Cellular-resolution gene expression profiling in the neonatal marmoset brain reveals dynamic species- and region-specific differences.

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)-based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.

Comparative anatomy of marmoset and mouse cortex from genomic expression.

Advances in mouse neural circuit genetics, brain atlases, and behavioral assays provide a powerful system for modeling the genetic basis of cognition and psychiatric disease. However, a critical limitation of this approach is how to achieve concordance of mouse neurobiology with the ultimate goal of understanding the human brain. Previously, the common marmoset has shown promise as a genetic model system toward the linking of mouse and human studies. However, the advent of marmoset transgenic approaches will require an understanding of developmental principles in marmoset compared to mouse. In this study, we used gene expression analysis in marmoset brain to pose a series of fundamental questions on cortical development and evolution for direct comparison to existing mouse brain atlas expression data. Most genes showed reliable conservation of expression between marmoset and mouse. However, certain markers had strikingly divergent expression patterns. The lateral geniculate nucleus and pulvinar in the thalamus showed diversification of genetic organization between marmoset and mouse, suggesting they share some similarity. In contrast, gene expression patterns in early visual cortical areas showed marmoset-specific expression. In prefrontal cortex, some markers labeled architectonic areas and layers distinct between mouse and marmoset. Core hippocampus was conserved, while afferent areas showed divergence. Together, these results indicate that existing cortical areas are genetically conserved between marmoset and mouse, while differences in areal parcellation, afferent diversification, and layer complexity are associated with specific genes. Collectively, we propose that gene expression patterns in marmoset brain reveal important clues to the principles underlying the molecular evolution of cortical and cognitive expansion.

Thalamocortical control of cell-type specificity drives circuits for processing whisker-related information in mouse barrel cortex.

Excitatory spiny stellate neurons are prominently featured in the cortical circuits of sensory modalities that provide high salience and high acuity representations of the environment. These specialized neurons are considered developmentally linked to bottom-up inputs from the thalamus, however, the molecular mechanisms underlying their diversification and function are unknown. Here, we investigated this in mouse somatosensory cortex, where spiny stellate neurons and pyramidal neurons have distinct roles in processing whisker-evoked signals. Utilizing spatial transcriptomics, we identified reciprocal patterns of gene expression which correlated with these cell-types and were linked to innervation by specific thalamic inputs during development. Genetic manipulation that prevents the acquisition of spiny stellate fate highlighted an important role for these neurons in processing distinct whisker signals within functional cortical columns, and as a key driver in the formation of specific whisker-related circuits in the cortex.

Digital gene atlas of neonate common marmoset brain.

Interest in the common marmoset (Callithrix jacchus) as a primate model animal has grown recently, in part due to the successful demonstration of transgenic marmosets. However, there is some debate as to the suitability of marmosets, compared to more widely used animal models, such as the macaque monkey and mouse. Especially, the usage of marmoset for animal models of human cognition and mental disorders, is still yet to be fully explored. To examine the prospects of the marmoset model for neuroscience research, the Marmoset Gene Atlas (https://gene-atlas.bminds.brain.riken.jp/) provides a whole brain gene expression atlas in the common marmoset. We employ in situ hybridization (ISH) to systematically analyze gene expression in neonate marmoset brains, which allows us to compare expression with other model animals such as mouse. We anticipate that these data will provide sufficient information to develop tools that enable us to reveal marmoset brain structure, function, cellular and molecular organization for primate brain research.

BTBD3 controls dendrite orientation toward active axons in mammalian neocortex.

Experience-dependent structural changes in the developing brain are fundamental for proper neural circuit formation. Here, we show that during the development of the sensory cortex, dendritic field orientation is controlled by the BTB/POZ domain-containing 3 (BTBD3). In developing mouse somatosensory cortex, endogenous Btbd3 translocated to the cell nucleus in response to neuronal activity and oriented primary dendrites toward active axons in the barrel hollow. Btbd3 also directed dendrites toward active axon terminals when ectopically expressed in mouse visual cortex or normally expressed in ferret visual cortex. BTBD3 regulation of dendrite orientation is conserved across species and cortical areas and shows how high-acuity sensory function may be achieved by the tuning of subcellular polarity to sources of high sensory activity.

Mouse in utero electroporation: controlled spatiotemporal gene transfection.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Dynamic spatiotemporal gene expression in embryonic mouse thalamus.

The anatomy of the mammalian thalamus is characterized by nuclei, which can be readily identified in postnatal animals. However, the molecular mechanisms that guide specification and differentiation of neurons in specific thalamic nuclei are still largely unknown, and few molecular markers are available for most of these thalamic subregions at early stages of development. We therefore searched for patterned gene expression restricted to specific mouse thalamic regions by in situ hybridization during the onset of thalamic neurogenesis (embryonic [E] days E10.5-E12.5). To obtain correct regional information, we used Shh as a landmark and compared spatial relationships with the zona limitans intrathalamica (Zli), the border of the p2 and p3 compartments of the diencephalon. We identified genes that are expressed specifically in the ventricular zone of the thalamic neuroepithelium and also identified a number of genes that already exhibited regional identity at E12.5. Although many genes expressed in the mantle regions of the thalamus at E12.5 showed regionally restricted patterns, none of these clearly corresponded to individual thalamic nuclei. We next examined gene expression at E15.5, when thalamocortical axons (TCAs) project from distinct regions of the thalamus and reach their targets in the cerebral cortex. Regionally restricted patterns of gene expression were again seen for many genes, but some regionally bounded expression patterns in the early postnatal thalamus had shifted substantially by E15.5. These findings reveal that nucleogenesis in the developing thalamus is associated with selective and complex changes in gene expression and provide a list of genes that may actively regulate the development of thalamic nuclei.

Region-specific gene expression in early postnatal mouse thalamus.

Previous studies in the developing mouse thalamus have demonstrated that regional identity is established during early stages of development (Suzuki-Hirano et al. J. Comp. Neurol. 2011;519:528-543). However, the developing thalamus often shows little resemblance to the anatomical organization of the postnatal thalamus, making it difficult to identify genes that might mediate the organization of thalamic nuclei. We therefore analyzed the expression pattern of genes that we have identified as showing regional expression in embryonic thalamus on postnatal days (P) 6-8 by using in situ hybridization. We also identified several genes expressed only in the postnatal thalamus with restricted expression in specific nuclei. We first demonstrated the selective expression of neurotransmitter-related genes (vGlut2, vGAT, D2R, and HTR2C), identifying the neurotransmitter subtypes of cells in this region, and we also demonstrated selective expression of additional genes in the thalamus (Steel, Slitrk6, and AI852580). In addition, we demonstrated expression of genes specific to somatosensory thalamic nuclei, the ventrobasal posterior nuclei (VP); a visual thalamic nucleus, the dorsal lateral geniculate nucleus (dLGN); and an auditory thalamic nucleus, the medial geniculate body (MGB) (p57Kip, Nr1d1, and GFRα1). We also identified genes that are selectively expressed in multiple different nuclei (Foxp2, Chst2, and EphA8). Finally, we demonstrated that several bone morphogenetic proteins (BMPs) and their inhibitors are expressed in the postnatal thalamus in a nucleus-specific fashion, suggesting that BMPs play roles in the postnatal thalamus unrelated to their known role in developmental patterning. Our findings provide important information for understanding the mechanisms of nuclear specification and connectivity during development, as well as their maintenance in adult thalamus.

A genomic atlas of mouse hypothalamic development.

The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.