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1.
Cells ; 12(15)2023 08 05.
Article in English | MEDLINE | ID: mdl-37566088

ABSTRACT

Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. We previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. We demonstrate that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.


Subject(s)
Amyotrophic Lateral Sclerosis , C9orf72 Protein , GTPase-Activating Proteins , Animals , Humans , Mice , ADP-Ribosylation Factor 1/metabolism , Amyotrophic Lateral Sclerosis/metabolism , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Drosophila/genetics , Drosophila/metabolism , RNA/metabolism , RNA, Messenger/genetics , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism
2.
Pharmacol Rep ; 73(2): 536-550, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33661518

ABSTRACT

BACKGROUND: ALS is an incurable neuromuscular degenerative disorder. A familiar form of the disease (fALS) is related to point mutations. The most common one is an expansion of a noncoding GGGGCC hexanucleotide repeat of the C9orf72 gene on chromosome 9p21. An abnormal translation of the C9orf72 gene generates dipeptide repeat proteins that aggregate in the brain. One of the classical approaches for developing treatment against protein aggregation-related diseases is to use chemical chaperones (CSs). In this work, we describe the development of novel 4-phenylbutyric acid (4-PBA) lysosome/ER-targeted derivatives. We assumed that 4-PBA targeting to specific organelles, where protein degradation takes place, might reduce the 4-PBA effective concentration. METHODS: Organic chemistry synthetic methods and solid-phase peptide synthesis (SPPS) were used for preparing the 4-PBA derivatives. The obtained compounds were evaluated in an ALS Drosophila model that expressed C9orf72 repeat expansion, causing eye degeneration. Targeting to lysosome was validated by the 19F-nuclear magnetic resonance (NMR) technique. RESULTS: Several synthesized compounds exhibited a significant biological effect by ameliorating the eye degeneration. They blocked the neurodegeneration of fly retina at different efficacy levels. The most active CS was compound 9, which is a peptide derivative and was targeted to ER. Another active compound targeted to lysosome was compound 4. CONCLUSIONS: Novel CSs were more effective than 4-PBA; therefore, they might be used as a new class of drug candidates to treat ALS and other protein misfolding disorders.


Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , C9orf72 Protein/genetics , Molecular Chaperones/pharmacology , Phenylbutyrates/pharmacology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , DNA Repeat Expansion/genetics , Disease Models, Animal , Drosophila melanogaster , Endoplasmic Reticulum/drug effects , Lysosomes/metabolism , Magnetic Resonance Imaging , Molecular Chaperones/chemical synthesis , Molecular Chaperones/chemistry , Phenylbutyrates/chemical synthesis , Phenylbutyrates/chemistry
3.
Sci Rep ; 7(1): 2033, 2017 05 17.
Article in English | MEDLINE | ID: mdl-28515487

ABSTRACT

Several of the identified genetic factors in Amyotrophic Lateral Sclerosis (ALS) point to dysfunction in RNA processing as a major pathogenic mechanism. However, whether a precise RNA pathway is particularly affected remains unknown. Evidence suggests that FUS, that is mutated in familial ALS, and SMN, the causative factor in Spinal Muscular Atrophy (SMA), cooperate to the same molecular pathway, i.e. regulation of alternative splicing, and that disturbances in SMN-regulated functions, either caused by depletion of SMN protein (as in the case of SMA) or by pathogenic interactions between FUS and SMN (as in the case of ALS) might be a common theme in both diseases. In this work, we followed these leads and tested their pathogenic relevance in vivo. FUS-associated ALS recapitulates, in transgenic mice, crucial molecular features that characterise mouse models of SMA, including defects in snRNPs distribution and in the alternative splicing of genes important for motor neurons. Notably, altering SMN levels by haploinsufficiency or overexpression does not impact the phenotypes of mouse or Drosophila models of FUS-mediated toxicity. Overall, these findings suggest that FUS and SMN functionally interact and that FUS may act downstream of SMN-regulated snRNP assembly in the regulation of alternative splicing and gene expression.

4.
Front Mol Neurosci ; 10: 85, 2017.
Article in English | MEDLINE | ID: mdl-28386218

ABSTRACT

Cells robustly reprogram gene expression during stress generated by protein misfolding and aggregation. In this condition, cells assemble the bulk of mRNAs into translationally silent stress granules (SGs), while they sustain the translation of specific mRNAs coding for proteins that are needed to overcome cellular stress. Alterations of this process are deeply associated to neurodegeneration. This is the case of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by a selective loss of motor neurons. Indeed, impairment of protein homeostasis as well as alterations of RNA metabolism are now recognized as major players in the pathogenesis of ALS. In particular, evidence shows that defective mRNA transport and translation are implicated. Here, we provide a review of what is currently known about altered mRNA translation in ALS and how this impacts on the ability of affected cells to cope with proteotoxic stress.

5.
Hum Mol Genet ; 23(15): 3907-22, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24599399

ABSTRACT

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by fragile skin forming blisters that heal invariably with scars. It is due to mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils connecting the cutaneous basement membrane to the dermis. Identical COL7A1 mutations often result in inter- and intra-familial disease variability, suggesting that additional modifiers contribute to RDEB course. Here, we studied a monozygotic twin pair with RDEB presenting markedly different phenotypic manifestations, while expressing similar amounts of collagen VII. Genome-wide expression analysis in twins' fibroblasts showed differential expression of genes associated with TGF-ß pathway inhibition. In particular, decorin, a skin matrix component with anti-fibrotic properties, was found to be more expressed in the less affected twin. Accordingly, fibroblasts from the more affected sibling manifested a profibrotic and contractile phenotype characterized by enhanced α-smooth muscle actin and plasminogen activator inhibitor 1 expression, collagen I release and collagen lattice contraction. These cells also produced increased amounts of proinflammatory cytokines interleukin 6 and monocyte chemoattractant protein-1. Both TGF-ß canonical (Smads) and non-canonical (MAPKs) pathways were basally more activated in the fibroblasts of the more affected twin. The profibrotic behaviour of these fibroblasts was suppressed by decorin delivery to cells. Our data show that the amount of type VII collagen is not the only determinant of RDEB clinical severity, and indicate an involvement of TGF-ß pathways in modulating disease variability. Moreover, our findings identify decorin as a possible anti-fibrotic/inflammatory agent for RDEB therapeutic intervention.


Subject(s)
Epidermolysis Bullosa Dystrophica/genetics , Fibroblasts/metabolism , Genotype , Phenotype , Skin/metabolism , Transforming Growth Factor beta/genetics , Twins, Monozygotic/genetics , Actins/genetics , Actins/metabolism , Adult , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Collagen Type VII/genetics , Collagen Type VII/metabolism , Epidermolysis Bullosa Dystrophica/metabolism , Epidermolysis Bullosa Dystrophica/pathology , Fibroblasts/pathology , Gene Expression Regulation , Genes, Recessive , Genetic Heterogeneity , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Male , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Severity of Illness Index , Signal Transduction , Skin/pathology , Smad Proteins/genetics , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism
6.
Development ; 141(2): 377-88, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24335253

ABSTRACT

During embryonic development, the rostral neuroectoderm is regionalized into broad areas that are subsequently subdivided into progenitor compartments with specialized identity and fate. These events are controlled by signals emitted by organizing centers and interpreted by target progenitors, which activate superimposing waves of intrinsic factors restricting their identity and fate. The transcription factor Otx2 plays a crucial role in mesencephalic development by positioning the midbrain-hindbrain boundary (MHB) and its organizing activity. Here, we investigated whether Otx2 is cell-autonomously required to control identity and fate of dorsal mesencephalic progenitors. With this aim, we have inactivated Otx2 in the Pax7(+) dorsal mesencephalic domain, previously named m1, without affecting MHB integrity. We found that the Pax7(+) m1 domain can be further subdivided into a dorsal Zic1(+) m1a and a ventral Zic1(-) m1b sub-domain. Loss of Otx2 in the m1a (Pax7(+) Zic1(+)) sub-domain impairs the identity and fate of progenitors, which undergo a full switch into a coordinated cerebellum differentiation program. By contrast, in the m1b sub-domain (Pax7(+) Zic1(-)) Otx2 is prevalently required for post-mitotic transition of mesencephalic GABAergic precursors. Moreover, genetic cell fate, BrdU cell labeling and Otx2 conditional inactivation experiments indicate that in Otx2 mutants all ectopic cerebellar cell types, including external granule cell layer (EGL) precursors, originate from the m1a progenitor sub-domain and that reprogramming of mesencephalic precursors into EGL or cerebellar GABAergic progenitors depends on temporal sensitivity to Otx2 ablation. Together, these findings indicate that Otx2 intrinsically controls different aspects of dorsal mesencephalic neurogenesis. In this context, Otx2 is cell-autonomously required in the m1a sub-domain to suppress cerebellar fate and promote mesencephalic differentiation independently of the MHB organizing activity.


Subject(s)
Cerebellum/embryology , Cerebellum/metabolism , Mesencephalon/embryology , Mesencephalon/metabolism , Otx Transcription Factors/metabolism , Animals , Body Patterning , Cell Differentiation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Female , Mice , Mice, Knockout , Mice, Mutant Strains , Mice, Transgenic , Mutation , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis , Organizers, Embryonic/embryology , Organizers, Embryonic/metabolism , Otx Transcription Factors/deficiency , Otx Transcription Factors/genetics , PAX7 Transcription Factor/metabolism , Pregnancy , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/metabolism
7.
Dev Biol ; 373(1): 176-83, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23117062

ABSTRACT

Understanding the molecular basis underlying the neurogenesis of mesencephalic-diencephalic Dopaminergic (mdDA) neurons is a major task fueled by their relevance in controlling locomotor activity and emotion and their involvement in neurodegenerative and psychiatric diseases. Increasing evidence suggests that mdDA neurons of the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) represent two main distinct neuronal populations, which, in turn, include specific neuronal subsets. Relevant studies provided important results on mdDA neurogenesis, but, nevertheless, have not yet clarified how the identity of mdDA neuronal subtypes is established and, in particular, whether neurogenic factors may direct progenitors towards the differentiation of specific mdDA neuronal subclasses. The transcription factor Otx2 is required for the neurogenesis of mesencephalic DA (mesDA) neurons and to control neuron subtype identity and sensitivity to the MPTP neurotoxin in the adult VTA. Here we studied whether Otx2 is required in mdDA progenitors for the generation of specific mdDA neuronal subtypes. We found that although expressed in virtually all mdDA progenitors, Otx2 is required selectively for the differentiation of VTA neuronal subtypes expressing Ahd2 and/or Calb but not for those co-expressing Girk2 and glyco-Dat. Moreover, mild over-expression of Otx2 in SNpc progenitors and neurons is sufficient to rescue En1 haploinsufficiency-dependent defects, such as progressive loss and increased MPTP sensitivity of SNpc neurons. Collectively, these data suggest that mdDA progenitors exhibit differential sensitivity to Otx2, which selectively influences the generation of a large and specific subset of VTA neurons. In addition, these data suggest that Otx2 and En1 may share similar properties and control survival and vulnerability to MPTP neurotoxin respectively in VTA and SNpc.


Subject(s)
Dopaminergic Neurons/physiology , Homeodomain Proteins/metabolism , Neurogenesis/physiology , Otx Transcription Factors/metabolism , Ventral Tegmental Area/cytology , Ventral Tegmental Area/embryology , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/administration & dosage , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Cell Count , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Immunohistochemistry , In Situ Hybridization , Mice
8.
Dev Neurobiol ; 71(8): 665-79, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21309083

ABSTRACT

Mesencephalic-diencephalic dopaminergic (mdDA) neurons play a relevant role in the control of movement, behavior, and cognition. Indeed loss and/or abnormal functioning of mdDA neurons are responsible for Parkinson's disease as well as for addictive and psychiatric disorders. In the last years a wealth of information has been provided on gene functions controlling identity, fate, and proliferation of mdDA progenitors. This review will focus on the role exerted by Otx genes in early decisions regulating sequential steps required for the neurogenesis of mesencephalic dopaminergic (mesDA) neurons. In this context, the regulatory network involving Otx functional interactions with signaling molecules and transcription factors required to promote or prevent the development of mesDA neurons will be analyzed in detail.


Subject(s)
Dopamine/metabolism , Mesencephalon/growth & development , Neurogenesis/physiology , Neurons/metabolism , Otx Transcription Factors/genetics , Humans , Mesencephalon/metabolism , Otx Transcription Factors/metabolism
9.
Mol Neurobiol ; 43(2): 107-13, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21086067

ABSTRACT

Mesencephalic and diencephalic dopaminergic (mdDA) progenitors generate two major groups of neurons corresponding to the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). MdDA neurons control motor, sensorimotor and motivated behaviour and their degeneration or abnormal functioning is associated to Parkinson's disease and psychiatric disorders. Although relevant advances have been made, the molecular basis controlling identity, survival and vulnerability to neurodegeneration of SNpc and VTA neurons remains poorly understood. Here, we will review recent findings on the role exerted by the transcription factor Otx2 in adult mdDA neurons. Otx2 expression is restricted to a relevant fraction of VTA neurons and absent in the SNpc. In particular, Otx2 is prevalently excluded from neurons of the dorsal-lateral VTA, which expressed Girk2 and high level of the dopamine transporter (Dat). Loss and gain of function mouse models revealed that Otx2 controls neuron subtype identity by antagonizing molecular and functional features of the dorsal-lateral VTA such as Girk2 and Dat expression as well as vulnerability to the parkinsonian MPTP toxin. Furthermore, when ectopically expressed in the SNpc, Otx2 suppresses Dat expression and confers efficient neuroprotection to MPTP toxicity by suppressing efficient DA uptake.


Subject(s)
Aging/metabolism , Dopamine/metabolism , Mesencephalon/metabolism , Neurons/metabolism , Otx Transcription Factors/metabolism , Animals , Diencephalon/metabolism
10.
Nat Neurosci ; 13(12): 1481-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21057506

ABSTRACT

Mesencephalic-diencephalic dopaminergic neurons control locomotor activity and emotion and are affected in neurodegenerative and psychiatric diseases. The homeoprotein Otx2 is restricted to ventral tegmental area (VTA) neurons that are prevalently complementary to those expressing Girk2 and glycosylated active form of the dopamine transporter (Dat). High levels of glycosylated Dat mark neurons with efficient dopamine uptake and pronounced vulnerability to Parkinsonian degeneration. We found that Otx2 controls neuron subtype identity by antagonizing molecular and functional features of dorsal-lateral VTA, such as Girk2 and Dat expression. Otx2 limited the number of VTA neurons with efficient dopamine uptake and conferred resistance to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Ectopic Otx2 expression also provided neurons of the substantia nigra with efficient neuroprotection to MPTP. These findings indicate that Otx2 is required to specify neuron subtype identity in VTA and may antagonize vulnerability to the Parkinsonian toxin MPTP.


Subject(s)
MPTP Poisoning/prevention & control , Neurons/classification , Neurons/physiology , Otx Transcription Factors/physiology , Ventral Tegmental Area/cytology , Ventral Tegmental Area/physiology , Animals , Embryonic Stem Cells/cytology , Embryonic Stem Cells/pathology , Embryonic Stem Cells/physiology , MPTP Poisoning/metabolism , MPTP Poisoning/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Neurons/pathology , Ventral Tegmental Area/pathology
11.
Nat Neurosci ; 13(6): 680-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20495559

ABSTRACT

Neurons usually migrate and differentiate in one particular encephalic vesicle. We identified a murine population of diencephalic neurons that colonized the telencephalic amygdaloid complex, migrating along a tangential route that crosses a boundary between developing brain vesicles. The diencephalic transcription factor OTP was necessary for this migratory behavior.


Subject(s)
Amygdala/embryology , Cell Movement/physiology , Diencephalon/embryology , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/physiology , Telencephalon/embryology , Amygdala/physiology , Animals , Cell Count , Diencephalon/physiology , Electroporation , Homeodomain Proteins/genetics , Hypothalamus/embryology , Hypothalamus/physiology , Immunohistochemistry , In Situ Hybridization , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/genetics , RNA Interference , Stem Cell Niche/embryology , Stem Cell Niche/physiology , Telencephalon/physiology
12.
Mol Cell Neurosci ; 45(1): 1-11, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20493948

ABSTRACT

The locus coeruleus (LC) which is the major noradrenergic nucleus in the brain develops under the influence of Bmps secreted by the roof plate and Fgf8 emitted from the mid-hindbrain organizer. We studied the development of the LC in different Bmp mouse mutants and report the absence of this nucleus in Bmp5(-/-);Bmp7(-/-) double knockouts. Notably, genes marking organizers and neuronal populations adjacent to the LC precursor field are unperturbed in Bmp5(-/-);Bmp7(-/-) animals. In addition, we found that in En1(+/Otx2) mutants in which the caudal Otx2 expression domain and thereby the mid-hindbrain organizer are shifted caudally, LC neurons are concomitantly reduced along with Bmp5/7. Complementing these results, Otx1(-/-);Otx2(+/-) mutants, in which the mid-hinbrain organizer is shifted rostrally, show a rostrally extended Bmp5 expression area and an increase in LC neurons. Taken together, our data indicate that LC development requires either Bmp5 or Bmp7, and one is able to compensate for the loss of the other. In addition, we conclude that the position of the mid-hindbrain organizer determines the size of the LC and propose that Bmp5/7 play an important role in mediating this organizer function.


Subject(s)
Bone Morphogenetic Protein 5/metabolism , Bone Morphogenetic Protein 7/metabolism , Locus Coeruleus/cytology , Locus Coeruleus/embryology , Mesencephalon/physiology , Norepinephrine/metabolism , Rhombencephalon/physiology , Animals , Apoptosis , Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Locus Coeruleus/metabolism , Mesencephalon/cytology , Mice , Mice, Knockout , Neurons/cytology , Neurons/physiology , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism , Rhombencephalon/cytology , Stem Cells/cytology , Stem Cells/physiology
13.
Int J Dev Biol ; 54(5): 939-45, 2010.
Article in English | MEDLINE | ID: mdl-19924631

ABSTRACT

Mesencephalic-diencephalic dopaminergic (mdDA) neurons control motor, sensorimotor and motivated behaviour and their degeneration or abnormal functioning is associated with important pathologies, such as Parkinsons disease and psychiatric disorders. Despite great efforts, the molecular basis and the genetic factors differentially controlling identity, survival and vulnerability to neurodegeneration of mdDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA) are poorly understood. We have previously shown that the transcription factor Otx2 is required for identity, fate and proliferation of mesencephalic DA (mesDA) progenitors. By using mouse models and immunohistochemistry, we have investigated whether Otx2 is expressed also in post-mitotic mdDA neurons. Our data reveal that Otx2 is expressed in post-mitotic mesDA neurons during mid-late gestation and in the adult brain. Remarkably, Otx2 expression is sharply excluded from mdDA neurons of the SN and is restricted to a relevant fraction of VTA neurons. Otx2+-TH+ neurons are concentrated to the ventral part of the VTA. Combined expression with other regionalized VTA markers shows that Otx2+-TH+ neurons are prevalently Girk2- and Calb+ and among these, those located in the medial and ventralmost portion of the VTA are also Ahd2+. These findings indicate that Otx2 represents the first transcription factor with a proven role in mdDA neurogenesis whose expression discriminates between SN and a relevant proportion of VTA neurons. This supports the possibility that Otx2 may act as a post-mitotic selector controlling functional features (e.g. identity and/or survival) of a relevant fraction of VTA neurons in the adult.


Subject(s)
Brain/metabolism , Neurons/metabolism , Otx Transcription Factors/metabolism , Ventral Tegmental Area/metabolism , Animals , Brain/embryology , Brain/growth & development , Dopamine/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Immunohistochemistry , Mice , Mutation , Neurons/cytology , Otx Transcription Factors/genetics , Time Factors , Ventral Tegmental Area/embryology , Ventral Tegmental Area/growth & development
14.
Mech Dev ; 126(10): 882-97, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19615442

ABSTRACT

Genetic and embryological experiments demonstrated that the visceral endoderm (VE) is essential for positioning the primitive streak at one pole of the embryo and head morphogenesis through antagonism of the Wnt and Nodal signaling pathways. The transcription factor Otx2 is required for VE anteriorization and specification of rostral neuroectoderm at least in part by controlling the expression of Dkk1 and Lefty1. Here, we investigated the relevance of the Otx2 transcriptional control in these processes. Otx2 protein is encoded by different mRNAs variants, which, on the basis of their transcription start site, may be distinguished in distal and proximal. Distal isoforms are prevalently expressed in the epiblast and neuroectoderm, while proximal isoforms prevalently in the VE. Selective inactivation of Otx2 variants reveals that distal isoforms are not required for gastrulation, but essential for maintenance of forebrain and midbrain identities; conversely, proximal isoforms control VE anteriorization and, indirectly, primitive streak positioning through the activation of Dkk1 and Lefty1. Moreover, in these mutants the expression of proximal isoforms is not affected by the lack of distal mRNAs and vice versa. Taken together these findings indicate that proximal and distal isoforms, whose expression is independently regulated in the VE and epiblast-derived neuroectoderm, functionally cooperate to provide these tissues with the sufficient level of Otx2 necessary to promote a normal development. Furthermore, we discovered that in the VE the expression of Otx2 isoforms is tightly controlled at single cell level, and we hypothesize that this molecular diversity may potentially confer specific functional properties to different subsets of VE cells.


Subject(s)
Endoderm/cytology , Head/embryology , Morphogenesis , Otx Transcription Factors/genetics , RNA, Messenger/genetics , Viscera/cytology , Animals , Base Sequence , DNA Primers , Gene Expression Profiling , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/genetics , Left-Right Determination Factors/genetics , Mesencephalon/embryology , Mice , Prosencephalon/embryology , Reverse Transcriptase Polymerase Chain Reaction
15.
Development ; 136(15): 2545-55, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19592574

ABSTRACT

Little is known about the cues controlling the generation of motoneuron populations in the mammalian ventral midbrain. We show that Otx2 provides the crucial anterior-posterior positional information for the generation of red nucleus neurons in the murine midbrain. Moreover, the homeodomain transcription factor Nkx6-1 controls the proper development of the red nucleus and of the oculomotor and trochlear nucleus neurons. Nkx6-1 is expressed in ventral midbrain progenitors and acts as a fate determinant of the Brn3a(+) (also known as Pou4f1) red nucleus neurons. These progenitors are partially dorsalized in the absence of Nkx6-1, and a fraction of their postmitotic offspring adopts an alternative cell fate, as revealed by the activation of Dbx1 and Otx2 in these cells. Nkx6-1 is also expressed in postmitotic Isl1(+) oculomotor and trochlear neurons. Similar to hindbrain visceral (branchio-) motoneurons, Nkx6-1 controls the proper migration and axon outgrowth of these neurons by regulating the expression of at least three axon guidance/neuronal migration molecules. Based on these findings, we provide additional evidence that the developmental mechanism of the oculomotor and trochlear neurons exhibits more similarity with that of special visceral motoneurons than with that controlling the generation of somatic motoneurons located in the murine caudal hindbrain and spinal cord.


Subject(s)
Cell Lineage , Homeodomain Proteins/metabolism , Motor Neurons/cytology , Motor Neurons/metabolism , Oculomotor Nerve/cytology , Red Nucleus/cytology , Red Nucleus/metabolism , Animals , Axons/metabolism , Cell Movement , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Mice , Mitosis , Models, Biological , Neurogenesis , Oculomotor Nerve/metabolism , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism , Stem Cells/cytology , Transcription Factor Brn-3A/metabolism , Trochlear Nerve/cytology
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