SHH signaling mediated by a prechordal and brain enhancer controls forebrain organization.

Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.

The Ciliopathy Gene Ftm/Rpgrip1l Controls Mouse Forebrain Patterning via Region-Specific Modulation of Hedgehog/Gli Signaling.

Primary cilia are essential for CNS development. In the mouse, they play a critical role in patterning the spinal cord and telencephalon via the regulation of Hedgehog/Gli signaling. However, despite the frequent disruption of this signaling pathway in human forebrain malformations, the role of primary cilia in forebrain morphogenesis has been little investigated outside the telencephalon. Here we studied development of the diencephalon, hypothalamus and eyes in mutant mice in which the Ftm/Rpgrip1l ciliopathy gene is disrupted. At the end of gestation, Ftm-/- fetuses displayed anophthalmia, a reduction of the ventral hypothalamus and a disorganization of diencephalic nuclei and axonal tracts. In Ftm-/- embryos, we found that the ventral forebrain structures and the rostral thalamus were missing. Optic vesicles formed but lacked the optic cups. In Ftm-/- embryos, Sonic hedgehog (Shh) expression was virtually lost in the ventral forebrain but maintained in the zona limitans intrathalamica (ZLI), the mid-diencephalic organizer. Gli activity was severely downregulated but not lost in the ventral forebrain and in regions adjacent to the Shh-expressing ZLI. Reintroduction of the repressor form of Gli3 into the Ftm-/- background restored optic cup formation. Our data thus uncover a complex role of cilia in development of the diencephalon, hypothalamus and eyes via the region-specific control of the ratio of activator and repressor forms of the Gli transcription factors. They call for a closer examination of forebrain defects in severe ciliopathies and for a search for ciliopathy genes as modifiers in other human conditions with forebrain defects.SIGNIFICANCE STATEMENT The Hedgehog (Hh) signaling pathway is essential for proper forebrain development as illustrated by a human condition called holoprosencephaly. The Hh pathway relies on primary cilia, cellular organelles that receive and transduce extracellular signals and whose dysfunctions lead to rare inherited diseases called ciliopathies. To date, the role of cilia in the forebrain has been poorly studied outside the telencephalon. In this paper we study the role of the Ftm/Rpgrip1l ciliopathy gene in mouse forebrain development. We uncover complex functions of primary cilia in forebrain morphogenesis through region-specific modulation of the Hh pathway. Our data call for further examination of forebrain defects in ciliopathies and for a search for ciliopathy genes as modifiers in human conditions affecting forebrain development.

Forebrain Ptf1a Is Required for Sexual Differentiation of the Brain.

The mammalian brain undergoes sexual differentiation by gonadal hormones during the perinatal critical period. However, the machinery at earlier stages has not been well studied. We found that Ptf1a is expressed in certain neuroepithelial cells and immature neurons around the third ventricle that give rise to various neurons in several hypothalamic nuclei. We show that conditional Ptf1a-deficient mice (Ptf1a cKO) exhibit abnormalities in sex-biased behaviors and reproductive organs in both sexes. Gonadal hormone administration to gonadectomized animals revealed that the abnormal behavior is caused by disorganized sexual development of the knockout brain. Accordingly, expression of sex-biased genes was severely altered in the cKO hypothalamus. In particular, Kiss1, important for sexual differentiation of the brain, was drastically reduced in the cKO hypothalamus, which may contribute to the observed phenotypes in the Ptf1a cKO. These findings suggest that forebrain Ptf1a is one of the earliest regulators for sexual differentiation of the brain.

Impact of Maternal Serotonin Transporter Genotype on Placental Serotonin, Fetal Forebrain Serotonin, and Neurodevelopment.

Biomarker, neuroimaging, and genetic findings implicate the serotonin transporter (SERT) in autism spectrum disorder (ASD). Previously, we found that adult male mice expressing the autism-associated SERT Ala56 variant have altered central serotonin (5-HT) system function, as well as elevated peripheral blood 5-HT levels. Early in gestation, before midbrain 5-HT projections have reached the cortex, peripheral sources supply 5-HT to the forebrain, suggesting that altered maternal or placenta 5-HT system function could impact the developing embryo. We therefore used different combinations of maternal and embryo SERT Ala56 genotypes to examine effects on blood, placenta and embryo serotonin levels and neurodevelopment at embryonic day E14.5, when peripheral sources of 5-HT predominate, and E18.5, when midbrain 5-HT projections have reached the forebrain. Maternal SERT Ala56 genotype was associated with decreased placenta and embryonic forebrain 5-HT levels at E14.5. Low 5-HT in the placenta persisted, but forebrain levels normalized by E18.5. Maternal SERT Ala56 genotype effects on forebrain 5-HT levels were accompanied by a broadening of 5-HT-sensitive thalamocortical axon projections. In contrast, no effect of embryo genotype was seen in concepti from heterozygous dams. Blood 5-HT levels were dynamic across pregnancy and were increased in SERT Ala56 dams at E14.5. Placenta RNA sequencing data at E14.5 indicated substantial impact of maternal SERT Ala56 genotype, with alterations in immune and metabolic-related pathways. Collectively, these findings indicate that maternal SERT function impacts offspring placental 5-HT levels, forebrain 5-HT levels, and neurodevelopment.

Cell migration in the developing rodent olfactory system.

The components of the nervous system are assembled in development by the process of cell migration. Although the principles of cell migration are conserved throughout the brain, different subsystems may predominantly utilize specific migratory mechanisms, or may display unusual features during migration. Examining these subsystems offers not only the potential for insights into the development of the system, but may also help in understanding disorders arising from aberrant cell migration. The olfactory system is an ancient sensory circuit that is essential for the survival and reproduction of a species. The organization of this circuit displays many evolutionarily conserved features in vertebrates, including molecular mechanisms and complex migratory pathways. In this review, we describe the elaborate migrations that populate each component of the olfactory system in rodents and compare them with those described in the well-studied neocortex. Understanding how the components of the olfactory system are assembled will not only shed light on the etiology of olfactory and sexual disorders, but will also offer insights into how conserved migratory mechanisms may have shaped the evolution of the brain.

The molecular and cellular signatures of the mouse eminentia thalami support its role as a signalling centre in the developing forebrain.

The mammalian eminentia thalami (EmT) (or thalamic eminence) is an embryonic forebrain structure of unknown function. Here, we examined the molecular and cellular properties of the mouse EmT. We first studied mRNA expression of signalling molecules and found that the EmT is a structure, rich in expression of secreted factors, with Wnts being the most abundantly detected. We then examined whether EmT tissue could induce cell fate changes when grafted ectopically. For this, we transplanted EmT tissue from a tau-GFP mouse to the ventral telencephalon of a wild type host, a telencephalic region where Wnt signalling is not normally active but which we showed in culture experiments is competent to respond to Wnts. We observed that the EmT was able to induce in adjacent ventral telencephalic cells ectopic expression of Lef1, a transcriptional activator and a target gene of the Wnt/ß-catenin pathway. These Lef1-positive;GFP-negative cells expressed the telencephalic marker Foxg1 but not Ascl1, which is normally expressed by ventral telencephalic cells. These results suggest that the EmT has the capacity to activate Wnt/ß-catenin signalling in the ventral telencephalon and to suppress ventral telencephalic gene expression. Altogether, our data support a role of the EmT as a signalling centre in the developing mouse forebrain.

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

Differential effects of prenatal chronic high-decibel noise and music exposure on the excitatory and inhibitory synaptic components of the auditory cortex analog in developing chicks (Gallus gallus domesticus).

Proper development of the auditory cortex depends on early acoustic experience that modulates the balance between excitatory and inhibitory (E/I) circuits. In the present social and occupational environment exposure to chronic loud sound in the form of occupational or recreational noise, is becoming inevitable. This could especially disrupt the functional auditory cortex development leading to altered processing of complex sound and hearing impairment. Here we report the effects of prenatal chronic loud sound (110-dB sound pressure level (SPL)) exposure (rhythmic [music] and arrhythmic [noise] forms) on the molecular components involved in regulation of the E/I balance in the developing auditory cortex analog/Field L (AuL) in domestic chicks. Noise exposure at 110-dB SPL significantly enhanced the E/I ratio (increased expression of AMPA receptor GluR2 subunit and glutamate with decreased expression of GABA(A) receptor gamma 2 subunit and GABA), whereas loud music exposure maintained the E/I ratio. Expressions of markers of synaptogenesis, synaptic stability and plasticity i.e., synaptophysin, PSD-95 and gephyrin were reduced with noise but increased with music exposure. Thus our results showed differential effects of prenatal chronic loud noise and music exposures on the E/I balance and synaptic function and stability in the developing auditory cortex. Loud music exposure showed an overall enrichment effect whereas loud noise-induced significant alterations in E/I balance could later impact the auditory function and associated cognitive behavior.

Evidence that descending cortical axons are essential for thalamocortical axons to cross the pallial-subpallial boundary in the embryonic forebrain.

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors.

Lhx2 and Lhx9 determine neuronal differentiation and compartition in the caudal forebrain by regulating Wnt signaling.

Initial axial patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs during gastrulation. After this patterning phase, further diversification within the brain is thought to proceed largely independently in the different primordia. However, mechanisms that maintain the demarcation of brain subdivisions at later stages are poorly understood. In the alar plate of the caudal forebrain there are two principal units, the thalamus and the pretectum, each of which is a developmental compartment. Here we show that proper neuronal differentiation of the thalamus requires Lhx2 and Lhx9 function. In Lhx2/Lhx9-deficient zebrafish embryos the differentiation process is blocked and the dorsally adjacent Wnt positive epithalamus expands into the thalamus. This leads to an upregulation of Wnt signaling in the caudal forebrain. Lack of Lhx2/Lhx9 function as well as increased Wnt signaling alter the expression of the thalamus specific cell adhesion factor pcdh10b and lead subsequently to a striking anterior-posterior disorganization of the caudal forebrain. We therefore suggest that after initial neural tube patterning, neurogenesis within a brain compartment influences the integrity of the neuronal progenitor pool and border formation of a neuromeric compartment.

Isolation of radial glia-like neural stem cells from fetal and adult mouse forebrain via selective adhesion to a novel adhesive peptide-conjugate.

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.

The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Transient expression of serotonin 5-HT4 receptors in the mouse developing thalamocortical projections.

The serotonin 5-HT(4) receptor (5-HT(4)-R) is an unusually complex G-protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5-HT(4)-Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT-PCR. The developing thalamocortical projections transiently expressed 5-HT(4)-Rs in the embryonic brain and the 5-HT(4)-R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13-17, the forebrain mRNA levels of the 5-HT(4(a))-R and 5-HT(4(b))-R splice variants increased nine- and fivefold, respectively, whereas the levels of the 5-HT(4(e))-R and 5-HT(4(f))-R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5-HT(4)-R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing.

Complex and dynamic patterns of Wnt pathway gene expression in the developing chick forebrain.

BACKGROUND: Wnt signalling regulates multiple aspects of brain development in vertebrate embryos. A large number of Wnts are expressed in the embryonic forebrain; however, it is poorly understood which specific Wnt performs which function and how they interact. Wnts are able to activate different intracellular pathways, but which of these pathways become activated in different brain subdivisions also remains enigmatic. RESULTS: We have compiled the first comprehensive spatiotemporal atlas of Wnt pathway gene expression at critical stages of forebrain regionalisation in the chick embryo and found that most of these genes are expressed in strikingly dynamic and complex patterns. Several expression domains do not respect proposed compartment boundaries in the developing forebrain, suggesting that areal identities are more dynamic than previously thought. Using an in ovo electroporation approach, we show that Wnt4 expression in the thalamus is negatively regulated by Sonic hedgehog (Shh) signalling from the zona limitans intrathalamica (ZLI), a known organising centre of forebrain development. CONCLUSION: The forebrain is exposed to a multitude of Wnts and Wnt inhibitors that are expressed in a highly dynamic and complex fashion, precluding simple correlative conclusions about their respective functions or signalling mechanisms. In various biological systems, Wnts are antagonised by Shh signalling. By demonstrating that Wnt4 expression in the thalamus is repressed by Shh from the ZLI we reveal an additional level of interaction between these two pathways and provide an example for the cross-regulation between patterning centres during forebrain regionalisation.

Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain.

The establishment of appropriate neural circuitry depends on the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival-all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization, and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus, and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits, with particular relevance to the social and emotional dimensions of behavior.

Newly identified patterns of Pax2 expression in the developing mouse forebrain.

BACKGROUND: The availability of specific markers expressed in different regions of the developing nervous system provides a useful tool for the study of mouse mutants. One such marker, the transcription factor Pax2, is expressed at the midbrain-hindbrain boundary and in the cerebellum, spinal cord, retina, optic stalk, and optic chiasm. We recently described a group of diencephalic cells that express Pax2 as early as embryonic day (E) 10.5, and become part of the eminentia thalami by E11.5. The discovery of this previously undescribed cell population prompted us to examine Pax2 protein expression in the developing mouse forebrain in more detail. RESULTS: We determined the expression pattern of Pax2 in the forebrain of wild type mouse embryos between E10.5 and postnatal day (P) 15. Pax2 expression was detected in the septum of the basal forebrain, hypothalamus, eminentia thalami and in the subfornical organ. To evaluate Pax2 as a marker for septal cells, we examined Pax2 expression in Pax6Sey/Sey mutants, which have an enlarged septum. We found that Pax2 clearly marks a population of septal cells equivalent to that seen in wild types, indicating its utility as a marker of septal identity. These cells did not express the GABAergic marker calbindin nor the cholinergic marker choline acetyltransferase and were not detectable after P15. CONCLUSION: Pax2 is expressed in populations of cells within the developing septum, hypothalamus, and eminentia thalami. It seems especially useful as a marker of the telencephalic septum, because of its early, strong and characteristic expression in this structure. Further, its expression is maintained in the enlarged septum of Pax6Sey/Sey mutants.

Early mammillary pouch specification in the course of prechordal ventralization of the forebrain tegmentum.

The mammillary body, a ventral specialization of the caudal hypothalamus, lies close to the transition between epichordal and prechordal parts of the forebrain (Puelles and Rubenstein, 2003). This report examines its presumed causal connection with either prechordal or notochordal mesodermal induction, as well as the timing of its specification, in the context of early ventral forebrain patterning. It was recently found that the ephrin receptor gene EphA7 is selectively expressed in the mammillary pouch from early stages of development (HH14: García-Calero et al., 2006). We used mammillary EphA7 expression as well as ventral hypothalamic expression of the gene markers Nkx2.1 and Shh to analyze experimental effects on mammillary specification and morphogenesis after axial mesoderm ablation at stages HH4+ to HH6. Progressively delayed ablation of the prechordal plate revealed its sequential implication in molecular specification of the entire ventral forebrain, including the mammillary and tuberal regions of the hypothalamus. We observed differential contact requirements for induction by the prechordal plate of all the forebrain regions expressing Shh and Nkx2.1, including distant subpallial ones. In contrast, ablation of the anterior notochordal tip at these stages did not elicit significant patterning changes, particularly no effects on mammillary EphA7 expression or mammillary pouch development.

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

Expression of FOXP2 in the developing monkey forebrain: comparison with the expression of the genes FOXP1, PBX3, and MEIS2.

By using the developing monkey brain as a model for human development, we investigated the expression pattern of the FOXP2 gene, a member of the FOX family of transcription factors in the developing monkey brain, and compared its expression pattern with transcription factors PBX3, MEIS2, and FOXP1. We observed FOXP2 mRNA expression in several brain structures, including the striatum, the islands of Calleja and other basal forebrain regions, the cerebral cortex, and the thalamus. FOXP2 mRNA was preferentially expressed in striosomal compartments during striatal development. The striosomal expression was transient and developmentally down-regulated in a topographical order. Specifically, during the perinatal state, striosomal FOXP2 expression was detected in both the caudate nucleus and the putamen, although expression was more prominent in the caudate nucleus than in the putamen. Striosomal FOXP2 expression declined during the postnatal period, first in the putamen and later in the caudate nucleus. During the same period, we also detected PBX3 mRNA in the striosomal compartment of the developing monkey striatum. FOXP2, as well as PBX3 and MEIS2, was expressed in the islands of Calleja and other cell clusters of the basal forebrain. FOXP2, in combination with PBX3 and MEIS2, may play a pivotal role in the development of striosomal neurons of the striatum and the islands of Calleja.