Developmental mouse brain common coordinate framework

Abstract

3D brain atlases are key resources to understand the brain's spatial organization and promote interoperability across different studies. However, unlike the adult mouse brain, the lack of developing mouse brain 3D reference atlases hinders advancements in understanding brain development. Here, we present a 3D developmental common coordinate framework (DevCCF) spanning embryonic day (E)11.5, E13.5, E15.5, E18.5, and postnatal day (P)4, P14, and P56, featuring undistorted morphologically averaged atlas templates created from magnetic resonance imaging and co-registered high-resolution light sheet fluorescence microscopy templates. The DevCCF with 3D anatomical segmentations can be downloaded or explored via an interactive 3D web-visualizer. As a use case, we utilize the DevCCF to unveil GABAergic neuron emergence in embryonic brains. Moreover, we map the Allen CCFv3 and spatial transcriptome cell-type data to our stereotaxic P56 atlas. In summary, the DevCCF is an openly accessible resource for multi-study data integration to advance our understanding of brain development.

Fig. 1. DevCCF overview.

a Morphology and intensity averaged templates from light sheet fluorescence microscopy (LSFM; blue box) and magnetic resonance imaging (MRI; orange block) imaging. The LSFM template was aligned to the undistorted MRI template via multimodal registration. b Multimodal DevCCF with anatomical segmentations is established at four embryonic and three postnatal ages. c Sagittal slices of multimodal E15.5 data registered to the E15.5 DevCCF, each highlighting unique anatomical features. Data includes MRI DWI (red), MRI FA (orange), LSFM GABAergic neurons from Gad2-Cre;Ai14 mice (yellow), LSFM autofluorescence (green), in situ hybridization (ISH) Lhx2 and Foxp2 gene expression (blue), DevCCF annotations (violet), Compiled colocalized data from each data type, as labeled. Scale bar = 2 mm. a, b created in BioRender. Kim, Y. (2023) BioRender.com/q01q598.

Fig. 2. 3D Multimodal developmental mouse brain templates.

a 3D DevCCF MRI templates from T2-weighted contrasts. b DevCCF LSFM autofluorescence templates (coronal slice) before multimodal registration to the MRI template. c Multimodal registration of E15.5 LSFM (green) to MRI DWI (magenta) templates. Top: horizontal plane. Bottom: coronal plane. d DevCCF E15.5 multimodal templates with unique and complementary contrasts. Note distinct anatomical marks differentially highlighted: purple ‘X’ (trigeminal nerve root), red circle (choroidal tissue), orange star (zona limitans), yellow arrowhead (retroflex tract), blue asterisk (ventricle). e The CCFv3 template is registered to the P56 DevCCF. Images are MRI DWI template midline sagittal slice, co-registered MRI DWI, LSFM autofluorescence, and CCFv3 coronal template coronal slices, as labeled. Scale bars in a–c = 2 mm.

Fig. 3. Example of landmarks used for atlas parcellation at E13.5.

The E13.5 DevCCF includes 6 aligned 3D whole brain images: an LSFM Autofluorescence template, 4 MRI contrast templates, and developmental annotations. We show each template as horizontal (a–f), coronal (g–l), and sagittal (m–r) cross sections with a selected set of visible landmarks that guided atlas segmentation. Selected landmarks are noted, including the dark ventricles marked in the LSFM template (a, g, m), telencephalic intermediate and superficial layers with inverse contrasts in DWI and FA templates (c, d), the telencephalic periventricular zone in the T2w template (e, k, q), the retroflex tract that denotes the boundary between prosomeres (p)2 and 3 in DWI and FA templates (j, o, p), and distinct diencephalic prosomeres 1, 2, and 3 in MRI ADC and DWI templates (h, i), the cerebellum in all sagittal viewed templates (m–r). Scale bar = 3 mm. 6M abducens motor nucleus, 3M occulomotor nucleus. Annotation colors are defined in DevCCF ontology (Supplementary Data 2).

Fig. 4. Anatomical segmentations based on a developmental ontology.

a DevCCF ontology levels 0 through 5 displayed over an E15.5 sagittal slice. b Coronal MRI FA slices through the subpallium with anatomical segmentation overlays on the right hemisphere. Embryonic and postnatal brains are depicted at uniform scales, respectively. c 3D renderings of DevCCF annotations, not to scale. d–f 3D staining guide segmentations, d Midline sagittal MRI ADC section indicating location of DevCCF segmentations with aligned neurofilament staining section to guide delineation of cortical areas (red line, e) and fluorescence Nissl staining to delineate cerebellar layers (f). g Brain volume growth curve defined by DevCCF template volume (black open circles; source data in the source data file) and mean ± standard deviation of template generation input sample volumes (blue closed circles and shading; data in Table 2). Scale bars = 2 mm. Annotation colors in a, c are defined in DevCCF ontology (Supplementary Data 2). Annotation colors in b, d are semi-randomized to best visualize individual regions.

Fig. 5. Early emergence of GABAergic neurons in embryonic brains.

a Sagittal images of LSFM imaging from E11.5 Gad2-Cre;Ai14 mice show clusters of GAD2+ neurons (green, pseudo colored) in the subpallium (SPall), prosomere 3 (p3). The E13.5 (b) and E15.5 (c) brains show additional GAD2+ neurons in the cerebellar hemisphere (CbH). Averaged Gad2-Cre;Ai14 brains registered to the age matched DevCCF at E11.5 (d), E13.5 (e), and E15.5 (f). The first column is for the medial, the second for the intermediate, and the third for the lateral sagittal planes. g Average GAD2+ signals from E11.5 (n = 7), E13.5 (n = 7), and E15.5 (n = 5) brains with 3D rendering overlay on DevCCF templates. Note rapid expansion of GAD2+ neurons at E13.5 and E15.5 from initial clusters at E11.5. h In addition to local expansion, GAD2+ neurons migrate to deep (1) and superficial areas (2) of the pallium to establish cortical interneurons. i Quantification of GAD2+ signals (mean % area filled with signal ± population standard deviation) in DevCCF segmentations at E11.5, E13.5, and E15.5 (source data provided in the source data file). Scale bars = 2 mm. CbH cerebellar hemisphere, H hindbrain, MTg midbrain tegmentum, OB olfactory bulb, PHy peduncular hypothalamus, PHyA PHy alar plate, p prosomere, r0 rhombomere 0, r1B rhombomere 1 basal plate, SeSPall septal subpallium, SPall subpallium, Str striatum.

Fig. 6. Integrating CCFv3 with DevCCF for enhanced anatomical mapping.

a CCFv3 annotations (left hemisphere) and DevCCF P56 annotations (right hemisphere, https://atlas.brain-map.org/) overlayed on the DevCCF P56 template across five slices from anterior (left) to posterior (right). b Sagittal slices with CCFv3 annotations (left, https://atlas.brain-map.org/), DevCCF (middle), and DevCCF label boundaries overlaid on CCFv3 annotations (right). Neuromeric boundaries of the prosomere (p)1, p2, p3 highlighted with white dashed lines over colored CCFv3 annotations demonstrate shared and non-overlapping boundaries of CCFv3 and DevCCF annotations. Annotation colors in a, b are semi-randomized to differentiate annotations. c Sankey diagram illustrates matched structural relationship between DevCCF and CCFv3 annotations. Size of individual areas represents logarithmic scale of regional volume. d MERFISH spatial transcriptome data with three representative genes (left) and cell type classifications (right). e Heatmap of cell-type distribution in CCFv3 (top) and DevCCF segmentations (bottom). Heatmap values show proportion of cells in a region relative to the total number of cells in the subclass. Cell-types are ordered by their parent class, denoted by color bars on the x-axis). f Registered spatial transcriptome with CCFv3 segmentation (left) and DevCCF segmentation (right). Colors are the same as in c. g Registered cell type data show the same GABA neurons in the reticular thalamus (Left, white arrow) and zona incerta (Left, green arrow) as a part of the p3 while hypothalamic glutamatergic cell type (Left, red arrow) as a part of terminal hypothalamus (Thy). Source data for plots is provided in source data file.

Fig. 7. Web Visualization for DevCCF.

a Layer panel allows layer selection with a right click and hiding by selecting the eye icon to the left of the layer. b Once selected, the layer edit tool enables user modification of layer color, contrast, and brightness. c The ontology viewer allows search and selection of individual segmentations and parent regions by ontological layer. When a region is selected in the viewer or ontology tool, the region’s metadata is displayed including region name, abbreviation, and ID. The ontology tool may be dragged to any location in the viewer. Neuroglancer allows users to visualize either individual E15.5 LSFM autofluorescence in d, MRI MTR template in e, or their overlay f. E15.5 DevCCF templates overlaid (g) and segmentation (h, i). Black dashed line in i denotes sagittal slice location of g, h. Annotation colors defined in DevCCF ontology (Supplementary Data 2).