH1 transcription, with the predominant site being an evolutionarily conserved initiator (INR) element. Transient transfection studies provide evidence for a generalized enhancer region that had high activity in all cell lines tested. Restriction of hASH1 expression to NE tumor cells depends on two tissue-specific repressor regions, present in the proximal and distal (> 13.5 kb) 5'-flanking region. Understanding the mechanisms of tissue-specific control of hASH1 gene expression provides a useful model to explore regulatory cascades influencing both normal nervous system development and the NE phenotype of tumors such as MTC and SCLC (Chen, 1997b).

In Drosophila and in vertebrates, the achaete-scute family of basic helix-loop-helix transcription factors plays a critical developmental role in neuronal commitment and differentiation. Relatively little is known, however, about the transcriptional control of neural features in cells outside a neuronal context. A minority of normal bronchial epithelial cells and many lung cancers, especially small-cell lung cancer, exhibit a neuroendocrine phenotype that may reflect a common precursor cell population. Human achaete-scute homolog-1 (hASH1) is shown to be selectively expressed in normal fetal pulmonary neuroendocrine cells, as well as in the diverse range of lung cancers with neuroendocrine features. Strikingly, newborn mice bearing a disruption of the ASH1 gene have no detectable pulmonary neuroendocrine cells. Depletion of this transcription factor from lung cancer cells by antisense oligonucleotides results in a significant decrease in the expression of neuroendrocrine markers. Thus, a homolog of Drosophila neural fate determination genes seems to be necessary for progression of lung epithelial cells through a neuroendocrine differentiation pathway that is a feature of small-cell lung cancer, the most lethal form of human lung cancer (Borges, 1997).

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. This study shows that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A. GFI1 colocalizes with chromogranin A and calcitonin-gene-related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation (Kazanjian, 2004).

Transcription factor Achaete Scute-Like 2 controls intestinal stem cell fate

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. The existence of a long-lived pool of cycling stem cells has been demonstrated defined by intestinal stem cell marker Lgr5 expression; these cells are found intermingled with post-mitotic Paneth cells at crypt bottoms. A gene signature has been determined for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate (van der Flier, 2009).

Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors

Myogenic regulatory factors (MRFs), including Myf5, MyoD and Myog. (see Drosophila Nautilus), are muscle-specific transcription factors that orchestrate myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for the self-renewal and maintenance of muscle stem cells (satellite cells). This study defines Ascl2 (see Drosophila Achaete) as a novel inhibitor of MRFs. During mouse development, Ascl2 is transiently detected in a subpopulation of Pax7+ MyoD+ progenitors (myoblasts) that become Pax7+ MyoD- satellite cells prior to birth, but is not detectable in postnatal satellite cells. Ascl2 knockout in embryonic myoblasts decreases both the number of Pax7+ (see Drosophila Paired) cells and the proportion of Pax7+ MyoD- cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts and impairs the regeneration of injured muscles. Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimers with classical E-proteins to sequester their transcriptional activity on MRF genes. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs and facilitates the generation of postnatal satellite cells (Wang, 2017).

Mammalian Achaete Homologs: General effects of mutation on neural development

Notch signaling has a central role in cell fate specification and differentiation. Evidence is provided that the Mash1 (bHLH) and Dlx1 and Dlx2 (homeobox) transcription factors have complementary roles in regulating Notch signaling, which in turn mediates the temporal control of subcortical telencephalic neurogenesis in mice. Progressively more mature subcortical progenitors (P1, P2 and P3) are defined through their combinatorial expression of MASH1 and DLX2, as well as the expression of proliferative and postmitotic cell markers at E10.5-E11.5. In the absence of Mash1, Notch signaling is greatly reduced and 'early' VZ progenitors (P1 and P2) precociously acquire SVZ progenitor (P3) properties. Comparing the molecular phenotypes of the delta-like 1 and Mash1 mutants, suggests that Mash1 regulates early neurogenesis through Notch-and Delta-dependent and -independent mechanisms. While Mash1 is required for early neurogenesis (E10.5), Dlx1 and Dlx2 are required to downregulate Notch signaling during specification and differentiation steps of 'late' progenitors (P3). Dlx1/2 function appears to be required to specify and differentiate P3 progenitors by repressing the genes that are normally expressed in VZ progenitor cells (e.g. Mash1, Gsh1/2, Lhx2, COUP-TF1) and by activating genes expressed in the SVZ (e.g. Dlx5, Dlx6 and SCIP/Oct6) and MZ (e.g. Drd2). It is suggested that alternate cell fate choices in the developing telencephalon are controlled by coordinated functions of bHLH and homeobox transcription factors through their differential affects on Notch signaling (Yun, 2002).

Dlx1/2 mutants exhibit increased levels of Hes5 expression, implying that differentiation may be blocked due to increased levels of Notch signaling. At E11.5 Dll1 (a Delta homolog) and Mash1 expression are elevated in the SVZ; these abnormalities become more severe at later stages. As MASH1 and DLX2 are co-expressed in some progenitors (P3), a potential mechanism underlying this phenotype would be that Dlx1 and Dlx2 repress Mash1 expression (directly or indirectly) as P3 cells mature. In Dlx1/2 mutants, failure to downregulate Mash1 expression would lead to elevated levels of Dll1 expression; this, in turn, would increase Notch signaling and Hes5 expression in adjacent cells (Yun, 2002).

Mash1, a mammalian homolog of the Drosophila proneural genes of the achaete-scute complex, is transiently expressed throughout the developing peripheral autonomic nervous system and in subsets of cells in the neural tube. In the mouse, targeted mutation of Mash1 has revealed a role in the development of parts of the autonomic nervous system and of olfactory neurons, but no discernible phenotype in the brain has yet been reported. The adrenergic and noradrenergic centres of the brain are missing in Mash1 mutant embryos, whereas most other brainstem nuclei are preserved. Indeed, the present data, together with the previous results, show that except in cranial sensory ganglia, Mash1 function is essential for the development of all central and peripheral neurons that express noradrenergic traits, either transiently or permanently. In particular, in the absence of MASH1, these neurons fail to initiate expression of the noradrenaline biosynthetic enzyme dopamine ß-hydroxylase (DBH). All these neurons normally express the homeodomain transcription factor Phox2a, a positive regulator of the dopamine ß-hydroxylase gene; indeed, a subset of them depend on Phox2a for their survival. Expression of Phox2a is abolished or massively altered in the Mash1-/- mutants, both in the noradrenergic centers of the brain and in peripheral autonomic ganglia. These results suggest that MASH1 controls noradrenergic differentiation at least in part by controlling expression of Phox2a and point to fundamental homologies in the genetic circuits that determine the noradrenergic phenotype in the central and peripheral nervous system. These results strongly support the idea that MASH1 is required not only for conferring pan-neuronal properties, but also for the implementation of specific aspects of neuronal differention and, in particular, for expression of the noradrenergic phenotype. Promotion of expression of Phox2a, which has been shown to be a positive regulator of the DBH gene in vivo, appears to be a key step controlled by MASH1 in these cells (Hirsch, 1998).

A novel paired homeodomain protein, PHD1, most closely related to C. elegans unc-4, has been identified by a differential RT-PCR method. Unc-4 has no paired domain (See Drosophila Paired) and is thus grouped separately from paired-homeodomains into a prd-like class. PHD1 is expressed in a narrow layer adjacent to the ventricular zone of the dorsal spinal cord, immediately following expression of MASH1 but preceding overt neuronal differentiation. Some cells coexpressing MASH1 and PHD1 can be seen, suggesting that these two genes are sequentially activated within the same lineage. In the olfactory sensory epithelium, PHD1 expression not only follows but is dependent upon MASH1 function, suggesting that PHD1 acts downstream of MASH1. A sequential action of bHLH and paired homeodomain proteins is apparent in other neurogenic lineages and may be a general feature of both vertebrate and invertebate neurogenesis (Saito, 1996).

Mammalian Achaete Homologs: Eye, olfactory and ear development

Achaete Evolutionary homologs part 3/3 back to part 1/3


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

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