ABSTRACT
The extracellular matrix molecule reelin is a crucial molecule in CNS development, in particular in the cerebellum and cerebral cortex. In the cerebral cortex, reelin is provided by a small number of neurons located in the marginal zone (MZ). These neurons belong to the earliest neurons generated, but little is known about the molecular mechanisms of their specification. Here we describe that reelin-positive cells are strongly increased in the developing cortex of the Pax6 mutant mice Small eye. Shortly after the onset of reelin expression, the number of reelin- and calretinin-positive cells is doubled in the cortex of Pax6 mutants and this increase is further enhanced during development. In contrast, calbindin-positive cells in the MZ do not co-express reelin and are not altered in the Pax6 mutant cortex. The split of the preplate cells was also defective in the Pax6 mutant cortex, suggesting that the amount of reelin is crucial for positioning of the cortical plate between the MZ and subplate. We further show that Pax6 mutant cortical cells isolated in vitro do not develop an increase in reelin-positive cells, while cells isolated from the entire telencephalon do. Consistent with non-cell-autonomous mechanisms contributing to the increase in reelin-positive cells in the Pax6-deficient cortex, tangential migration of diverse cell types from the ventral telencephalon into the cortex is enhanced in the Pax6 mutant mice. Taken together, these experiments further elucidate how patterning of the forebrain by the transcription factor Pax6 regulates the specification of distinct neuronal subtypes in the cortical MZ.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Extracellular Matrix Proteins/metabolism , Neurons/metabolism , S100 Calcium Binding Protein G/metabolism , Animals , Calbindin 2 , Calbindins , Cell Differentiation/physiology , Cell Movement/physiology , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/growth & development , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Eye Proteins , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Morphogenesis , Nerve Tissue Proteins , Nervous System Malformations/genetics , Nervous System Malformations/metabolism , Neurons/cytology , Neurons/physiology , PAX6 Transcription Factor , Paired Box Transcription Factors , Reelin Protein , Repressor Proteins , Serine Endopeptidases , Signal TransductionABSTRACT
Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1-/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4+CD8+ (DP) thymocytes from Apaf-1-/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1-/- mice were crossed with the T-cell receptor (TCR)-alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha-/-Apaf-1-/- and TCR-alpha-/-Apaf-1+/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1-/- Db/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33+ thymocytes and an increased percentage of the autoreactive CD8+M33+ thymocytes in male, but not female Apaf-1-/- Db/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.
Subject(s)
Proteins/immunology , Thymus Gland/embryology , Animals , Apoptosis/immunology , Apoptotic Protease-Activating Factor 1 , Autoimmunity , Female , Fetus/cytology , Fetus/immunology , Genes, T-Cell Receptor alpha , Genes, T-Cell Receptor beta , Male , Mice , Mice, Knockout , Mice, Transgenic , Organ Culture Techniques , Proteins/genetics , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Thymus Gland/immunologySubject(s)
Molecular Biology/methods , Stem Cells/physiology , Animals , Embryo, Mammalian , Gene Library , Genes, Reporter , Mice , Stem Cells/cytologyABSTRACT
The paired box containing transcription factor Pax3 is a crucial regulator of dermomyotome and muscle development. However, the allelic series of Pax3/Splotch mutants also displays characteristic vertebral column malformations, which do not result from defective dorsoventral somite pattern. Rather, vertebral column and sclerotomal phenotypes are reminiscent of the phenotypes observed in the segmentation/somitogenesis mutants rachiterata and pudgy. Moreover, rostrocaudal somite pattern and somitic boundaries are disturbed in Splotch as monitored by the expression of Uncx4.1 and Lunatic fringe. Alterations in EphA4, Dll1, and Uncx4.1 expression are evident already in the condensing paraxial mesoderm, correlating with the first phase of Pax3 expression before and during somite formation. This finding suggests an early function of Pax3 during the formation of epithelial somites.
Subject(s)
DNA-Binding Proteins/physiology , Epithelium/embryology , Mesoderm/physiology , Mice, Mutant Strains/genetics , Transcription Factors , Animals , Embryo, Mammalian/physiology , Embryonic and Fetal Development , Mice/embryology , Mice, Inbred Strains , PAX3 Transcription Factor , Paired Box Transcription Factors , Phenotype , Spine/embryologyABSTRACT
Apaf1 has been described as the core of the apoptosome. Deficiency in murine Apaf1 leads to embryonic lethality with a phenotype affecting many aspects of developmental apoptosis. In the developing brain, Apaf1 is a death regulator of the neuronal founder cells. Combined intercrosses of mouse lines mutant for members of the mitochondrial death pathway are providing us with some clues about the relative regulation existing among neuronal cell populations. Apaf1-deficient embryos display an interesting phenotype in the inner ear and in limb development, which involves different caspase-dependent and -independent pathways. Moreover, APAF1 is mutated in human melanomas, and its depletion contributes to malignant transformation in a mouse model of cancer. This review has a double aim: the analysis of the alternatives taken by the embryo to bring into the suicidal program different cells at different stages, and the relevance of APAF1 in the onset and progression of cancer.
Subject(s)
Apoptosis , Embryonic and Fetal Development , Neoplasms/pathology , Proteins/metabolism , Animals , Apoptotic Protease-Activating Factor 1 , Brain/embryology , Ear, Inner/embryology , Humans , Melanoma/genetics , Melanoma/pathology , Mitochondria/physiology , Models, Biological , Multigene Family , Neurons/cytology , Neurons/metabolism , Phenotype , Proteins/geneticsABSTRACT
Recent reports have exposed the temporal and spatial functions of the transcription factor Pax6 in the developing vertebrate eye. Pax6 is demonstrated to play essential roles in successive steps triggering lens differentiation while in the retina it functions to maintain multipotency and proliferation of retinal progenitor cells. These findings, together with the identification of Pax6 protein partners and downstream targets, pave the way for future work aimed to understand the molecular mechanism of eye development.
Subject(s)
Eye/embryology , Homeodomain Proteins/physiology , Animals , Eye Proteins , Gene Expression Regulation, Developmental , Lens, Crystalline/embryology , Mice , Models, Biological , PAX6 Transcription Factor , Paired Box Transcription Factors , Repressor Proteins , Retina/embryology , Stem Cells/physiologyABSTRACT
We report an improved vector, pCAGGS-FLPe, for transient expression of the enhanced FLP recombinase in mouse ES cells and oocytes. In standard transfection experiments, about 6% of total ES colonies showed FLP recombination, albeit with mosaicism within each colony. After microinjection of pCAGGS-FLPe into oocytes, about one-third of heterozygotic mice born showed complete FLP recombination. Thus pCAGGS-FLPe presents two practical options for removal of FRT cassettes in mice.
Subject(s)
DNA Nucleotidyltransferases/genetics , Embryo, Mammalian/enzymology , Oocytes/enzymology , Amino Acid Sequence , Animals , Base Sequence , DNA Nucleotidyltransferases/metabolism , DNA Primers/chemistry , Female , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Developmental , Genetic Engineering/methods , Genetic Vectors , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , Molecular Sequence Data , Polymerase Chain Reaction , TransfectionABSTRACT
Retinal ganglion cell (RGC) axons grow towards the diencephalic ventral midline during embryogenesis guided by cues whose nature is largely unknown. We provide in vitro and in vivo evidence for a novel role of Sonic hedgehog (SHH) as a negative regulator of growth cone movement. SHH suppresses both the number and the length of neurites emerging from the chick retina but not from neural tube or dorsal root ganglia explants, without interfering with their rate of proliferation and differentiation. Similarly, retroviral-mediated ectopic expression of Shh along the chick visual pathway greatly interferes the growth of RGC axons. Upon SHH addition to grown neurites, the intracellular level of cAMP decreases, suggesting that the dampening of growth cone extension mediated by SHH may involve interaction with its receptor Patched which is expressed by RGC. Based on these findings, we propose that Shh expression at the chiasm border defines a constrained pathway within the ventral midline which serves to guide the progression of RGC axons.
Subject(s)
Retinal Ganglion Cells/ultrastructure , Trans-Activators/physiology , Animals , Axons/physiology , Axons/ultrastructure , Cell Differentiation , Cell Division , Chick Embryo , Cyclic AMP/metabolism , Growth Cones/physiology , Growth Cones/ultrastructure , Hedgehog Proteins , In Situ Hybridization , In Vitro Techniques , Models, Biological , Neurites/physiology , Neurites/ultrastructure , Optic Chiasm/embryology , Retinal Ganglion Cells/physiology , Trans-Activators/geneticsABSTRACT
The transcription factor Pax6 is required for eye morphogenesis in humans, mice and insects, and can induce ectopic eye formation in vertebrate and invertebrate organisms. Although the role of Pax6 has intensively been studied, only a limited number of genes have been identified that depend on Pax6 activity for their expression in the mammalian visual system. Using a large-scale in situ hybridization screen approach, we have identified a novel gene expressed in the mouse optic vesicle. This gene, Necab, encodes a putative cytoplasmic Ca(2+)-binding protein and coincides with Pax6 expression pattern in the neural ectoderm of the optic vesicle and in the forebrain pretectum. Remarkably, Necab expression is absent in both structures in Pax6 mutant embryos. By contrast, the optic vesicle-expressed homeobox genes Rx, Six3, Otx2 and Lhx2 do not exhibit an altered expression pattern. Using gain-of-function experiments, we show that Pax6 can induce ectopic expression of Necab, suggesting that Necab is a direct or indirect transcriptional target of Pax6. In addition, we have found that Necab misexpression can induce ectopic expression of the homeobox gene Chx10, a transcription factor implicated in retina development. Taken together, our results provide evidence that Necab is genetically downstream of Pax6 and that it is a part of a signal transduction pathway in retina development.
Subject(s)
Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/physiology , Eye Proteins/genetics , Eye Proteins/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Retina/embryology , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Genes, Homeobox , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Multigene Family , PAX6 Transcription Factor , Paired Box Transcription Factors , Repressor Proteins , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
Pax3 is a member of the paired-box-containing transcription factors. It is expressed in the developing somites, dorsal spinal cord, mesencephalon and neural crest derivatives. Several loss-of-function mutations are correlated with the Splotch phenotype in mice and Waardenburg syndrome in humans. Malformations include a lack of muscle in the limb, a failure of neural tube closure and dysgenesis of numerous neural crest derivatives. In this study we have used embryonic stem (ES) cells to generate a lacZ knock-in into the Pax3 locus. The Pax3 knock-in Splotch allele (Sp(2G)) was used to generate Pax3-deficient ES cells in order to investigate whether, in chimeric embryos, Pax3 is acting cell autonomously in the somites and the neural tube. We found that while Pax3 function is essential for the neuroepithelium and somites, a wild-type environment rescues mutant neural crest cells. In the two affected embryonic tissues, mutant and wild-type cells undergo segregation and do not intermingle. The contribution of mutant cells to the neural tube and the somites displayed temporal differences. All chimeric embryos showed a remarkable contribution of blue cells to the neural tube at all stages analyzed, indicating that the Pax3-deficient cells are not excluded from the neural epithelium while development proceeds. In contrast, this is not true for the paraxial mesoderm. The somite contribution of Pax3(-/-) ES cells becomes less frequent in older embryos as compared to controls with Pax3(+/-) ES cells. We propose that although Pax3 function is related to cell surface properties, its role may differ in various tissues. In fact, apoptosis was found in Pax3-deficient cells of the lateral dermomyotome but not in the neural tube.
Subject(s)
DNA-Binding Proteins/physiology , Neural Crest/cytology , Somites/cytology , Transcription Factors/physiology , Animals , Cell Line , Cell Membrane/metabolism , Cell Membrane/physiology , Cell Movement/physiology , Chick Embryo , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Olfactory Mucosa/cytology , PAX3 Transcription Factor , Paired Box Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The cerebral cortex is composed of a large variety of different neuron types. All cortical neurons, except some interneurons, are born in two proliferative zones, the cortical ventricular (VZ) and subventricular (SVZ) zones. The relative contribution of both proliferative zones to the generation of the diversity of the cortical neurons is not well understood. To further dissect the underlying mechanism, molecular markers specific for the SVZ are required. Towards this end we performed a subtraction of cDNA libraries, generated from E15.5 and E18.5 mouse cerebral cortex. A novel cDNA, Svet1, was cloned which was specifically expressed in the proliferating cells of the SVZ but not the VZ. The VZ is marked by the expression of the Otx1 gene. Later in development, Svet1 and Otx1 were expressed in subsets of cells of upper (II-IV) and deep (V-VI) layers, respectively. In the reeler cortex, where the layers are inverted, Svet1 and Otx1 label precursors of the upper and deeper layers, respectively, in their new location. Interestingly, in the Pax6/small eye mutant, Svet1 activity was abolished in the SVZ and in the upper part of the cortical plate while the Otx1 expression domain remained unchanged. Therefore, using Svet1 and Otx1 as cell-type-specific molecular markers for the upper and deep cortical layers we conclude that the Sey mutation affects predominantly the differentiation of the SVZ cells that fail to migrate into the cortical plate. The abnormality of the SVZ coincides with the absence of upper layer cells in the cortex. Taken together our data suggest that while the specification of deep cortical layers occurs in the ventricular zone, the SVZ is important for the proper specification of upper layers.
Subject(s)
Cerebral Cortex/cytology , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/physiology , Neurons/cytology , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Transcription Factors , Animals , Base Sequence , Cell Adhesion Molecules, Neuronal/genetics , Cell Differentiation , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Cloning, Molecular , DNA, Complementary , Extracellular Matrix Proteins/genetics , Gene Expression , Homeodomain Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mitosis , Molecular Sequence Data , Mutagenesis , Nerve Tissue Proteins/genetics , Neurons/metabolism , Nuclear Proteins/genetics , Occipital Lobe/embryology , Otx Transcription Factors , Reelin Protein , Serine EndopeptidasesABSTRACT
We describe a comparative lung expression analysis of the murine Irx1 and Irx2 genes. At embryonic day 8.5 (E8.5), the Irx1 and Irx2 expression starts in the foregut region, where the laryngo-tracheal groove will form. The expression is prominent in the lung epithelium during glandular development. It declines at the end of the canalicular phase. We further compare the Irx1 and Irx2 expression domains to Gli1, 2, 3 and Mash1. Their homologues in Drosophila melanogaster are known as regulative partners of the iroquois complex. The Irx and Gli genes are coexpressed in the developing lungs at the same time. Their transcripts are not localised in the same cells but adjacent to each other in either mesenchymal or epithelial structures. It is thought that the lung development is regulated by the mesenchymal/epithelial interactions.
Subject(s)
Gene Expression , Homeodomain Proteins/genetics , Lung/embryology , Nerve Tissue Proteins , Repressor Proteins , Transcription Factors/genetics , Xenopus Proteins , Animals , Basic Helix-Loop-Helix Transcription Factors , DNA-Binding Proteins/genetics , Epithelium/embryology , Epithelium/metabolism , Gene Expression Profiling , Genes, Homeobox , Homeodomain Proteins/metabolism , In Situ Hybridization , Kruppel-Like Transcription Factors , Lung/metabolism , Mesoderm/metabolism , Mice , Oncogene Proteins/genetics , Trans-Activators , Transcription Factors/metabolism , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3ABSTRACT
Irx1 and Irx2 are members of the murine Iroquois homeobox (Irx) gene family. In this study, we describe the dynamic expression pattern of these genes during limb development with a focus on digit formation. We further present a comparative expression analysis with Gli genes (Gli1, Gli2, Gli3). Gli1, Gli2, and Gli3 were suggested for candidate regulators of the Irx genes. The expression was studied between E11.5 and E14.5 when the digits are being formed. Irx1 and Irx2 reproduce the developmental program of the digits in time and space and the Irx1 provides an early and excellent marker for this process. Our analysis also indicates that the expression of Irx1, Gli1 and Irx2, Gli2 are relative to each other. In contrast, Gli3 exhibits a different expression pattern.
Subject(s)
Gene Expression , Homeodomain Proteins/genetics , Nerve Tissue Proteins , Repressor Proteins , Toes/embryology , Transcription Factors/genetics , Xenopus Proteins , Animals , DNA-Binding Proteins/genetics , Forelimb/embryology , Forelimb/metabolism , Gene Expression Profiling , Genes, Homeobox , Hindlimb/embryology , Hindlimb/metabolism , Kruppel-Like Transcription Factors , Mice , Oncogene Proteins/genetics , Trans-Activators , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3ABSTRACT
Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1(-/-) mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1(-/-) phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1(-/-) progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.
Subject(s)
Carrier Proteins/physiology , Cation Transport Proteins , Copper/metabolism , Fetus/metabolism , Molecular Chaperones , Neuropeptides/physiology , Animals , Congenital Abnormalities/etiology , Copper Transport Proteins , Female , Fetal Death/etiology , Fetal Growth Retardation/etiology , Homeostasis , Male , Mice , Neuropeptides/deficiency , Phenotype , PregnancyABSTRACT
Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The Spnr mRNA is expressed at high levels in testis, ovary, and brain and is present in these tissues in multiple forms. We have generated a gene trap allele of the murine Spnr, named Spnr(+/GT). Spnr(GT/GT) mutants show a high rate of mortality, reduced weight, and an abnormal clutching reflex. In addition to minor anatomical abnormalities in the brain, males exhibit defects in spermatogenesis that include a thin seminiferous epithelium and disorganization of spermatogenesis. Most of the sperm from mutant males display defects in the flagellum and consequently show decreased motility and transport within the oviducts. Furthermore, sperm from mutant males achieve in vitro fertilization less frequently. Our findings suggest that SPNR plays an important role in normal spermatogenesis and sperm function. Thus, the Spnr(GT/GT) mutant male mouse provides a unique model for some human male infertility cases.
Subject(s)
Microtubule-Associated Proteins/deficiency , Spermatids/physiology , Spermatogenesis/physiology , Alleles , Animals , Base Sequence , Biological Transport, Active , Brain/abnormalities , DNA Primers/genetics , Dendrites/metabolism , Female , Humans , Male , Mice , Microscopy, Electron , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Mutation , Phenotype , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Spermatids/ultrastructure , Spermatogenesis/genetics , Spermatozoa/abnormalities , Testis/abnormalitiesABSTRACT
The molecular mechanisms mediating the retinogenic potential of multipotent retinal progenitor cells (RPCs) are poorly defined. Prior to initiating retinogenesis, RPCs express a limited set of transcription factors implicated in the evolutionary ancient genetic network that initiates eye development. We elucidated the function of one of these factors, Pax6, in the RPCs of the intact developing eye by conditional gene targeting. Upon Pax6 inactivation, the potential of RPCs becomes entirely restricted to only one of the cell fates normally available to RPCs, resulting in the exclusive generation of amacrine interneurons. Our findings demonstrate furthermore that Pax6 directly controls the transcriptional activation of retinogenic bHLH factors that bias subsets of RPCs toward the different retinal cell fates, thereby mediating the full retinogenic potential of RPCs.
Subject(s)
Homeodomain Proteins/metabolism , Retina/embryology , Retina/metabolism , Stem Cells/metabolism , Animals , Cell Line , Cell Lineage , Chick Embryo , Clone Cells/cytology , Eye Proteins , Gene Targeting , Helix-Loop-Helix Motifs , Homeodomain Proteins/genetics , Interneurons/cytology , Interneurons/metabolism , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Morphogenesis , Neurotransmitter Agents/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors , Phenotype , Qa-SNARE Proteins , Repressor Proteins , Retina/cytology , Stem Cells/cytology , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
We describe the sequence and expression pattern of Sp5, a novel member of the vertebrate Sp1 transcription factor gene family which consists of at least five members. This gene family is characterized by a highly conserved domain which is formed by three Zn fingers, which bind to the GC box or the GT/CACC box in the promoter of many genes. These boxes are important cis-acting elements required for the expression of the respective genes. In vitro experiments indicate that the Sp1 transcription factors act by influencing the methylation state of the DNA, or by direct interactions with other promoter specific transcription factors. Despite intensive research, the results from in vivo experiments, including targeted gene inactivation, have been difficult to explain. This may be due to possible redundancies and interferences with other transcription factors of this gene family. Here, we report the isolation of the mouse Sp5 gene, a novel Sp1 homolog. Its sequence indicates that Sp5 is a possible link between Sp1 and the closely related BTEB/KLF gene family. We provide detailed information of its highly dynamic expression pattern during mouse embryogenesis in the developing brain, the spinal cord, the trigeminal ganglia, the somites and additional sites outside the nervous system starting from embryonic day 7.25 (E7.25) up to E10.5.
Subject(s)
Brain/embryology , DNA-Binding Proteins/biosynthesis , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Transcription Factors , Amino Acid Sequence , Animals , Brain/metabolism , DNA Methylation , DNA-Binding Proteins/genetics , Gastrula/metabolism , In Situ Hybridization , Mice , Models, Genetic , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Somites/metabolism , Spinal Cord/metabolism , Time Factors , Tissue Distribution , Trigeminal Ganglion/metabolism , Zinc FingersABSTRACT
Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. We have previously shown that Cdx1 is a direct retinoid target gene, suggesting that Cdx1 may convey some of the effects of retinoid signaling. However, RA appears to be essential for only early stages of Cdx1 expression, and therefore other factors must be involved in maintaining later stages of expression. Based on function and pattern of expression, Wnt family members, in particular Wnt3a, are candidates for regulation of expression of Cdx1. Consistent with this, we confirm prior results which demonstrated that Cdx1 can be directly regulated by Wnt signaling, and identify functional LEF/TCF response motifs essential for this response. We also find that Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and present data demonstrating that Wnt3a and RA synergize strongly to activate Cdx1. Finally, we show that Cdx1 positively regulates its own expression. These data prompt a model whereby retinoid and Wnt signaling function directly and synergistically to initiate Cdx1 expression in the caudal embryo. Expression is then maintained, at least in part, by an autoregulatory mechanism at later stages.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Xenopus Proteins , Zebrafish Proteins , Animals , Base Sequence , CDX2 Transcription Factor , Genes, Reporter , In Situ Hybridization , Mice , Models, Biological , Molecular Sequence Data , Organ Culture Techniques , Plasmids/metabolism , Promoter Regions, Genetic , Protein Structure, Tertiary , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Retinoic Acid/metabolism , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Time Factors , Transfection , Tretinoin/metabolism , Wnt Proteins , Wnt3 Protein , Wnt3A Protein , XenopusABSTRACT
Exogenous retinoic acid (RA) can evoke vertebral homeosis when administered during late gastrulation. These vertebral transformations correlate with alterations of the rostral limit of Hox gene expression in the prevertebrae, suggesting that retinoid signaling regulates the combinatorial expression of Hox genes dictating vertebral identity. Conversely, loss of certain RA receptors (RARs) results in anterior homeotic transformations principally affecting the cervical region. Despite these observations, the relationship between retinoid signaling, somitic Hox expression, and vertebral patterning is poorly understood. The members of the murine Cdx family (Cdx1, Cdx2, and Cdx4) are the homologues of Drosophila caudal and encode homeobox-containing transcription factors. Cdx1 homozygous null mutants exhibit anterior homeotic transformations, some of which are reminiscent of those in RARgamma null offspring. In Cdx1 mutants, these transformations occur concomitant with posteriorized prevertebral expression of certain Hox genes. Cdx1 has recently been demonstrated to be a direct RA target, suggesting an indirect means by which retinoid signaling may impact vertebral patterning. To further investigate this relationship, a complete allelic series of Cdx1-RARgamma mutants was generated and the skeletal phenotype assessed either following normal gestation or after administration of RA. Synergistic interactions between these null alleles were observed in compound mutants, and the full effects of exogenous RA on vertebral morphogenesis required Cdx1. These findings are consistent with a role for RA upstream of Cdx1 as regards axial patterning. However, exogenous RA attenuated several defects inherent to Cdx1 null mutants. This finding, together with the increased phenotypic severity of RARgamma-Cdx1 double null mutants relative to single nulls, suggests that these pathways also function in parallel, likely by converging on common targets.
Subject(s)
Body Patterning/physiology , Homeodomain Proteins/physiology , Receptors, Retinoic Acid/physiology , Vertebrates/embryology , Animals , Base Sequence , DNA Primers , Gene Expression Regulation, Developmental/genetics , Genes, Homeobox , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor gammaABSTRACT
Distinct sets of precursor cells generate the mammalian cerebral cortex. During neurogenesis most precursors are specified to generate a single cell type and only few are multipotent. The cell-intrinsic molecular determinants of these distinct lineages are not known. Here we describe that retroviral transduction of the transcription factor Emx2 in precursors from the cerebral cortex results in a significant increase of large clones that are generated mostly by symmetric cell divisions and contain multiple cell types, comprising neurons and glial cells. Thus, Emx2 is the first cell-intrinsic determinant able to instruct CNS precursors towards a multipotential fate. To evaluate the role of endogenous Emx2 in cortical precursors, we examined cell division in Emx2-/- mice. These analyses further supported the role of endogenous Emx2 in the regulation of symmetric cell divisions in the developing cortex.