Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding.

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

Focused ultrasound as a novel strategy for noninvasive gene delivery to retinal Müller glia.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

Capicua regulates neural stem cell proliferation and lineage specification through control of Ets factors.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.

Zebrin II Is Ectopically Expressed in Microglia in the Cerebellum of Neurogenin 2 Null Mice.

Zebrin II/aldolase C expression in the normal cerebellum is restricted to a Purkinje cell subset and is the canonical marker for stripes and zones. This spatial restriction has been confirmed in over 30 species of mammals, birds, fish, etc. In a transgenic mouse model in which the Neurogenin 2 gene has been disrupted (Neurog2-/-), the cerebellum is smaller than normal and Purkinje cell dendrites are disordered, but the basic zone and stripe architecture is preserved. Here, we show that in the Neurog2-/- mouse, in addition to the normal Purkinje cell expression, zebrin II is also expressed in a population of cells with a morphology characteristic of microglia. This identity was confirmed by double immunohistochemistry for zebrin II and the microglial marker, Iba1. The expression of zebrin II in cerebellar microglia is not restricted by zone or stripe or lamina. A second zone and stripe marker, PLCß4, does not show the same ectopic expression. When microglia are compared in control vs. Neurog2-/- mice, no difference is seen in apparent number or distribution, suggesting that the ectopic zebrin II immunoreactivity in Neurog2-/- cerebellum reflects an ectopic expression rather than the invasion of a new population of microglia from the periphery. This ectopic expression of zebrin II in microglia is unique as it is not seen in numerous other models of cerebellar disruption, such as in Acp2-/- mice and in human pontocerebellar hypoplasia. The upregulation of zebrin II in microglia is thus specific to the disruption of Neurog2 downstream pathways, rather than a generic response to a cerebellar disruption.

Plag1 and Plagl2 have overlapping and distinct functions in telencephalic development.

The Plag gene family has three members; Plagl1/Zac1, which is a tumor suppressor gene, and Plag1 and Plagl2, which are proto-oncogenes. All three genes are known to be expressed in embryonic neural progenitors, and Zac1 regulates proliferation, neuronal differentiation and migration in the developing neocortex. Here we examined the functions of Plag1 and Plagl2 in neocortical development. We first attempted, and were unable to generate, E12.5 Plag1;Plagl2 double mutants, indicating that at least one Plag1 or Plagl2 gene copy is required for embryonic survival. We therefore focused on single mutants, revealing a telencephalic patterning defect in E12.5 Plagl2 mutants and a proliferation/differentiation defect in Plag1 mutant neocortices. Specifically, the ventral pallium, a dorsal telencephalic territory, expands into the ventral telencephalon in Plagl2 mutants. In contrast, Plag1 mutants develop normal regional territories, but neocortical progenitors proliferate less and instead produce more neurons. Finally, in gain-of-function studies, both Plag1 and Plagl2 reduce neurogenesis and increase BrdU-uptake, indicative of enhanced proliferation, but while Plagl2 effects on proliferation are more immediate, Plag1 effects are delayed. Taken together, we found that the Plag proto-oncogenes genes are essential regulators of neocortical development and although Plag1 and Plagl2 functions are similar, they do not entirely overlap. This article has an associated First Person interview with the first author of the paper.

A non-canonical role for the proneural gene Neurog1 as a negative regulator of neocortical neurogenesis.

Neural progenitors undergo temporal identity transitions to sequentially generate the neuronal and glial cells that make up the mature brain. Proneural genes have well-characterised roles in promoting neural cell differentiation and subtype specification, but they also regulate the timing of identity transitions through poorly understood mechanisms. Here, we investigated how the highly related proneural genes Neurog1 and Neurog2 interact to control the timing of neocortical neurogenesis. We found that Neurog1 acts in an atypical fashion as it is required to suppress rather than promote neuronal differentiation in early corticogenesis. In Neurog1-/- neocortices, early born neurons differentiate in excess, whereas, in vitro, Neurog1-/- progenitors have a decreased propensity to proliferate and form neurospheres. Instead, Neurog1-/- progenitors preferentially generate neurons, a phenotype restricted to the Neurog2+ progenitor pool. Mechanistically, Neurog1 and Neurog2 heterodimerise, and while Neurog1 and Neurog2 individually promote neurogenesis, misexpression together blocks this effect. Finally, Neurog1 is also required to induce the expression of neurogenic factors (Dll1 and Hes5) and to repress the expression of neuronal differentiation genes (Fezf2 and Neurod6). Neurog1 thus employs different mechanisms to temper the pace of early neocortical neurogenesis.

Hamartoma-like lesions in the mouse retina: an animal model of Pten hamartoma tumour syndrome.

PTEN hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with PTEN germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized 'normal' cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in Pten, together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a Pten conditional knockout (cKO) using a retinal-specific Pax6::Cre driver that leads to a nearly complete deletion of Pten in the peripheral retina but produces a mosaic of 'wild-type' and Pten cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the Pten cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies.This article has an associated First Person interview with the first author of the paper.

Neurog2 and Ascl1 together regulate a postmitotic derepression circuit to govern laminar fate specification in the murine neocortex.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.

Regulation of Brn3b by DLX1 and DLX2 is required for retinal ganglion cell differentiation in the vertebrate retina.

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.

Pten Regulates Retinal Amacrine Cell Number by Modulating Akt, Tgfß, and Erk Signaling.

UNLABELLED: All tissues are genetically programmed to acquire an optimal size that is defined by total cell number and individual cellular dimensions. The retina contains stereotyped proportions of one glial and six neuronal cell types that are generated in overlapping waves. How multipotent retinal progenitors know when to switch from making one cell type to the next so that appropriate numbers of each cell type are generated is poorly understood. Pten is a phosphatase that controls progenitor cell proliferation and differentiation in several lineages. Here, using a conditional loss-of-function strategy, we found that Pten regulates retinal cell division and is required to produce the full complement of rod photoreceptors and amacrine cells in mouse. We focused on amacrine cell number control, identifying three downstream Pten effector pathways. First, phosphoinositide 3-kinase/Akt signaling is hyperactivated in Pten conditional knock-out (cKO) retinas, and misexpression of constitutively active Akt (Akt-CA) in retinal explants phenocopies the reduction in amacrine cell production observed in Pten cKOs. Second, Akt-CA activates Tgfß signaling in retinal explants, which is a negative feedback pathway for amacrine cell production. Accordingly, Tgfß signaling is elevated in Pten cKO retinas, and epistatic analyses placed Pten downstream of TgfßRII in amacrine cell number control. Finally, Pten regulates Raf/Mek/Erk signaling levels to promote the differentiation of all amacrine cell subtypes, which are each reduced in number in Pten cKOs. Pten is thus a positive regulator of amacrine cell production, acting via multiple downstream pathways, highlighting its diverse actions as a mediator of cell number control. SIGNIFICANCE STATEMENT: Despite the importance of size for optimal organ function, how individual cell types are generated in correct proportions is poorly understood. There are several ways to control cell number, including readouts of organ function (e.g., secreted hormones reach functional levels when enough cells are made) or counting of cell divisions or cell number. The latter applies to the retina, where cell number is regulated by negative feedback signals, which arrest differentiation of particular cell types at threshold levels. Herein, we show that Pten is a critical regulator of amacrine cell number in the retina, acting via multiple downstream pathways. Our studies provide molecular insights into how PTEN loss in humans may lead to uncontrolled cell division in several pathological conditions.

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.

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 Ascl1's 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 Ascl1's 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 Neurog2's 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.

Gene expression is dynamically regulated in retinal progenitor cells prior to and during overt cellular differentiation.

The retina is comprised of one glial and six neuronal populations that are generated from a multipotent pool of retinal progenitor cells (RPCs) during development. To give rise to these different cell types, RPCs undergo temporal identity transitions, displaying distinct gene expression profiles at different stages of differentiation. Little, however, is known about temporal differences in RPC identities prior to the onset of overt cellular differentiation, during the period when a retinal identity is gradually acquired. Here we examined the sequential onset of expression of regional markers (i.e., homeodomain transcription factors) and cell fate determinants (i.e., basic-helix-loop-helix transcription factors and neurogenic genes) in RPCs from the earliest appearance of a morphologically-distinct retina. By performing a comparative analysis of the expression of a panel of 27 homeodomain, basic-helix-loop-helix and Notch pathway genes between embryonic day (E) 8.75 and postnatal day (P) 9, we identified six distinct RPC molecular profiles. At E8.75, the earliest stage assayed, murine RPCs expressed five homeodomain genes and a single neurogenic gene (Pax6, Six3, Six6, Rx, Otx2, Hes1). This early gene expression profile was remarkably similar to that of 'early' RPCs in the amphibian ciliary marginal zone (CMZ), where RPCs are compartmentalised according to developmental stage, and homologs of Pax6, Six3 and Rx are expressed in the 'early' stem cell zone. As development proceeds, expression of additional homeodomain, bHLH and neurogenic genes was gradually initiated in murine RPCs, allowing distinct genetic profiles to also be defined at E9.5, E10.5, E12.5, E15.5 and P0. In addition, RPCs in the postnatal ciliary margin, where retinal stem cells are retained throughout life, displayed a unique molecular signature, expressing all of the early-onset genes as well as several late-onset markers, indicative of a 'mixed' temporal identity. Taken together, the identification of temporal differences in gene expression in mammalian RPCs during pre-neurogenic developmental stages leads to new insights into how regional identities are progressively acquired during development, while comparisons at later stages highlight the dynamic nature of gene expression in temporally distinct RPC pools.

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.

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.