HLH Transcription Factors Research Articles

Sohlh2 promotes pulmonary fibrosis via repression of p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway

Disturbance in the redox balance of alveolar epithelial cells (AECs) was considered as a causal factor for pulmonary fibrosis. The regulatory mechanisms of redox hemostasis in the development of pulmonary fibrosis remain largely unknown. Using a type II AEC-specific Sohlh2 conditional knock-in (CKI) mouse model, we found that Sohlh2, a basic HLH transcription factor, accelerated age-related pulmonary fibrosis. High-fat diet (HFD) resulted in a tremendous increase in lung inflammation and fibrotic changes in the lung tissues of Sohlh2 CKI mice. Sohlh2 overexpression led to a significant rise of intracellular ROS and apoptosis in the lung, mouse primary AECIIs, and human A549 cells, which was attenuated by ROS inhibitor (NAC). Sohlh2 enhanced oxidative stress via repressing p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway. p62, a direct target of Sohlh2, mediated Sohlh2 effects on ROS generation and apoptosis in A549 cells. Hence, our findings elucidate a pivotal mechanism underlying oxidative stress-induced pulmonary fibrosis, providing a framework for aging-related disorder interventions.

Identification and Characterization of PRE Genes in Moso Bamboo (Phyllostachys edulis).

Basic helix-loop-helix (bHLH)/HLH transcription factors are involved in various aspects of the growth and development of plants. Here, we identified four HLH genes, PePRE1-4, in moso bamboo plants that are homologous to Arabidopsis PRE genes. In bamboo seedlings, PePRE1/3 were found to be highly expressed in the internode and lamina joint by using quantitative RT-PCR analysis. In the elongating internode of bamboo shoots, PePRE genes are expressed at higher levels in the basal segment than in the mature top segment. Overexpression of PePREs (PePREs-OX) in Arabidopsis showed longer petioles and hypocotyls, as well as earlier flowering. PePRE1 overexpression restored the phenotype due to the deficiency of AtPRE genes caused by artificial micro-RNA. PePRE1-OX plants showed hypersensitivity to propiconazole treatment compared with the wild type. In addition, PePRE1/3 but not PePRE2/4 proteins accumulated as punctate structures in the cytosol, which was disrupted by the vesicle recycling inhibitor brefeldin A (BFA). PePRE genes have a positive function in the internode elongation of moso bamboo shoots, and overexpression of PePREs genes promotes flowering and growth in Arabidopsis. Our findings provided new insights about the fast-growing mechanism of bamboo shoots and the application of PRE genes from bamboo.

Transcriptomic Time-Series Analyses of Gene Expression Profile During Zygotic Embryo Development in Picea mongolica

Zygotic embryogenesis is a critical process during seed development in gymnosperms. However, knowledge on the genome-wide transcriptional activation that guides this process in conifers is limited, especially in Picea mongolica. This tree species is endemic to semiarid habitats of Inner Mongolia in China. To extend what is known about the molecular events underpinning its zygotic embryogenesis, comparative transcriptomic analyses of gene expression in zygotic embryos were performed by RNA sequencing in P. mongolica. Our results showed that most changes in transcript levels occurred in the early embryonic pattering determination and formation of mature embryos. Transcripts related to embryogenic competence, cell division pattern, hormones, and stress response genes were identified during embryogenesis. Auxin is essential for early embryo patterning and pre-cotyledon embryonic formation. However, ABA is a major regulator of embryo maturation. Moreover, we found that methylation-related gene expression is associated with activation of early-stage embryos, late embryogenesis abundant proteins, and storage/energy-related genes with late and mature embryos. Furthermore, network analysis revealed stage-specific and multistage gene expression clusters during embryogenesis. WOX, MYB, AP2, and HLH transcription factors seem more relevant to embryogenesis in different stages. Our results provide large-scale and comprehensive transcriptome data for embryo development in P. mongolica. These data will lay a foundation for the protection and utilization of P. mongolica resources.

Inhibitor of Differentiation 4 (ID4) represses mammary myoepithelial differentiation via inhibition of HEB

SummaryInhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition.

BackgroundThe overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies.MethodsGlioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro.ResultsThe induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone.ConclusionsGliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex

The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting “acidless” varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.

Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo

BackgroundMelanoma is one of the most invasive and aggressive types of cancer with a very poor prognosis. Surgery remains the most efficient treatment prior melanoma invasion and metastasis formation. However, therapy becomes a challenge once the cancer cells colonized other tissues. At present, there are two main classes of therapies acting with a certain efficiency on metastatic melanoma: immune check point inhibitors (anti-PD1/PDL1) and targeted therapy such as Vemurafenib. Unfortunately, these therapies are not fully responsive, induce resistance and/or generate unwanted side effects. In this respect, it is important to continue to discover new cancer therapeutics. Here, we show that daphnane diterpenes type of compounds can prevent melanoma metastasis by inhibiting metastasis-associated matrix metalloproteinases expression without cytotoxicity.MethodsEvaluation of the anti-metastasis effect of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) and its bioactive component gnidilatidin was carried out in vitro using B16 murine melanoma cells and in vivo using male C57BL/6 J mice. Global gene expression in B16 cells was done using DNA microarray, validated using real-time PCR, to further understand the effect of daphnane diterpenes, specifically daphnane diterpenoid gnidilatidin.ResultsOral administration of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) suppressed MMP2 and MMP9 expression, decreasing lung tumor in mice injected with B16 murine melanoma cells. Validation of these observations in vitro showed reduced B16 cells migration, adhesion, and invasion. Results of microarray analysis of B16 cells treated with daphnane diterpenoid gnidilatidin from TH revealed an upregulation of tumor suppressor Egr1 while inhibiting metastasis-associated genes Id2 and Sytl2 expression. A downregulation of the melanoma oncogene microphthalmia-associated transcription factor (Mitf) was observed, and most likely caused by the inhibition of Id2, a gene that regulated HLH transcription factors such as MITF and also reported to promote tumor cell migration and invasion.ConclusionsDaphnane diterpenes have inhibitory effect on the metastatic potential of B16 melanoma cells, and the results of this study provided evidence for their potential for use in the prevention and inhibition of melanoma metastasis.

Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals.

Germline removal provokes longevity in several species and shifts resources towards survival and repair. Several Caenorhabditis elegans transcription factors regulate longevity arising from germline removal; yet, how they work together is unknown. Here we identify a Myc-like HLH transcription factor network comprised of Mondo/Max-like complex (MML-1/MXL-2) to be required for longevity induced by germline removal, as well as by reduced TOR, insulin/IGF signalling and mitochondrial function. Germline removal increases MML-1 nuclear accumulation and activity. Surprisingly, MML-1 regulates nuclear localization and activity of HLH-30/TFEB, a convergent regulator of autophagy, lysosome biogenesis and longevity, by downregulating TOR signalling via LARS-1/leucyl-transfer RNA synthase. HLH-30 also upregulates MML-1 upon germline removal. Mammalian MondoA/B and TFEB show similar mutual regulation. MML-1/MXL-2 and HLH-30 transcriptomes show both shared and preferential outputs including MDL-1/MAD-like HLH factor required for longevity. These studies reveal how an extensive interdependent HLH transcription factor network distributes responsibility and mutually enforces states geared towards reproduction or survival.

Zinc-finger and helix-loop-helix transcription factors regulate Purkinje neuron neurogenesis and cerebellar corticogenesis

Many regulatory genes have been pinpointed as orchestrators of cerebellar development, from the onset of neurogenesis to the patterning of the adult cerebellar cortex, with a special reference to the development of cerebellar Purkinje cells (PCs). PCs provide the sole output from cerebellar cortical circuits, where each PC integrates myriads of presynaptic inputs, both inhibitory and excitatory. In the murine cerebellar primordium PCs are generated from a pool of ventricular zone progenitors facing the fourth ventricle between embryonic day (E) 10.5 and 13.5. This progenitor pool expands in the ventricular zone (VZ) through symmetric cell division until E10.5, when a gradual switch to asymmetric cell division occurs, regulated by Notch1 (Lutolf et al., 2002) and its interactor (Masserdotti et al., 2010), the Zn-finger TF Zfp423 (Alcaraz et al., 2006; Warming et al., 2006; Croci et al., submitted). Zfp423 was recently implicated in Joubert syndrome and cerebellar vermis hypoplasia (Chaki et al., 2012). Allelic mutations of Zfp423 produce distinct alterations in PC development (Croci et al., submitted). PCs arise from a pool of progenitors positive for the basic-helix-loop-helix transcription factors (TFs) neurogenin (Ngn) 1 and 2 (Zordan et al., 2008; Lundell et al., 2009). Ngn2 regulates cell cycle progression and dendritic arbor generation in PC precursors (Florio et al., 2012). PCs also express HLH transcription factors of the Olf/EBF family. In Ebf2 -/- mutants, PC migration and survival are affected (Croci et al., 2006). Neonatal PC death is due to local downregulation of Igf1 gene expression (Croci et al., 2011). Finally, EBF2 regulates cortical patterning in the adult cerebellum, regulating its subdivision into alternate parasagittal stripes of distinct PC subtypes. Indeed, EBF2 is required to repress the zebrin II+ phenotype in postnatal PCs (Croci et al., 2006; Chung et al., 2008).

Abstract LB-62: Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin

Abstract Basal-like breast cancers (BLBC) are heterogenous, poorly differentiated and present with limited therapeutic opportunities and a poor prognosis. We have investigated the role of the HLH transcription factor ID4 in breast stem cells and basal breast cancer aetiology. Using an Id4GFP knock-in mouse and single cell gene expression studies we show that ID4 defines a subset of Cd24+Cd29hi basal mammary epithelial cells enriched for stem cell gene expression and mammary repopulating capacity. Homozygous deletion of Id4 followed by mammary transplantation reveals a cell-autonomous requirement for Id4 in mammary ductal morphogenesis in vivo. shRNA-mediated depletion of Id4 in the Comma-D mammary stem cell line in vitro shows that Id4 is required for epithelial cell proliferation. Conversely, over-expression of Id4 maintains the stem cell pool by restricting luminal differentiation of Comma-D cells. This effect of Id4 is mediated via suppression of BrCa1 expression and the Notch-Elf5 axis. In clinical breast cancer specimens, ID4 expression is absent in luminal tumours but very high in approximately half of all BLBCs, a proportion of which possess ID4 gene amplification. These tumours are characterized by unique transcriptomes with many features of mammary stem cells. Furthermore, ID4-positive BLBCs have a very poor prognosis across multiple independent tissue cohorts. This study identifies ID4 as a master regulator of mammary stem cell homeostasis and suggests that a subset of aggressive BLBCs derive from a primitive mammary stem or progenitor cell with basal characteristics. Citation Format: Simon Junankar, Laura Baker, Kyuson Yun, Alexander Swarbrick. Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-62. doi:10.1158/1538-7445.AM2014-LB-62

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

Retinoid Acid Specifies Neuronal Identity through Graded Expression of Ascl1

SummaryCell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors [1]. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals [2, 3], two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively [4–7]. These neurons have different physiological properties, functions, and connectivity [8, 9]. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1 [10], whereas V3 neurons express Sim1 [4]. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.

Interactions between HLH and bHLH Factors Modulate Light-Regulated Plant Development

Phytochromes (Phy) and phytochrome-interacting factor (PIF) transcription factors constitute a major signaling module that controls plant development in response to red and far-red light. A low red:far-red ratio is interpreted as shading by neighbor plants and induces cell elongation-a phenomenon called shade-avoidance syndrome (SAS). PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation (ChIP) assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 (PAR1OX) are insensitive to gibberellin (GA) or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid (BR) and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PAR1OX. These results indicate that PAR1-PRE1 and PAR1-PIF4 heterodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.

Abstract 90: The Atheroprotective Effects of Id3 Are Linked to B-1a Cell Proliferation and Plasma Levels of E06

Background: The HLH transcription factor, Id3, is essential for B cell-mediated atheroprotection in mice and a functionally significant single nucleotide polymorphism in the human ID3 gene at rs11574 is associated with carotid intimal medial thickness in humans. Yet, the mechanisms mediating Id3 atheroprotection are poorly understood. Recent studies provide clear evidence that B cell effects on atherosclerosis are subset dependent; with B2 cells promoting and B-1a cells attenuating atherosclerosis in a sIgM-dependent manner. Innate, natural IgM antibodies, such as E06, recognize oxidized phospholipids, are produced by B-1a cells, and inhibit atherogenesis. Therefore, we hypothesized that Id3 would regulate plasma levels of E06 IgM and atheroprotective B-1a cells. Methods & Results: Id3 -/- ApoE -/- mice (n = 9) at 8 weeks old had lower plasma levels of E06 compared to control Id3 +/+ ApoE -/- mice (n = 9) (1500 vs. 2750 RLU, p < 0.05) as measured by ELISA. This was not due to lower total IgM (270 vs. 100 ug/mL respectively, p < 0.05). ApoB-100 levels were not different (1500 vs 1600 RLU, n.s.). Consistent with plasma data, the number of B-1a cells, assessed by flow cytometry, was lower in Id3 -/- ApoE -/- (n = 5) compared to Id3 +/+ ApoE -/- mice (n = 4) (0.8 x10 5 vs 3.0 x10 5 , p < 0.05). There was no difference in transcript levels, as measured by real-time PCR, of E06 or sIgM in fluorescence-activated cell sorted B-1a cells. Furthermore, we found decreased homeostatic proliferation, measured by in situ CFSE dilution, of B-1a cells in Id3 -/- ApoE -/- mice (n = 5) compared to control mice (n = 5) (30.1% vs. 50.0%, p = 0.001). Additionally, humans with the functionally significant polymorphism in ID3 had lower plasma levels of IgM to MDA-LDL (n = 97, trend with p = 0.08) and no difference in IgG as measured by ELISA. Conclusion: Taken together, these data suggest a novel role for Id3 in regulating B-1a cell proliferation, resulting in reduced plasma levels of the atheroprotective natural antibody, E06. Moreover, the data suggest that the ID3 polymorphism in humans at rs11574 may link protective IgM to modified lipids with vascular disease.

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

Local insulin-like growth factor I expression is essential for Purkinje neuron survival at birth

IGF1, an anabolic and neuroprotective factor, promotes neuronal survival by blocking apoptosis. It is released into the bloodstream by the liver, or synthesized locally by muscles and neural cells, acting in an autocrine or paracrine fashion. Intriguingly, genetic studies conducted in invertebrate and murine models also suggest that an excess of IGF1 signaling may trigger neurodegeneration. This emphasizes the importance of gaining a better understanding of the mechanisms controlling IGF1 regulation and gene transcription. In the cerebellum, Igf1 expression is activated just before birth in a subset of Purkinje cells (PCs). Mice carrying a null mutation for HLH transcription factor EBF2 feature PC apoptosis at birth. We show that Igf1 is sharply downregulated in Ebf2 null PCs starting before the onset of PC death. In vitro, EBF2 binds a conserved distal Igf1 promoter region. The pro-survival PI3K signaling pathway is strongly inhibited in mutant cerebella. Finally, Ebf2 null organotypic cultures respond to IGF1 treatment by inhibiting PC apoptosis. Consistently, wild type slices treated with an IGF1 competitor feature a sharp increase in PC death. Our findings reveal that IGF1 is required for PC survival in the neonatal cerebellum, and identify a new mechanism regulating its local production in the CNS.

Engineering of Dominant Active Basic Helix-Loop-Helix Proteins That Are Resistant to Negative Regulation by Postnatal Central Nervous System Antineurogenic Cues

Neural precursor cells (NPCs) are present in most regions of the adult central nervous system (CNS). Using NPCs in a therapeutical perspective, that is, to regenerate CNS tissue after injury or in neurodegenerative diseases, will require the efficient manipulation of their fate. Proneural gene overexpression in NPCs represents a promising strategy to promote neuronal differentiation. The activity of the proneural proteins is, however, context-dependent and can be inhibited/modulated by binding with other bHLH (basic helix-loop-helix) or HLH transcription factors. In this study, we show that the two proneural proteins, Ngn2 and Mash1, are differentially sensitive to negative regulation by gliogenic factors or a gliogenic substrate (i.e., postnatal spinal cord slices). Coexpressing E-proteins with proneural proteins was efficient to rescue proneural proteins neurogenic activity, suggesting a central role for E-protein sequestration in mediating postnatal CNS gliogenic inhibition. Tethering of proneural proteins with E47 further insulated Mash1 from negative environmental influences whereas this strategy was not successful with Ngn2, suggesting that mechanisms of inhibition differ in between these two proneural proteins. Our results demonstrate that a better understanding of proneural protein modulation by environmental cues is a prerequisite to develop innovative approaches that will permit the manipulation of the fate of NPCs in the adult CNS after trauma or disease.

18. Real-time analyses of HLH transcription factors

18. Real-time analyses of HLH transcription factors

A key role for the HLH transcription factor EBF2COE2,O/E-3 in Purkinje neuron migration and cerebellar cortical topography

Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.