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In JoVE (1)
Other Publications (19)
- Genes & Development
- Current Opinion in Neurobiology
- Neuron
- The EMBO Journal
- Developmental Biology
- Development (Cambridge, England)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Neuron
- The EMBO Journal
- Genes & Development
- Cerebral Cortex (New York, N.Y. : 1991)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Molecular and Cellular Neurosciences
- Neural Development
- Molecular and Cellular Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Cerebral Cortex (New York, N.Y. : 1991)
- Neural Development
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Articles by Carol Schuurmans in JoVE
JoVE Neuroscience
Effektiv Gene Delivery i flera CNS territorierna Använda I Utero Elektroporation
1Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, 2Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary
I livmodern elektroporation möjliggör snabb genen leverans i ett rumsligt och tidsmässigt-kontrollerat sätt i utvecklingsländerna centrala nervsystemet (CNS). Här beskriver vi en mycket anpassningsbar i livmodern elektroporering protokoll som kan användas för att leverera uttryck konstruktioner i flera embryonala CNS-domäner, inklusive telencephalon, diencephalon och näthinnan.
Other articles by Carol Schuurmans on PubMed
Divergent Functions of the Proneural Genes Mash1 and Ngn2 in the Specification of Neuronal Subtype Identity
The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity.
Molecular Mechanisms Underlying Cell Fate Specification in the Developing Telencephalon
The cellular properties of neural progenitor cells have been best characterized in the telencephalon, the most complex region of the vertebrate brain. In recent years, several transcription factors, including Mash1, Ngn1/2, Pax6 and Emx1/2, and signaling molecules, such as Notch and bone morphogenetic proteins, have emerged as important players in key areas of telencephalic development. These include the specification of positional identity, the proliferation of neural stem cells and their commitment to a neuronal or glial fate, and the differentiation of layer-specific neuronal phenotypes in the cerebral cortex.
Neurogenin2 Specifies the Connectivity of Thalamic Neurons by Controlling Axon Responsiveness to Intermediate Target Cues
Many lines of evidence indicate that important traits of neuronal phenotype, such as cell body position and neurotransmitter expression, are specified through complex interactions between extrinsic and intrinsic genetic determinants. However, the molecular mechanisms specifying neuronal connectivity are less well understood at the transcriptional level. Here we demonstrate that the bHLH transcription factor Neurogenin2 cell autonomously specifies the projection of thalamic neurons to frontal cortical areas. Unexpectedly, Ngn2 determines the projection of thalamic neurons to specific cortical domains by specifying the responsiveness of their axons to cues encountered in an intermediate target, the ventral telencephalon. Our results thus demonstrate that in parallel to their well-documented proneural function, bHLH transcription factors also contribute to the specification of neuronal connectivity in the mammalian brain.
Sequential Phases of Cortical Specification Involve Neurogenin-dependent and -independent Pathways
Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.
A Screen for Downstream Effectors of Neurogenin2 in the Embryonic Neocortex
Neurogenin (Ngn) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effectors remain poorly defined. We set out to identify Ngn2 effectors in the cortex using a subtractive hybridization screen and identified several regionally expressed genes that were misregulated in Ngn2 and Ngn1;Ngn2 mutants. Included were genes down-regulated in germinal zone progenitors (e.g., Nlgn1, Unc5H4, and Dcc) and in postmitotic neurons in the cortical plate (e.g., Bhlhb5 and NFIB) and subplate (e.g., Mef2c, srGAP3, and protocadherin 9). Further analysis revealed that Ngn2 mutant subplate neurons were misspecified and that thalamocortical afferents (TCAs) that normally target this layer instead inappropriately projected towards the germinal zone. Strikingly, EphA5 and Sema3c, which encode repulsive guidance cues, were down-regulated in the Ngn2 and Ngn1;Ngn2 mutant germinal zones, providing a possible molecular basis for axonal targeting defects. Thus, we identified several new components of the differentiation cascade(s) activated downstream of Ngn1 and Ngn2 and provided novel insights into a new developmental process controlled by these proneural genes. Further analysis of the genes isolated in our screen should provide a fertile basis for understanding the molecular mechanisms underlying corticogenesis.
Conserved and Acquired Features of Neurogenin1 Regulation
The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution.
Members of the Plag Gene Family Are Expressed in Complementary and Overlapping Regions in the Developing Murine Nervous System
In the developing nervous system, cell fate specification and proliferation are tightly coupled events, ensuring the coordinated generation of the appropriate numbers and correct types of neuronal and glial cells. While it has become clear that tumor suppressor genes and oncogenes are key regulators of cell division in tumor cells, their role in normal cellular and developmental processes is less well understood. Here we present a comparative analysis of the expression profiles of the three members of the pleiomorphic adenoma gene (Plag) family, which encode zinc finger transcription factors previously characterized as tumor suppressors (Zac1) or oncogenes (Plag1, Plag-l2). We focused our analysis on the developing nervous system of mouse where we found that the Plag genes were expressed in both unique and overlapping patterns in the central and peripheral nervous systems, and in olfactory and neuroendocrine lineages. Based on their patterns of expression, we suggest that members of the Plag gene family might control cell fate and proliferation decisions in the developing nervous system and propose that deciphering these functions will help to explain why their inappropriate inactivation/activation leads to tumor formation.
Phosphorylation of Neurogenin2 Specifies the Migration Properties and the Dendritic Morphology of Pyramidal Neurons in the Neocortex
The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.
A Cell-autonomous Requirement for the Cell Cycle Regulatory Protein, Rb, in Neuronal Migration
Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon-specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early-born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal-Retzius neurons. In the ventral telencephalon, although calbindin- and Lhx6-expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell-autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell-autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.
P27kip1 Independently Promotes Neuronal Differentiation and Migration in the Cerebral Cortex
The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27(Kip1) plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27(Kip1) involve distinct activities, which are independent of its role in cell cycle regulation. p27(Kip1) promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27(Kip1) promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27(Kip1) plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.
A Role for Proneural Genes in the Maturation of Cortical Progenitor Cells
We showed previously that the proneural genes Neurogenin1 (Ngn1) and Ngn2 are required to specify the phenotypes of early- and not late-born neurons in the neocortex, acting in part through repression of Mash1, a third cortically expressed proneural gene. The precise timing of Ngn1/2 specification activity was unexpected given these genes are expressed throughout cortical development, prompting us to search for a later function. Here we reveal that Ngn2 and Mash1 are expressed in a dynamic fashion, acquiring a cell cycle-biased, nonoverlapping distribution, with preferential expression in prospective basal progenitors, during mid corticogenesis. We also identified a new function for Ngn2 during this latter period, demonstrating that it is required to regulate the transit of cortical progenitors from the ventricular zone (VZ) to the subventricular zone. Notably, Ngn2 regulates progenitor maturation at least in part through repression of Mash1 as misexpression of Mash1 strongly enhanced progenitor cell exit from the VZ. Significantly, the ability of Mash1 to promote progenitor cell maturation occurred independently of its ability to respecify cortical cells and is thus a novel function for Mash1. Taken together, these data support a model whereby Ngn2 and Mash1 function together to regulate the zonal distribution of progenitors in the developing neocortex.
Zac1 Promotes a Müller Glial Cell Fate and Interferes with Retinal Ganglion Cell Differentiation in Xenopus Retina
The timing of cell cycle exit is tightly linked to cell fate specification in the developing retina. Accordingly, several tumor suppressor genes, which are key regulators of cell cycle exit in cancer cells, play critical roles in retinogenesis. Here we investigated the role of Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, in retinal development. Strikingly, in gain-of-function assays in Xenopus, mouse Zac1 promotes proliferation and apoptosis at an intermediate stage of retinogenesis. Zac1 also influences cell fate decisions, preferentially promoting the differentiation of tumor-like clusters of abnormal neuronal cells in the ganglion cell layer, as well as inducing the formation of supernumerary Müller glial cells at the expense of other cell types. Thus Zac1 has the capacity to influence cell cycle exit, and cell fate specification and differentiation decisions by retinal progenitors, suggesting that further functional studies will uncover new insights into how retinogenesis is regulated.
Validating in Utero Electroporation for the Rapid Analysis of Gene Regulatory Elements in the Murine Telencephalon
With the ultimate goal of understanding how genetic modules have evolved in the telencephalon, we set out to modernize the functional analysis of cross-species cis-regulatory elements in mouse. In utero electroporation is rapidly replacing transgenesis as the method of choice for gain- and loss-of-function studies in the murine telencephalon, but the application of this technique to the analysis of transcriptional regulation has yet to be fully explored and exploited. To empirically define the developmental stages required to target specific populations of neurons in the dorsal telencephalon, or pallium, which gives rise to the neocortex in mouse, we performed a temporal and spatial analysis of the migratory properties of electroporated versus birth-dated cells. Next, we compared the activities of two known Ngn2 enhancers via transgenesis and in utero electroporation, demonstrating that the latter technique more faithfully reports the endogenous telencephalic expression pattern observed in an Ngn2lacZ knock-in line. Finally, we used this approach to test the telencephalic activities of a series of deletion constructs comprised of the zebrafish ER81 upstream regulatory region, allowing us to identify a previously uncharacterized enhancer that displays cross-species activity in the murine piriform cortex and lateral neocortex, yet not in more medial domains of the forebrain. Taken together, our data supports the contention that in utero technology can be exploited to rapidly examine the architecture and evolution of pallial-specific cis-regulatory elements.
STAT5A/B Activity is Required in the Developing Forebrain and Spinal Cord
Formation of the CNS requires the coordination and integration of processes such as cell proliferation, neuronal differentiation, neuronal migration, axon tract formation and synaptogenesis, all of which must occur at precise times and places during development. Although growth factors are known to play a role in regulating many of these processes, very little is known of the signaling events immediately downstream of ligand-receptor interactions in the developing CNS. Here we present evidence that STAT5, an important mediator of cytokine signaling, is required for some aspects of CNS development. We show that phosphorylated and hence activated forms of STAT5 (pSTAT5) are expressed in a temporally restricted manner in a subset of early-born telencephalic neurons and axons. Accordingly, Stat5 mutants have reduced numbers of interneurons in the cortical marginal zone, suggestive of migration defects. Moreover, corticofugal axons develop aberrantly in Stat5 mutants, indicative of a role for pSTAT5 in axon guidance. Notably, pSTAT5 is also expressed in commissural axons in the embryonic spinal cord, where it is also required for their guidance. Taken together, we provide the first evidence that STAT5 is a key effector molecule in the developing mammalian CNS.
Zac1 Functions Through TGFbetaII to Negatively Regulate Cell Number in the Developing Retina
Organs are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the Zac1 zinc finger transcription factor and that encoding the cytokine TGFbetaII, in the developing retina.
Basic Helix-loop-helix Transcription Factors Cooperate to Specify a Cortical Projection Neuron Identity
Several transcription factors are essential determinants of a cortical projection neuron identity, but their mode of action (instructive versus permissive) and downstream genetic cascades remain poorly defined. Here, we demonstrate that the proneural basic helix-loop-helix (bHLH) gene Ngn2 instructs a partial cortical identity when misexpressed in ventral telencephalic progenitors, inducing ectopic marker expression in a defined temporal sequence, including early (24 h; Nscl2), intermediate (48 h; BhlhB5), and late (72 h; NeuroD, NeuroD2, Math2, and Tbr1) target genes. Strikingly, cortical gene expression was much more rapidly induced by Ngn2 in the dorsal telencephalon (within 12 to 24 h). We identify the bHLH gene Math3 as a dorsally restricted Ngn2 transcriptional target and cofactor, which synergizes with Ngn2 to accelerate target gene transcription in the cortex. Using a novel in vivo luciferase assay, we show that Ngn2 generates only approximately 60% of the transcriptional drive in ventral versus dorsal telencephalic domains, an activity that is augmented by Math3, providing a mechanistic basis for regional differences in Ngn2 function. Cortical bHLH genes thus cooperate to control transcriptional strength, thereby temporally coordinating downstream gene expression.
Neural Stem Cell Self-renewal Requires the Mrj Co-chaperone
The Mrj co-chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj(-/-) embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin-walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down-regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj(-/-) neuroepithelial cells. Furthermore, Mrj(-/-) neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self-renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi-1, a self-renewal protein. Furthermore, unlike in Mrj(-/-) placentas, intermediate filament-containing aggregates do not accumulate in Mrj(-/-) neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self-renewal.
Ascl1 Participates in Cajal-Retzius Cell Development in the Neocortex
Cajal-Retzius cells are essential pioneer neurons that guide neuronal migration in the developing neocortex. During development, Cajal-Retzius cells arise from distinct progenitor domains that line the margins of the dorsal telencephalon, or pallium. Here, we show that the proneural gene Ascl1 is expressed in Cajal-Retzius cell progenitors in the pallial septum, ventral pallium, and cortical hem. Using a short-term lineage trace, we demonstrate that it is primarily the Ascl1-expressing progenitors in the pallial septum and ventral pallium that differentiate into Cajal-Retzius cells. Accordingly, we found a small, albeit significant reduction in the number of Reelin(+) and Trp73(+) Cajal-Retzius cells in the Ascl1(-/-) neocortex. Conversely, using a gain-of-function approach, we found that Ascl1 induces the expression of both Reelin, a Cajal-Retzius marker, and Tbr1, a marker of pallial-derived neurons, in a subset of early-stage pallial progenitors, an activity that declines over developmental time. Taken together, our data indicate that the proneural gene Ascl1 is required and sufficient to promote the differentiation of a subset of Cajal-Retzius neurons during early neocortical development. Notably, this is the first study that reports a function for Ascl1 in the pallium, as this gene is best known for its role in specifying subpallial neuronal identities.
Zac1 Plays a Key Role in the Development of Specific Neuronal Subsets in the Mouse Cerebellum
The cerebellum is composed of a diverse array of neuronal subtypes. Here we have used a candidate approach to identify Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, as a new player in the transcriptional network required for the development of a specific subset of cerebellar nuclei and a population of Golgi cells in the cerebellar cortex.
