The detection of specific RNA molecules in embryonic tissues has wide research applications including studying gene expression dynamics in brain development and evolution. Recent advances in sequencing technologies have introduced new animal models to explore the molecular principles underlying the assembly and diversification of brain circuits between different amniote species. Here, we provide a step-by-step protocol for a versatile in situ hybridization method that is immediately applicable to a range of amniote embryos including zebra finch and Madagascar ground gecko, two new model organisms that have rapidly emerged for comparative brain studies over recent years. The sensitive detection of transcripts from low to high abundance expression range using the same platform enables direct comparison of gene of interest among different amniotes, providing high-resolution spatiotemporal information of gene expression to dissect the molecular principles underlying brain evolution.

The development of a multicellular organism involves time-dependent changes in molecular and cellular states; therefore 'time' is an indispensable mathematical parameter of ontogenesis. Regardless of their inextricable relationship, there is a limited number of events for which the output of developmental phenomena primarily uses temporal cues that are generated through multilevel interactions between molecules, cells, and tissues. In this review, we focus on neural stem cells, which serve as a faithful decoder of temporal cues to transmit biological information and generate specific output in the developing nervous system. We further explore the identity of the temporal information that is encoded in neural development, and how this information is decoded into various cellular fate decisions.

Electroporation is a useful technique to study gene function during development but its broad application is hampered due to the expensive equipment needed. We describe the construction of a transportable, simple and inexpensive electroporator delivering square pulses with varying length and amplitude. The device was successfully used for in utero electroporation in mouse with a performance comparable to that of commercial products.

Information processing in the cerebral cortex requires the activation of diverse neurons across layers and columns, which are established through the coordinated production of distinct neuronal subtypes and their placement along the three-dimensional axis. Over recent years, our knowledge of the regulatory mechanisms of the specification and integration of neuronal subtypes in the cerebral cortex has progressed rapidly. In this review, we address how the unique cytoarchitecture of the neocortex is established from a limited number of progenitors featuring neuronal identity transitions during development. We further illuminate the molecular mechanisms of the subtype-specific integration of these neurons into the cerebral cortex along the radial and tangential axis, and we discuss these key features to exemplify how neocortical circuit formation accomplishes economical connectivity while maintaining plasticity and evolvability to adapt to environmental changes.

The prevailing view of upper-layer (UL) neurogenesis in the cerebral cortex is that progenitor cells undergo successive rounds of asymmetric cell division that restrict the competence and production of UL neurons later in development. However, the recent discovery of UL fate-committed early progenitors raises an alternative perspective concerning their ontogeny. To investigate the emergence of UL progenitors, we manipulated the timing and extent of cortical neurogenesis in vivo in mice. We demonstrated that UL competence is tightly linked to deep-layer (DL) neurogenesis and that this sequence is determined primarily through derepression of Fezf2 by Foxg1 within a closed transcriptional cascade. We further demonstrated that the sequential acquisition of UL competence requires negative feedback, which is propagated from postmitotic DL neurons. Thus, neocortical progenitors integrate intrinsic and extrinsic cues to generate UL neurons through a system that controls the sequence of DL and UL neurogenesis and to scale the production of intracortical projection neurons based on the availability of their subcortical projection neuron counterparts during cortical development and evolution.

The functional integrity of the neocortical circuit relies on the precise production of diverse neuron populations and their assembly during development. In recent years, extensive progress has been made in the understanding of the mechanisms that control differentiation of each neuronal type within the neocortex. In this review, we address how the elaborate neocortical cytoarchitecture is established from a simple neuroepithelium based on recent studies examining the spatiotemporal mechanisms of neuronal subtype specification. We further discuss the critical events that underlie the conversion of the stem amniotes cerebrum to a mammalian-type neocortex, and extend these key findings in the light of mammalian evolution to understand how the neocortex in humans evolved from common ancestral mammals. (C) 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

The elaborate cytoarchitecture of the mammalian neocortex requires the timely production of its constituent pyramidal neurons and interneurons and their disposition in appropriate layers. Numerous chemotropic factors present in the forebrain throughout cortical development play important roles in the orchestration of these events. The Roundabout (Robo) family of receptors and their ligands, the Slit proteins, are expressed in the developing forebrain, and are known to play important roles in the generation and migration of cortical interneurons. However, few studies have investigated their function(s) in the development of pyramidal cells. Here, we observed expression of Robo1 and Slit genes (Slit1, Slit2) in cells lining the telencephalic ventricles, and found significant increases in progenitor cells (basal and apical) at embryonic day (E)12.5 and E14.5 in the developing cortex of Robo1(-/-), Slit1(-/-), and Slit1(-/-)/Slit2(-/-), but not in mice lacking the other Robo or Slit genes. Using layer-specific markers, we found that both early- and late-born pyramidal neuron populations were significantly increased in the cortices of Robo1(-/-) mice at the end of corticogenesis (E18.5). The excess number of cortical pyramidal neurons generated prenatally appears to die in early postnatal life. The observed increase in pyramidal neurons was due to prolonged proliferative activity of their progenitors and not due to changes in cell cycle events. This finding, confirmed by in utero electroporation with Robo1 short hairpin RNA (shRNA) or control constructs into progenitors along the ventricular zone as well as in dissociated cortical cell cultures, points to a novel role for Robo1 in regulating the proliferation and generation of pyramidal neurons.

Laminar organization is a key feature of the mammalian cerebral cortex, but the mechanisms by which final positioning and "inside-out" distribution of neurons are determined remain largely unknown. Here, we demonstrate that Robo1, a member of the family of Roundabout receptors, regulates the correct positioning of layers II/III pyramidal neurons in the neocortex. Specifically, we used RNA interference in mice to suppress the expression of Robo1 in a subset of layers II/III neurons, and observed the positions of these cells at distinct developmental stages. In contrast to control neurons that migrated toward the pial surface by P1, Robo1-suppressed neurons exhibited a delay in entering the cortical plate at respective stages. Unexpectedly, after the first postnatal week, these neurons were predominantly located in the upper part of layers II/III, in contrast to control cells that were distributed throughout these layers. Sequential electroporation studies revealed that Robo1-suppressed cells failed to establish the characteristic inside-out neuronal distribution and, instead, they accumulated beneath the marginal zone regardless of their birthdate. These results demonstrate that Robo receptors play a crucial role in neocortical lamination and particularly in the positioning of layers II/III pyramidal neurons.

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion.

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.

The precise stereotypic projections of pyramidal neurons within the six-layered cortex of mammals are key in allowing this structure to attain its high level of function. Recent studies have provided the first indications that postmitotic transcription factors are required for the formation and maintenance of both corticofugal and intracortical pyramidal cell populations. Here, we discuss these new findings in the context of our present understanding of cortical cell specification.

Cajal-Retzius (CR) cells, the earliest-born neurons in the neocortex, arise from discrete sources within the telencephalon, including the dorsal midline and the pallial-subpallial boundary (PSB). In particular, the cortical hem, a region of high bone morphogenetic proteins (BMPs) and Wnt (wingless-type MMTV integration site family) expression but lacking in Foxg1 (forkhead box G1) is a major source of CR neurons. Whether CR cells from distinct origins arise from disparate developmental processes or share a common mechanism is unclear. To elucidate the molecular basis of CR cell development, we assessed the role of both Foxg1 and dorsal midline signaling in the production of cortical hem- and PSB-derived CR cells. We demonstrate that the loss of Foxg1 results in the overproduction of both of these CR populations. However, removal of Foxg1 at embryonic day 13, although expanding the number of CR cells with a PSB phenotype, does not result in an expansion of BMPs or Wnts in the dorsomedial signaling center. Conversely, loss of the dorsal midline ligands as observed in Gli3 (glioma-associated oncogene homolog 3) mutants results in the loss of the cortical hem-derived CR character but does not affect the specification of PSB-derived CR cells. Hence, our findings demonstrate that, although the specification of cortical hem-derived CR cells is dependent on signaling from the dorsal midline, Foxg1 functions to repress the generation of both cortical hem- and PSB-derived CR cells.

Innervation of the neocortex by the thalamus is dependent on the precise coordination of spatial and temporal guidance cues. In this issue of Cell, work by López-Bendito et al.(2006) reveals that tangentially migrating cells within the ventral telencephalon are essential for axonal navigation between the thalamus and the neocortex, a process apparently mediated by Neuregulin-1/ErbB4 short- and long-range signaling.

During mammalian cerebral corticogenesis, progenitor cells become progressively restricted in the types of neurons they can produce. The molecular mechanism that determines earlier versus later born neuron fate is unknown. We demonstrate here that the generation of the earliest born neurons, the Cajal-Retzius cells, is suppressed by the telencephalic transcription factor Foxg1. In Foxg1 null mutants, we observed an excess of Cajal-Retzius neuron production in the cortex. By conditionally inactivating Foxg1 in cortical progenitors that normally produce deep-layer cortical neurons, we demonstrate that Foxg1 is constitutively required to suppress Cajal-Retzius cell fate. Hence, the competence to generate the earliest born neurons during later cortical development is actively suppressed but not lost.

The winged helix gene Brain factor-1 (BF1) has a pleiotropic role in the development of the cerebral hemispheres of the brain. Mice lacking BF1 have defects in the morphogenesis of the structures of the dorsal telencephalon (e.g., neocortex) and the ventral telencephalon (e.g., the basal ganglia). This study focuses on the functions of BF1 in the dorsal telencephalon. We showed previously that telencephalic progenitor cells lacking BF1 differentiate into neurons prematurely. Here, we demonstrate that the loss of BF1 also results in an early lengthening of the cell cycle in neocortical progenitors. To investigate the mechanisms by which BF1 regulates progenitor cell proliferation and differentiation in the developing brain, we have replaced the endogenous BF1 protein with a DNA binding defective form of BF1 in mice, BF1(NHAA). The BF1(NHAA) protein restores the growth of the dorsal telencephalon, by improving the proliferation of progenitor cells. However, the BF1(NHAA) protein does not correct the early neuronal differentiation associated with the loss of BF1. In contrast, replacement of endogenous BF1 with wild-type BF1 corrects the defects in both the proliferation and differentiation of neocortical progenitors. These results demonstrate that BF1 controls progenitor cell proliferation and differentiation in the neocortex through distinct DNA binding-independent and binding-dependent mechanisms.

Ascorbic acid (AsA) is highly concentrated in vitreous in bovine, human as well as in other species. In order to evaluate the role of ascorbic acid as an ocular neovascularization inhibitor, we examined the effect of ascorbic acid on growth and survival of cultured vascular endothelial cells. When added to culture medium, high concentration of ascorbic acid (0.3 mM<) reduced viability of bovine aortic endothelial cells (BAEC) within 24 hr. Morphology of ascorbic acid treated endothelial cells demonstrated that fragmentation of nuclei does not accompany during this incubation period, suggesting that ascorbic acid induces vascular endothelial cell death in a non-apoptotic manner. To further confirm that this event was not specific on BAEC, bovine retinal endothelial cells (BREC) and human aortic endothelial cells (HAEC) were tested for ascorbic acid cytotoxicity. Ascorbic acid induced cell death in all three types of cells, but the dose required for induction of cell death differed, human endothelial cells were apparently more resistant to ascorbic acid cytotoxicity than bovine cells. Decrease in viability of BAEC exposed to ascorbic acid were partially inhibited by exposure to low oxygen concentration (O-2 = 1%). Addition of vascular endothelial growth factor (VEGF) stimulated proliferation of both BAEC and BREC, but cc-addition of ascorbic acid reduced VEGF-induced endothelial cell proliferation. These results show that ascorbic acid modulates endothelial cell behavior in vitro and suggest that it is a negative regulator for ocular neovascularization.

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor. (C) 1999 Academic Press.