PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction

Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.     

The neuronal stem cell of the olfactory epithelium

The vertebrate olfactory epithelium (OE) is a system in which behavior of neuronal progenitor cells can be observed and manipulated easily. It is morphologically and functionally similar to embryonic germinal neuroepithelia, but is simpler in that it produces large numbers of a single type of neuron, the olfactory receptor neuron (ORN). The OE is amenable to tissue culture, gene transfer, and in vivo surgical approaches, and these have been exploited in experiments aimed at understanding the characteristics of OE neuronal progenitor cells. This has led to the realization that the ORN lineage contains at least three distinct stages of proliferating neuronal progenitor cells (including a stem cell), each of which represents a point at which growth control can be exerted. Neurogenesis proceeds continually in the OE, and studies in vivo have shown that this is a regulated process that serves to maintain the number of ORNs at a particular level. These studies suggest that OE neuronal progenitors—which are in close physical proximity to ORNs—can “read” the number of differentiated neurons in their environment and regulate production of new neurons accordingly. Putative neuronal stem cells of the OE have been identified in vitro, and studies of these cells indicate that ORNs produce a signal that feeds back to inhibit neurogenesis. This inhibitory signal may be exerted at the level of the stem cell itself. Recent studies to identify this signal, as well as endogenous stimulatory signals that may be important in regulating OE neurogenesis, are also discussed. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 190–205, 1998     

Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.     

Neuronal degeneration and regeneration induced by axotomy in the olfactory epithelium of Xenopus laevis

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific‐ neuronal death through apoptosis between 24 and 48h post‐injury. In concordance, there was a progressive decrease of the mature‐ORN marker OMP until it was completely absent 72h post‐injury. On the other hand, neurogenesis was evident 48h post‐injury by an increase in the number of proliferating basal cells as well as NCAM‐180– GAP‐43+ immature neurons. Mature ORNs were replenished 21 days post‐injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308–1320, 2017     

The Ginkgo biloba extract modulates the balance between proliferation and differentiation in the olfactory epithelium of adult mice following bulbectomy.

Abstract - The adult olfactory receptor neurons (ORNs), located in the olfactory epithelium (OE) are permanently renewed thanks to neuronal progenitors present in the deep part of the OE, the globose basal cells (GBCs). Following the ablation of their synaptic target, the olfactory bulb (OB), ORNs degenerate by apoptosis and a wave of neurogenesis, including proliferation of GBCs and neuronal differentiation of their progeny, restores the olfactory function. The Ginkgo biloba extract (EGb 761) (Beaufour Ipsen, France) was administered to adult mice at the doses of 50 or 100 mg/kg, following bilateral bulbectomy and its effects on the expression of PCNA, reflecting the number of proliferating GBCs and on growth associated protein 43 (GAP-43), expressed by differentiating neurons were measured by Western blotting. PCNA expression peaked 9 days post-bulbectomy in untreated animals, but 7 days post-lesion in EGb 761-treated animals. A simultaneous reduction in GAP-43 expression suggested that EGb 761 may temporarily favor the proliferation of GBCs rather than their entry into the differentiation pathway. Probably as a consequence of the earlier onset of the neurogenetic response to bulbectomy, neuronal differentiation was enhanced in the OE, 3 weeks post-bulbectomy. These data suggest that EGb 761 may have beneficial effects upon neurogenesis in the OE through changing the balance between proliferation and differentiation.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.     

CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.     

Differential requirements for different subfamilies of the mammalian SWI/SNF chromatin remodeling enzymes in myoblast cell cycle progression and expression of the Pax7 regulator

The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) families of ATP-dependent chromatin remodeling enzymes are established co-regulators of gene expression. mSWI/SNF complexes can be assembled into three major subfamilies: BAF (BRG1 or BRM-Associated Factor), PBAF (Polybromo containing BAF), or ncBAF (non-canonical BAF) that are distinguished by the presence of mutually exclusive subunits. The mechanisms by which each subfamily contributes to the establishment or function of specific cell lineages are poorly understood. Here, we determined the contributions of the BAF, ncBAF, and PBAF complexes to myoblast proliferation via knock down (KD) of distinguishing subunits from each complex. KD of subunits unique to the BAF or the ncBAF complexes reduced myoblast proliferation rate, while KD of PBAF-specific subunits did not affect proliferation. RNA-seq from proliferating KD myoblasts targeting Baf250A (BAF complex), Brd9 (ncBAF complex), or Baf180 (PBAF complex) showed mis-regulation of a limited number of genes. KD of Baf250A specifically reduced the expression of Pax7, which is required for myoblast proliferation, concomitant with decreased binding of Baf250A to and impaired chromatin remodeling at the Pax7 gene promoter. Although Brd9 also bound to the Pax7 promoter, suggesting occupancy by the ncBAF complex, no changes were detected in Pax7 gene expression, Pax7 protein expression or chromatin remodeling at the Pax7 promoter upon Brd9 KD. The data indicate that the BAF subfamily of the mSWI/SNF enzymes is specifically required for myoblast proliferation via regulation of Pax7 expression.     

Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits

Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.     

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.     

SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development

ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.