MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1

BACKGROUND: Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs. RESULTS: Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. CONCLUSIONS: Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances

EDF-1, a novel gene product down-regulated in human endothelial cell differentiation.

Abstract Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encodingEDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridgingfactor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiationin vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation

Mmot1, a New Helix-Loop-Helix Transcription Factor Gene Displaying a Sharp Expression Boundary in the Embryonic Mouse Brain

Several genetic factors have been proven to contribute to the specification of the metencephalic-mesencephalic territory, a process that sets the developmental foundation for prospective morphogenesis of the cerebellum and mesencephalon. However, evidence stemming from genetic and developmental studies performed in man and various model organisms suggests the contribution of many additional factors in determining the fine subdivision and differentiation of these central nervous system regions. In man, the cerebellar ataxias/aplasias represent a large and heterogeneous family of genetic disorders. Here, we describe the identification by differential screening and the characterization of Mmot1, a new gene encoding a DNA-binding protein strikingly similar to the helix-loop-helix factor Ebf/Olf1. Throughout midgestation embryogenesis, Mmot1is expressed at high levels in the metencephalon, mesencephalon, and sensory neurons of the nasal cavity. In vitro DNA binding data suggest some functional equivalence of Mmot1 and Ebf/Olf1, possibly accounting for the reported lack of olfactory or neural defects in Ebf −/− knockout mutants. The isolation of Mmot1 and of an additional homolog in the mouse genome defines a novel, phylogenetically conserved mammalian family of transcription factor genes of potential relevance in studies of neural development and its aberrations

Epithelial V-like Antigen (EVA), a Novel Member of the Immunoglobulin Superfamily, Expressed in Embryonic Epithelia with a Potential Role as Homotypic Adhesion Molecule in Thymus Histogenesis

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis

Overall Lack of Regulated Secretion in a PC12 Variant Cell Clone

Abstract A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and α-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-β-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation

Neural stem cells engrafted in the adult brain fuse with endogenous neurons.

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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)

Domain-specific regulation of cerebellar morphogenesis by Zfp423 / ZNF423, a gene implicated in Joubert syndrome and cerebellar vermis hypoplasia

The Zfp423 gene encodes a 30-Zn-finger transcription factor that acts as a scaffold for the assembly of complex transcriptional and cellular machineries regulating neural development. While null Zfp423 mutants feature a sharp decrease in the total number of cerebellar Purkinje cells (PCs), the underlying mechanisms remain unclear. Mutations of the human homolog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), associated with other signs of classical ciliopathy outside the central nervous system. To further characterize the role of ZFP423 in cerebellar neurogenesis, we have performed morphological, cellular and molecular studies on two mutant mouse lines carrying allelic in-frame deletions of Zfp423. While both lines exhibit cerebellar hypoplasia, considerable differences are observed between the two mutants, with respect to neural progenitor differentiation, cell survival and morphogenesis. The results of this in vivo and in vitro structure-function analysis point to domain- and context-specific roles played by ZFP423 in different aspects of cerebellar development, and contribute to our understanding of its role as a disease / modifier gene in JS, CVH and other ciliopathies

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments

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Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50–60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS