Chiaki Ohtaka-Maruyama

Birds evolved large, cognitively capable forebrains independently of mammals, yet comparative analyses of avian brain organization have been constrained by the lack of standardized resources capable of resolving internal parcellation and long-range connectivity across species. Here, we present a comparative MRI resource spanning 16 avian species representing major clades and diverse ecological niches. We analyzed high-resolution T2-weighted and diffusion-weighted datasets suitable for direct interspecific comparison. T2-weighted morphometry revealed pronounced region-specific variation in internal brain architecture, including lineage-dependent differences in the relative prominence of major brain divisions and commissural structures, supporting a pattern of mosaic diversification rather than uniform scaling. To validate MRI-derived anatomical boundaries, we compared MRI parcellations with complementary histological analyses in three representative taxa (the large-billed crow, gentoo penguin, and mandarin duck), demonstrating close correspondence between MRI-defined borders and cytoarchitectonic transitions identified by Nissl staining, as well as major myelinated compartments visualized by Luxol Fast Blue staining. Moreover, diffusion MRI tractography and fractional anisotropy (FA) mapping further revealed both conserved and species-specific features of large-scale brain organization. Seed-based tractography of the optic lobe, dorsal cortex, cerebellum, and anterior cortex in chick, gentoo penguin, and large-billed crow revealed conserved within-compartment trajectory patterns alongside marked region-specific interspecific differences, particularly in optic-lobe-associated long-range trajectories. Whole-brain FA maps revealed complementary variation in regional microstructural organization across taxa. Together, this comparative MRI framework provides a cross-validated foundation for linking internal brain anatomy and long-range connectivity to ecological and evolutionary diversification in birds, with broader applications to comparative neuroanatomy across amniotes.

Subplate neurons (SpNs) are among the earliest‐born and maturing neurons in the developing cerebral cortex. They arise from multiple origins and can be classified into several subgroups based on morphology, connectivity, and gene expression. These neurons play essential roles in cortical circuit formation, yet their cellular diversity and transcriptional dynamics remain incompletely understood. Here, we characterized transcriptomic profiles of SpN subpopulations in embryonic mouse cortex using Lpar1‐EGFP and NeuroD1/Cre‐ERT2 (D1B) reporter lines. We applied complementary approaches of gene expression profiling, including bulk microarray analysis, single‐cell RNA sequencing (scRNA‐seq), and Visium spatial transcriptomics. At embryonic day 17 (E17), scRNA‐seq identified 10 distinct Lpar1‐EGFP ‐positive SpN clusters, which spatial transcriptomics mapped to specific cortical regions. While many markers showed enrichment within the subplate region, others extended into the hippocampus and ventral pallium (including the amygdala, claustrum, and endopiriform nucleus). Integrated analysis of Lpar1‐EGFP and D1B lines revealed both overlapping and unique gene expression signatures, highlighting dynamic markers of subplate identity. Comparisons between E15 and E17 datasets showed substantial transcriptional shifts, suggesting rapid developmental changes in SpN subgroups. Validation with in situ hybridization and RNAscope confirmed the selectivity of key markers, including Cryab , Cdh13 , Nr4a2, and Lmo3 . Together, these findings provide a molecular framework for further classifying SpN subtypes and identifying candidate markers for transient versus persistent populations, thereby advancing our understanding of early cortical development.

Birds evolved large, cognitively capable forebrains independently of mammals, yet comparative analyses of avian brain organization have been constrained by the lack of standardized resources capable of resolving internal parcellation and long-range connectivity across species. Here, we present a comparative MRI resource spanning 16 avian species representing major clades and diverse ecological niches. We analyzed high-resolution T2-weighted and diffusion-weighted datasets suitable for direct interspecific comparison. T2-weighted morphometry revealed pronounced region-specific variation in internal brain architecture, including lineage-dependent differences in the relative prominence of major brain divisions and commissural structures, supporting a pattern of mosaic diversification rather than uniform scaling. To validate MRI-derived anatomical boundaries, we compared MRI parcellations with complementary histological analyses in three representative taxa (the large-billed crow, gentoo penguin, and mandarin duck), demonstrating close correspondence between MRI-defined borders and cytoarchitectonic transitions identified by Nissl staining, as well as major myelinated compartments visualized by Luxol Fast Blue staining. Moreover, diffusion MRI tractography and fractional anisotropy (FA) mapping further revealed both conserved and species-specific features of large-scale brain organization. Seed-based tractography of the optic lobe, dorsal cortex, cerebellum, and anterior cortex in chick, gentoo penguin, and large-billed crow revealed conserved within-compartment trajectory patterns alongside marked region-specific interspecific differences, particularly in optic-lobe-associated long-range trajectories. Whole-brain FA maps revealed complementary variation in regional microstructural organization across taxa. Together, this comparative MRI framework provides a cross-validated foundation for linking internal brain anatomy and long-range connectivity to ecological and evolutionary diversification in birds, with broader applications to comparative neuroanatomy across amniotes.

Datasets for the study of developing Non‐Human Primate (NHP) brain are scarce. Some of the reasons are related to the financial and technical obstacles that make the production of these datasets logistically challenging. NHP embryos of different and verified developmental ages require timed pregnancies and the maintenance of expensive and highly regulated breeding colonies. Here, we document our efforts to provide digital datasets of rhesus macaque ( Macaca mulatta ) embryonic brain histological series of sections that can be used and reused for research. These efforts fall well within the primary mission of the MacBrain Resource Center (MBRC, https://medicine.yale.edu/neuroscience/macbrain/mission/ ), which targets a reduction in the need to sacrifice animals, improved efficiency in the use of resources, the use of male (M) and female (F) specimens to conduct developmental research, and increase archival and sharing of materials for the advancement of neuroscience. First, we describe the already existent tritiated thymidine ( ³ H‐TdR) cases in MBRC Collection 1 and the electron microscopy (EM) blocks of Collection 5, which together contain >100 specimens of different developmental ages. We also report methodological details on, as of July 2025, n = 24 histo‐ and immunohistochemically processed embryonic brains that enrich Collection 6 with thousands of coronal sections stained for n = 38 different cellular and fiber markers. These datasets provide NHP brain histo‐ and immunohistology for comparison with other species and hence help close a gap in the availability of suitable materials for comparative evolution and neurodevelopmental studies. Through examples, we illustrate how different materials are currently being used in de novo research. Finally, we advocate the absolute need to continue using NHPs in the study of neurodevelopment because of the unparalleled molecular, genetic, and anatomical similarities between these animal models and humans.

Understanding how neural progenitors generate diverse neuronal subtypes is central to developmental and evolutionary neuroscience. Lineage tracing is a key tool for dissecting these processes, but existing methods are often constrained by stochastic expression, recombination bias, or reliance on transgenic models—limitations that restrict their application in non-model organisms. Here, we present an enhanced iOn switch system optimized for evo-devo research. Leveraging its modular, integration-based transgene expression logic, we developed a tunable labeling strategy that enables both sparse and dense labeling within a single framework. We further expanded the system’s fluorescent palette and introduced subcellular targeting options to support multiclonal analysis and stable time-lapse imaging. Finally, we demonstrate the versatility of this approach across a wide range of vertebrate species, including mouse, guinea pig, rat, chick, turtle, and zebrafish, thereby establishing the iOn switch system as a flexible and accessible tool for evo-devo research.

The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in nonmammalian amniotes, such as reptiles and birds. In these animal groups, the spatio‐temporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)‐mediated gene targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian‐specific CC formation.

Subplate neurons (SpNs) are among the earliest-born neurons in the mammalian neocortex and play key roles in radial migration and transient circuit formation. It has long been assumed that most SpNs undergo extensive postnatal cell death, leaving only a small remnant population that contributes to layer 6b (L6b) in adulthood. However, the extent to which SpNs actually persist as L6b neurons has remained unresolved, partly because previous studies lacked quantitative, whole-cortex analyses that account for postnatal cortical expansion. Here, we performed a comprehensive birthdating analysis using multiple EdU injections spanning the entire neurogenic window of SpNs in mice, combined with whole-neocortex 3D tissue clearing to measure subplate and L6b volumes. This approach allowed us to directly estimate the total number and distribution of SpN-derived neurons in adulthood. We found that most early-born SpNs persist as L6b neurons, and that the apparent postnatal reduction in SpN density reflects tangential cortical expansion rather than neuronal loss. Moreover, surviving SpNs comprise diverse neuronal subtypes reminiscent of those present at early postnatal stages. Together, these findings demonstrate that, in rodents, the majority of adult L6b neurons originate from SpNs, revising the long-held view that the subplate is a largely transient neuronal population.

The avian pallium is comprised of several nuclear regions (four main areas) and differs significantly from the mammalian telencephalon, which is a six‐layered structure. Although it is known that cells with identical features at the gene expression level can exist in different regions, it remains unclear whether cells with identical features arise from the same region. In this study, we examined the cell lineages produced from each region by employing the recently developed integration‐coupled gene expression On (iOn) switch and performing whole‐brain imaging by light‐sheet fluorescence microscopy (LSFM). We found that cells born in each area migrate across regions and, therefore, cells with identical characteristics are not always generated from the same progenitor regions. Furthermore, an examination of cell migration patterns in the dorsal ventricular ridge revealed that, as in mammals, cells preferably migrate along radial fibers in the early stages of migration, but radial fibers are not required for later migration. These results support the developmental convergence models recently proposed to describe cell lineage.

Understanding brain evolution requires detailed comparative analyses of brain structures across species. However, high-resolution anatomical and connective data remain limited for most vertebrates beyond a few well-studied model organisms. To address this gap, we collected postmortem brain samples from a range of vertebrates, primarily small amniotes, and performed magnetic resonance imaging and histological staining. Here, we present the “Animal Brain Collection (ABC),” a freely accessible database that enables researchers to examine and compare cellular and tissue-level brain architectures across species. This resource provides a foundation for cross-species investigations of brain structure and development, offering new opportunities for research into the diversity and evolution of vertebrate brains.

The neocortical development process includes cell proliferation, differentiation, migration, and maturation, supported by precise genetic regulation. To understand these processes at the cellular and molecular levels, it is necessary to characterize the fundamental anatomical structures by gene expression. However, markers established in the adult brain sometimes behave differently in the fetal brain, actively changing during development. The spatial transcriptome is a powerful analytical method that enables sequence analysis while retaining spatial information. However, a deeper understanding of these data requires computational estimation, including integration with single-cell transcriptome data and aggregation of spots at the single-cell cluster level. The application of such analysis to biomarker discovery has only begun recently, and its application to the developing fetal brain is largely unexplored. In this study, we performed a spatial transcriptome analysis of the developing mouse brain to investigate spatio-temporal regulation of gene expression during development. Using these data, we conducted an integrated study with publicly available mouse data sets. Our data-driven analysis identified novel molecular markers of the choroid plexus, piriform cortex, and thalamus. Furthermore, we identified a novel molecular marker that can determine the dorsal endopiriform nucleus (DEn) of the developmental stage in the claustrum/DEn complex.