Rajiv Dixit

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2–Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2–E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2–Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2–E47 heterodimerization by GSK3 is a central component of the neuronal differentiation “clock” that coordinates the timing and tempo of neocortical neurogenesis in mouse.

RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis.

Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1s fate specification properties in murine cortical progenitors–at higher RAS/ERK levels, Ascl1+ progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1s GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

Neurog1 and Neurog2 Control Two Waves of Neuronal Differentiation in the Piriform Cortex

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genes Neurog1 and Neurog2 are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find that Neurog1 has a unique early role in reducing CR cell neurogenesis by tempering Neurog2s proneural activity. In addition, Neurog1 and Neurog2 have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase, Neurog1 and Neurog2 are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation of Trp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement. Neurog1 and Neurog2 thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.

Neurog2 Simultaneously Activates and Represses Alternative Gene Expression Programs in the Developing Neocortex

Progenitor cells undergo a series of stable identity transitions on their way to becoming fully differentiated cells with unique identities. Each cellular transition requires that new sets of genes are expressed, while alternative genetic programs are concurrently repressed. Here, we investigated how the proneural gene Neurog2 simultaneously activates and represses alternative gene expression programs in the developing neocortex. By comparing the activities of transcriptional activator (Neurog2-VP16) and repressor (Neurog2-EnR) fusions to wild-type Neurog2, we first demonstrate that Neurog2 functions as an activator to both extinguish Pax6 expression in radial glial cells and initiate Tbr2 expression in intermediate neuronal progenitors. Similarly, we show that Neurog2 functions as an activator to promote the differentiation of neurons with a dorsal telencephalic (i.e., neocortical) identity and to block a ventral fate, identifying 2 Neurog2-regulated transcriptional programs involved in the latter. First, we show that the Neurog2-transcriptional target Tbr2 is a direct transcriptional repressor of the ventral gene Ebf1. Secondly, we demonstrate that Neurog2 indirectly turns off Etv1 expression, which in turn indirectly regulates the expression of the ventral proneural gene Ascl1. Neurog2 thus activates several genetic off-switches, each with distinct transcriptional targets, revealing an unappreciated level of specificity for how Neurog2 prevents inappropriate gene expression during neocortical development.

Cell-Type Specific Roles for PTEN in Establishing a Functional Retinal Architecture

Background The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture. Methodology/Principal Findings In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam. Conclusions/Significance We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.

Zac1 plays a key role in the development of specific neuronal subsets in the mouse cerebellum

BackgroundThe 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.ResultsWe found that Zac1 has a complex expression profile in the developing cerebellum, including in two proliferating progenitor populations; the cerebellar ventricular zone and the external granular layer overlying posterior cerebellar lobules IX and X. Zac1 is also expressed in some postmitotic cerebellar neurons, including a subset of GABAergic interneurons in the medial cerebellar nuclei. Notably, GABAergic interneurons in the cerebellar nuclei are derived from the cerebellar ventricular zone, where Zac1 is also expressed, consistent with a lineage relationship between these two Zac1+ populations. Zac1 is also expressed in a small subset of cells in the posterior vermis, including some neurogranin-immunoreactive (NG+) Golgi cells, which, based on short-term birthdating, are derived from the EGL, where Zac1 is also expressed. However, Zac1+ cells and NG+ Golgi cells in the cerebellar cortex also display unique properties, as they are generated within different, albeit overlapping, time windows. Finally, consistent with the expression profile of Zac1, two conspicuous abnormalities were found in the cerebellum of Zac1 null mice: the medial cerebellar nuclei, and not the others, were significantly reduced in size; and the number of Golgi cells in cerebellar lobule IX was reduced by approximately 60% compared to wild-type littermates.ConclusionsThe data presented here indicate that the tumor suppressor gene Zac1 is expressed in a complex fashion in the developing cerebellum, including in two dividing progenitor populations and in specific subsets of postmitotic neurons, including Golgi cells and GABAergic neurons in the medial nuclei, which require Zac1 for their differentiation. We thus conclude that Zac1 is a critical regulator of normal cerebellar development, adding a new transcriptional regulator to the growing list of factors involved in generating neuronal diversity in the developing cerebellum.

Efficient gene delivery into multiple CNS territories using in utero electroporation.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described(6). Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells. To date, in utero electroporation has been most widely used for the analysis of neocortical development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3(rd) ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

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.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Zac1 Regulates the Differentiation and Migration of Neocortical Neurons via Pac1

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. SIGNIFICANCE STATEMENT Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader–Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.