Coordination of Neuron Production in Mouse and Human Cerebral Cortex by the Homolog of Drosophila Mastermind Protein.

The coordination of progenitor self-renewal, neuronal production, and migration is essential to the normal development and evolution of the cerebral cortex. Numerous studies have shown that the Notch, Wnt/beta-catenin, and Neurogenin pathways contribute separately to progenitor expansion, neurogenesis, and neuronal migration, but it is unknown how these signals are coordinated. In vitro studies suggested that the mastermind-like 1 (MAML1) gene, homologue of the Drosophila mastermind, plays a role in coordinating the aforementioned signaling pathways, yet its role during cortical development remains largely unknown. Here we show that ectopic expression of dominant-negative MAML (dnMAML) causes exuberant neuronal production in the mouse cortex without disrupting neuronal migration. Comparing the transcriptional consequences of dnMAML and Neurog2 ectopic expression revealed a complex genetic network controlling the balance of progenitor expansion versus neuronal production. Manipulation of MAML and Neurog2 in cultured human cerebral stem cells exposed interactions with the same set of signaling pathways. Thus, our data suggest that evolutionary changes that affect the timing, tempo, and density of successive neuronal layers of the small lissencephalic rodent and large convoluted primate cerebral cortex depend on similar molecular mechanisms that act from the earliest developmental stages.

Transcriptomics of critical period of visual cortical plasticity in mice.

In contrast to the prenatal development of the cerebral cortex, when cell production, migration, and layer formation dominate, development after birth involves more subtle processes, such as activity-dependent plasticity that includes refinement of synaptic connectivity by its stabilization and elimination. In the present study, we use RNA-seq with high spatial resolution to examine differential gene expression across layers 2/3, 4, 5, and 6 of the mouse visual cortex before the onset of the critical period of plasticity [postnatal day 5 (P5)], at its peak (P26), and at the mature stage (P180) and compare it with the prefrontal association area. We find that, although genes involved in early developmental events such as cell division, neuronal migration, and axon guidance are still prominent at P5, their expression largely terminates by P26, when synaptic plasticity and associated signaling pathways become enriched. Unexpectedly, the gene expression profile was similar in both areas at this age, suggesting that activity-dependent plasticity between visual and association cortices are subject to the same genetic constraints. Although gene expression changes follow similar paths until P26, we have identified 30 regionally enriched genes that are prominent during the critical period. At P180, we identified several hundred differentially expressed gene isoforms despite subsiding levels of gene expression differences. This result indicates that, once genetic developmental programs cease, the remaining morphogenetic processes may depend on posttranslational events.

Evolutionary genomics. Evolutionary changes in promoter and enhancer activity during human corticogenesis.

Human higher cognition is attributed to the evolutionary expansion and elaboration of the human cerebral cortex. However, the genetic mechanisms contributing to these developmental changes are poorly understood. We used comparative epigenetic profiling of human, rhesus macaque, and mouse corticogenesis to identify promoters and enhancers that have gained activity in humans. These gains are significantly enriched in modules of coexpressed genes in the cortex that function in neuronal proliferation, migration, and cortical-map organization. Gain-enriched modules also showed correlated gene expression patterns and similar transcription factor binding site enrichments in promoters and enhancers, suggesting that they are connected by common regulatory mechanisms. Our results reveal coordinated patterns of potential regulatory changes associated with conserved developmental processes during corticogenesis, providing insight into human cortical evolution.

Diversity of cortical interneurons in primates: the role of the dorsal proliferative niche.

Evolutionary elaboration of tissues starts with changes in the genome and location of the stem cells. For example, GABAergic interneurons of the mammalian neocortex are generated in the ventral telencephalon and migrate tangentially to the neocortex, in contrast to the projection neurons originating in the ventricular/subventricular zone (VZ/SVZ) of the dorsal telencephalon. In human and nonhuman primates, evidence suggests that an additional subset of neocortical GABAergic interneurons is generated in the cortical VZ and a proliferative niche, the outer SVZ. The origin, magnitude, and significance of this species-specific difference are not known. We use a battery of assays applicable to the human, monkey, and mouse organotypic cultures and supravital tissue to identify neuronal progenitors in the cortical VZ/SVZ niche that produce a subset of GABAergic interneurons. Our findings suggest that these progenitors constitute an evolutionary novelty contributing to the elaboration of higher cognitive functions in primates.

The evolution of lineage-specific regulatory activities in the human embryonic limb.

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

POU-III transcription factors (Brn1, Brn2, and Oct6) influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex.

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.

Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA sequencing.

Characterizing the genetic programs that specify development and evolution of the cerebral cortex is a central challenge in neuroscience. Stem cells in the transient embryonic ventricular and subventricular zones generate neurons that migrate across the intermediate zone to the overlying cortical plate, where they differentiate and form the neocortex. It is clear that not one but a multitude of molecular pathways are necessary to progress through each cellular milestone, yet the underlying transcriptional programs remain unknown. Here, we apply differential transcriptome analysis on microscopically isolated cell populations, to define five transcriptional programs that represent each transient embryonic zone and the progression between these zones. The five transcriptional programs contain largely uncharacterized genes in addition to transcripts necessary for stem cell maintenance, neurogenesis, migration, and differentiation. Additionally, we found intergenic transcriptionally active regions that possibly encode unique zone-specific transcripts. Finally, we present a high-resolution transcriptome map of transient zones in the embryonic mouse forebrain.

Timing of neurogenesis is a determinant of olfactory circuitry.

An odorant receptor map in mammals that is constructed by the glomerular coalescence of sensory neuron axons in the olfactory bulb is essential for proper odor information processing. How this map is linked with olfactory cortex is unknown. Using a battery of methods, including various markers of cell division in combination with tracers of neuronal connections and time-lapse live imaging, we found that early- and late-generated mouse mitral cells became differentially distributed in the dorsal and ventral subdivisions of the odorant receptor map. In addition, the late-generated mitral cells extended substantially stronger projections to the olfactory tubercle than did the early-generated cells. Together, these data indicate that the odorant receptor map is developmentally linked to the olfactory cortices in part by the birthdate of mitral cells. Thus, different olfactory cortical regions become involved in processing information from distinct regions of the odorant receptor map.

Decision by division: making cortical maps.

In the past three decades, mounting evidence has revealed that specification of the basic cortical neuronal classes starts at the time of their final mitotic divisions in the embryonic proliferative zones. This early cell determination continues during the migration of the newborn neurons across the widening cerebral wall, and it is in the cortical plate that they attain their final positions and establish species-specific cytoarchitectonic areas. Here, the development and evolutionary expansion of the neocortex is viewed in the context of the radial unit and protomap hypotheses. A broad spectrum of findings gave insight into the pathogenesis of cortical malformations and the biological bases for the evolution of the modern human neocortex. We examine the history and evidence behind the concept of early specification of neurons and provide the latest compendium of genes and signaling molecules involved in neuronal fate determination and specification.

New horizons for the subplate zone and its pioneering neurons.

Transitional neuronal layers are a hallmark of the prenatal and neonatal brain yet their contribution to the development of higher functions is not clear. Evidence accumulated over the last 3 decades shows that early connectivity and functional activity start in a transitional layer called the subplate zone (SPZ). The SPZ is host to a heterogeneous population of neurons and its evolutionary complexity peaked in the human brain. In this issue of Cerebral Cortex, three reports (Hoerder-Suabedissen et al., 2008; McKellar and Shatz, 2008; Moore et al., 2008) present new data and evidence in three species (mouse, rat, human) as to the function of the SPZ, to the heterogeneity of its cellular composition, and to the genetic basis of its development.

Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling.

Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular "antennae" critically required to modulate ALNP behavior.

The stumpy gene is required for mammalian ciliogenesis.

Cilia are present on nearly all cell types in mammals and perform remarkably diverse functions. However, the mechanisms underlying ciliogenesis are unclear. Here, we cloned a previously uncharacterized highly conserved gene, stumpy, located on mouse chromosome 7. Stumpy was ubiquitously expressed, and conditional loss in mouse resulted in complete penetrance of perinatal hydrocephalus (HC) and severe polycystic kidney disease (PKD). We found that cilia in stumpy mutant brain and kidney cells were absent or markedly deformed, resulting in defective flow of cerebrospinal fluid. Stumpy colocalized with ciliary basal bodies, physically interacted with gamma-tubulin, and was present along ciliary axonemes, suggesting that stumpy plays a role in ciliary axoneme extension. Therefore, stumpy is essential for ciliogenesis and may be involved in the pathogenesis of human congenital malformations such as HC and PKD.

Nestin-CreER mice reveal DNA synthesis by nonapoptotic neurons following cerebral ischemia hypoxia.

The standard method of detecting neurogenesis uses bromodeoxyuridine (BrdU) to label DNA synthesis followed by double labeling with neuronal markers. However, DNA synthesis may occur in events unrelated to neurogenesis including aneuploidy and abortive cell cycle reentry. Hence, it is important to confirm neurogenesis with methods other than BrdU incorporation. To this end, we have generated transgenic nestin-CreER mice that express tamoxifen-inducible Cre recombinase under the control of a nestin enhancer. When crossed with a ubiquitous Enhanced Green Fluorescent Protein (EGFP)-Cre-reporter line, the bitransgenic animals can reveal the nestin-positive progenitors and their progeny with EGFP after tamoxifen induction. This system has many applications including visualization of embryonic neural progenitors, detection of postnatally transformed radial glial cells, and labeling adult neural progenitors in the subventricular zone (SVZ). To examine the contribution of SVZ progenitors to cell replacement after stroke, tamoxifen-induced mice were challenged with focal ischemia or combined ischemia-hypoxia followed by BrdU injection. This analysis revealed only very few EGFP-positive cells outside the SVZ after focal ischemia but robust DNA synthesis by hippocampal neurons without immediate cell death following ischemia-hypoxia. These results suggest that the nestin-CreER system is a useful tool for detecting embryonic and adult neurogensis. They also confirm the existence of nonproliferative DNA synthesis by old neurons after experimental brain injury.

Translocation of synaptically connected interneurons across the dentate gyrus of the early postnatal rat hippocampus.

Most neurons in the developing mammalian brain migrate to their final destinations by translocation of the cell nucleus within their leading process and immature bipolar body that is devoid of synaptic connections. Here, we used a combination of immunohistochemistry at light- and electron-microscopic (EM) levels and time-lapse imaging in slice cultures to analyze migration of synaptically interconnected, cholecystokinin-immunopositive [CCK(+)] interneurons in the dentate gyrus in the rat hippocampus during early postnatal ages. We observed dynamic morphogenetic transformation of the CCK(+) interneurons, from a horizontal bipolar shape situated in the molecular layer, through a transitional triangular and then vertical bipolar form that they acquire while traversing the granular layer to finally assume an adult-like pyramidal-shaped morphology on entering the hilus. Immunostaining with anti-glial fibrillary acidic protein and three-dimensional reconstructions from serial EM images indicate that, unlike granule cells, which migrate from the hilus to the granular layer, interneurons traverse this layer in the opposite direction without apparent surface-mediated guidance of the radial glial cells. Importantly, the somas, dendrites, and axons of the CCK(+) transitional forms maintain old and acquire new synaptic contacts while migrating across the dentate plate. The migration of synaptically interconnected neurons that may occur in response to local functional demand represents a novel mode of cell movement and form of neuroplasticity.

Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones.

The proliferative ventricular zone (VZ) is the main source of projection neurons for the overlying cerebral neocortex. The number and diversity of neocortical neurons is determined, in part, by factors controlling the proliferation and specification of VZ cells during embryonic development. We used a variety of methods, including in utero electroporation with specific cellular markers, computer-assisted serial EM cell reconstruction, and time-lapse multiphoton imaging to characterize the molecular and morphological characteristics of the VZ constituents and to capture their behavior during cell division. Our analyses reveal at least two types of dividing cells in the VZ: (1) radial glial cells (RGCs) that span the entire neocortical wall and maintain contact both at the ventricular and pial surfaces throughout mitotic division, and (2) short neural precursors (SNPs) that possess a ventricular endfoot and a basal process of variable length that is retracted during mitotic division. These two precursor cell classes are present concomitantly in the VZ, but their relative number changes over the course of cortical neurogenesis. Moreover, the SNPs are morphologically, ultrastructurally and molecularly distinct from dividing RGCs. For example, SNPs are marked by their preferential expression of the tubulin alpha-1 promoter whereas RGCs instead express the glutamate-aspartate transporter and brain lipid binding protein promoters. In contrast to recent studies that suggest that RGCs are the sole type of VZ precursor, the present study indicates that the VZ in murine dorsal telencephalon is similar to that in human and nonhuman primates, because it contains multiple types of neuronal precursors.

Developmental expression of matrix metalloproteinases 2 and 9 and their potential role in the histogenesis of the cerebellar cortex.

The development of the cerebellar cortex depends on intrinsic genetic programs and orchestrated cell-cell/cell-matrix interactions. Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in these interactions. MMP-2 and MMP-9 are involved in diverse neuronal functions including migration, process extension, and synaptic plasticity. We investigated the spatiotemporal pattern of expression/activity of MMP-2/MMP-9 in the developing cerebellum and their role in the histogenesis of the cerebellar cortex. The levels of transcripts of MMP-2/MMP-9 were measured with real-time quantitative polymerase chain reaction. An initial decrease in MMP-2/MMP-9 transcripts was observed between postnatal days 3 (PD3) and PD6, and the mRNA levels remained relatively constant thereafter. Zymographic analysis revealed that the expression/activity of MMP-2/MMP-9 persisted longer than their transcripts; the downregulation occurred around PD9, suggesting a mechanism of translational or post-translational regulation. The gelatinase activity was localized in the external granule layer (EGL) and the internal granule layer during PD3-PD12. The immunoreactivity of MMP-2 was mainly localized in the EGL, the Bergmann glial fibers, and the Purkinje cell layer (PCL), whereas MMP-9 immunoreactivity was detected intensively in the PCL and the extracellular space of the molecular layer. Expression of MMP-9 was relatively weak in the EGL. The immunoreactivity of MMP-2/MMP-9 became undetectable after PD21. A similar expression pattern of MMP-2/MMP-9 was observed in organotypic cerebellar slice cultures. Exposure of organotypic slices to a specific MMP-2/MMP-9 inhibitor significantly increased the thickness of the EGL and concurrently decreased the number of migrating granule neurons in the molecular layer. Thus, MMP-2 and MMP-9 play a role in the postnatal cerebellar morphogenesis.

Increased morphological diversity of microglia in the activated hypothalamic supraoptic nucleus.

Microglia are the immune cells of the CNS. In the normal adult mammalian brain, the majority of these cells is quiescent and exhibits a ramified morphology. Microglia are perhaps best known for their swift transformation to an activated ameboid morphology in response to pathological insults. Here we have observed the responsiveness of these cells to events surrounding the normal activation of neurosecretory neurons in the hypothalamic supraoptic nucleus (SON), a well studied model of structural plasticity in the CNS. Neurons in the SON were activated by substituting 2% saline for drinking water. Brain sections were collected from four experimental groups [controls (C), 2 d-dehydrated (2D), 7 d-dehydrated (D7), and 7 d-dehydrated/21 d-rehydrated animals (R21)] and stained with Isolectin-B4-HRP to visualize microglial cells. Based on morphological criteria, we quantified ramified, hypertrophied, and ameboid microglia using unbiased stereological techniques. Statistical analyses showed significant increases in the number of hypertrophied microglia in the D2 and D7 groups. Moreover, there was a significant increase in the number of ameboid microglia in the D7 group. No changes were seen across conditions in the number of ramified cells, nor did we observe any significant phenotypic changes in a control area of the cingulate gyrus. Hence, increased morphological diversity of microglia was found specifically in the SON and was reversible with the cessation of stimulation. These results indicate that phenotypic plasticity of microglia may be a feature of the normal structural remodeling that accompanies neuronal activation in addition to the activation that accompanies brain pathology.