Protracted neuronal recruitment in the temporal lobes of young children

Abstract

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs^1-4. The human EC continues to develop during childhood⁵, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5⁺RELN⁺ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5⁺RELN⁺ cells^6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

Fig. 1. Stream of migratory neurons in the perinatal human temporal lobe.

a, Optical section of a 7.6-mm-thick stack of cleared medial temporal lobe at birth, stained with TOPRO and imaged using light-sheet microscopy. The EC stream can be identified as a multilayered lamina of dense clusters of nuclei extending medially from the tLV. Three-dimensional (3D) reconstruction of these clusters highlights their extension along the anterior–posterior axis (see Supplementary Video 1). b, Coronal section at 38 GW, showing DCX⁺ immunostained cells in the EC stream. HP, hippocampus. c, Deconvolution image at birth, showing a dense cluster of DCX⁺ cells co-expressing PSA-NCAM (arrowheads) with elongated nuclei (DAPI). d, Coronal maps of the EC stream (purple arrows) at 38 GW; mapped sections spaced by around 1 mm from the anterior tip of the basolateral amygdala (BLA). e, Sagittal maps of the EC stream (purple arrows) at 10 postnatal days; sections spaced by around 1 mm from the medial end of the tLV. f, Left, transmission electron microscopy (TEM) image of immature neurons in the human EC stream at birth, showing densely packed neurons (red, N) with compacted chromatin and fusiform morphology, surrounded by astrocytes (blue, A); note the presence of ependymal cells (cyan, E; see also Fig. 3). Middle, higher magnification of a neuron with adherens junctions (arrowheads, inset). Right, many cells in the EC stream had features of migratory neurons: a centrosome (inset) opposite a trailing process with an adhesion point (arrowhead). Scale bars, 1 mm (a,b,d,e); 100 µm (b bottom); 10 µm (c, f left); 1 µm (f middle and right); 200 nm (f middle and right insets). D, dorsal; L, lateral; A, anterior.

Fig. 2. The EC stream supplies migratory neurons until the age of two to three years.

a, Maps of DCX⁺PSA-NCAM⁺ cell clusters in coronal sections of the human medial temporal lobe from birth to 24 months of age. Bottom right box: DCX⁺PSA-NCAM⁺ immunostained cell clusters are shown at 11 months of age. b, Maps of the orientation of DCX⁺ neurons in coronal sections from birth to three years of age, indicating the location of radial (orthogonal to the cortical surface; green) and tangentially oriented (yellow) neurons in the EC. c, Number of neurons with radial and tangential orientations in subcortical white matter and in the EC between birth and three years of age, counted in 20-μm sections. d, Maps of DCX⁺ cells in the macaque medial temporal lobe between birth and six months of age, and (inset) an example of DCX⁺ immunostained cells. e, DCX⁺ immunostaining in the macaque EC stream region indicated in d (box) in at birth and at three months of age. Scale bars, 1 mm (a maps, b, d maps); 100 µm (e); 10 µm (a bottom right stains, d top left).

Fig. 3. The EC stream extends along a radial scaffold next to a fused extension of the ventricle.

a, At 18 GW, coronal sections show Ki-67⁺SOX2⁺ cells prominent in the CGE and lining the ventricle walls. Ventrally, the medial wall facing the hippocampus and lateral wall facing the cortex are open (dotted line). DG, dentate gyrus. b, At 22 GW, the medial and lateral ventricular zone (VZ) are in close proximity. c, At 22 GW, in an adjacent section (boxed region in b), FOXJ1⁺ cells are present in the medial wall VZ, but not on the opposite wall. Note the network of vimentin⁺ fibres of RG on the lateral wall (inset). d, At birth, the ventricular walls in the EC stream region have fused together, with some remaining FOXJ1⁺ ependymal cells (arrowheads), but no open ventricle. Note the multiple layers of medially oriented DCX⁺ cells flanked by vimentin⁺ fibres turning away from the ventricle (arrow). Right, at seven months, clusters of DCX⁺ cells remain in the EC stream, but have decreased in size. Some FOXJ1⁺ ependymal cells (arrowheads) and vimentin⁺ fibres remain. e, TEM of five ependymal cells (E) in the EC stream at birth, containing multiple long cilia (yellow arrowheads), ciliary basal bodies (magenta arrowheads) and cell–cell junctions typical of ependymal cells (magenta arrows). f, Ultrastructural detail of an immature migratory neuron (red, N) in the EC stream at birth surrounded by glial fibres and cell bodies (blue, A). Adherens junctions (magenta arrows) are visible next to glial fibres (magenta arrowheads). Scale bars, 1 mm (a,b); 100 µm (c left, d); 10 µm (c inset, e left, f top left); 1 µm (e right, f right and bottom).

Fig. 4. The postnatal EC stream mainly supplies migrating CGE-derived interneurons.

a, Tissue section showing a microdissected region of the EC stream from a two-week-old sample (dotted box). Sub, subiculum. b, Unsupervised clustering of nuclei in the EC stream. OL, oligodendrocytes; OPCs, oligodendrocyte precursor cells; Im, immature; pre-OL, pre-oligodendrocytes. c,d, Gene-expression feature plots (c) and relative abundance (d) of immature excitatory (TBR1⁺DCX⁺) and inhibitory (DLX2⁺GAD2⁺DCX⁺) neurons and MGE-derived (LHX6⁺) or CGE-derived NR2F2⁺ interneurons. e, At birth, the tLV lateral wall contains many DCX⁺DLX2⁺ cells that are TBR1^–. f, EC stream DCX⁺ cells (at birth) co-stained for DLX2 and TBR1 or DLX2 and COUPTFII. g, Left, SCGN⁺ cell clusters in the EC stream at 38 GW, birth and 7 postnatal months. Right, DCX⁺SCGN⁺ cells at birth in the temporal lobe V-SVZ, EC stream and extending into the EC. h, The human EC at 38 GW immunostained for DCX and DLX2 or TBR1. i, The human EC between birth and two years of age, immunostained for SCGN. j, The human EC at 38 GW and two years of age, showing DCX⁺ cells co-stained with COUPTFII, SP8, LHX6 or NKX2.1 (arrowheads). k, Percentage of DCX⁺ cells co-stained with COUPTFII, SP8, LHX6 or NKX2.1 in the EC between birth and two years of age. Large dots, mean; small dots, sampled images; bars, s.e.m. of images for each individual; n = 5 individuals in three independent experiments. l, Top two rows: macaque EC stream region at birth, containing DCX⁺ cells co-stained with DLX2 (arrows) that are COUPTFII⁻. Bottom row: this region contains individually migratory DCX⁺SP8⁺ cells (arrows). Scale bars, 3 mm (a); 100 µm (e overview, g left, h left, i left, l); 20 µm (e inset, g right panels, i insets, j); 10 µm (f, h right panels).

Fig. 5. Progenitor cells in the CGE and EC stream region continue dividing at birth.

a, Coronal map (at birth) of Ki-67⁺ cells (green), blood vessels (red) and DCX⁺ cell clusters (cyan) along the tLV and extending along the EC stream. Boxes show the anatomical locations of subsequent panels. b, Ki-67⁺vimentin⁺ RG (arrow) at the medial end of the EC stream region at birth. c, At birth, Ki-67⁺vimentin⁺ cell bodies located proximal to the tLV are either HOPX⁺ (arrowhead) or HOPX^– (arrow). d, Lineage trajectory analysis using Monocle reveals a bifurcation emerging from the cluster of RG into an oligodendrocyte lineage (blue, top) and a neurogenic lineage of excitatory cells (green, bottom); on the right, expression of key maturation-associated genes across pseudotime. e, Ki-67⁺COUPTFII⁺ cells (arrows) and DCX⁺COUPTFII⁺ cells (arrowheads) located in the CGE region of the temporal lobe at birth. f, TEM within the CGE region at birth (near boxed inset in e) showing a pair of recently divided cells completing cytokinesis (top boxed region; left of the two magnified panels) near a cell with ultrastructural features of a migratory neuron (bottom boxed region; right of the two magnified panels). g,h, Quantification of the density of Ki-67⁺ cells in the CGE (g) from 22 GW to birth and in the EC stream (h) from 28 GW to 11 postnatal months. Data points are the average section density at each age; bars are mean ± s.e.m., n = 4 individuals (g) and n = 6 individuals (h) in three independent experiments. Scale bars: 1 mm (a); 100 µm (b, c top, e left); 10 µm (c bottom, e right, f left); 2 µm (f right).

Fig. 6. The EC stream mainly supplies CGE-derived LAMP5⁺ interneurons.

a, Samples collected for snRNA-seq. b–e, Uniform manifold approximation and projection (UMAP) plots of maturing interneurons showing the sample origin (b), donor age (c), subpallial lineages (d) and mature neuronal types (e). f, Heat map of the top differentially expressed (DE) protein-coding genes in each cell identity shown in d,e. g, Top row, main maturation trajectories inferred from Monocle. Bottom row, histograms showing the contributions of each sample origin to each trajectory along pseudotime. h, Number of cells from the EC stream in each of the trajectories shown in g. i, Spatial transcriptomic label transfer identifies superficial- and deep-layer LAMP5⁺ subpopulations. j, Volcano plot of a DE analysis (quasi-likelihood F-test based on pseudobulk counts aggregated by donor) between superficial-layer (blue) and deep-layer (magenta) LAMP5⁺ interneurons. k, Module scores for superficial- and deep-layer LAMP5⁺ cells. Density lines drawn for non-EC stream cells (grey; see Extended Data Fig. 13). l, At birth, LAMP5⁺DCX⁺ (arrows) and LAMP5⁺COUPTFII⁺ (arrowheads) young neurons are present in the EC stream and in the EC. m, At birth, RELN⁺DCX⁺COUPTFII⁺ cells are present in streams of migrating neurons close to the ventral temporal lobe VZ. In the EC, many RELN⁺ cells express COUPTFII and a subset are DCX⁺ (arrow). n, Top, maps of cells in the EC at birth co-expressing markers of CGE-derived (COUPTFII⁺) interneuron subpopulations: LAMP5, RELN, calretinin (CR) and VIP. Bottom, their frequency distribution across cortical layers. Scale bars, 50 µm (l); 1 mm (n); 100 µm (m top right); 20 µm (m bottom right).

Extended Data Fig. 1. The EC stream of migratory neurons extends medially from the temporal lateral ventricle as a multilayered lamina along the anterior–posterior axis.

a, Three orientations of the cleared tissue block in Fig. 1a stained with TOPRO and imaged using a light-sheet microscope. Dense chains emerging from the ventricle can be seen escaping from the ventricle in horizontal view (arrowheads). Reconstruction of the EC stream is shown from a ventral view facing anterior-medially. b, Immunostained sections of the EC stream at birth showing dense clusters of DCX⁺PSA-NCAM⁺ cells. c, Map at 10 days postnatal showing the location of insets (1–2) with a cross section of DAB immunostained PSA-NCAM⁺ cell clusters. Boxes 3 and 4 show a location next to the ventricle with DAB immunostained PSA-NCAM⁺ cells in a continuous layer next to the ventricle and in distinct clusters at varying distances from the ventricle. d, Map at birth of DCX⁺PSA-NCAM⁺ cell clusters in a coronal series of sections of the medial temporal lobe. Clusters distributed along a medial-lateral orientation are present ventral to the basolateral amygdala between the EC and medial temporal gyrus (i-iv). The EC stream (arrow) is visible from the first section containing the temporal lobe ventricle and at every section examined posteriorly across the hippocampus (iii-viii). Note at levels iii and iv an additional laterally oriented migratory stream (green arrowhead). e, TEM of immature neurons in the human EC stream at birth showing densely packed neurons (red, N) with compacted chromatin and fusiform morphology (inset), astrocytes (blue, A) and microglia (yellow, M). Scale bars: 1 mm (a, d), 100 µm (b left panels, c 3, 4), 20 µm (c 1,2), 10 µm (b right 1,2), 2 µm (e). Abbreviations: BLA basolateral amygdala; EC: entorhinal cortex; FuG: fusiform gyrus; HP: hippocampus; ITG: inferior temporal gyrus; MTG: medial temporal gyrus, STG: superior temporal gyrus; sub: subiculum; tLV: temporal lobe lateral ventricle.

Extended Data Fig. 2. Individual DCX⁺ neurons migrate into the EC from birth to three years of age.

a, Maps of DCX⁺ neurons (green) in coronal sections between birth and 3 years. b, DCX⁺ cell orientations between birth and 3 years of age in the same sections shown in Fig. 2b. Here colours depict the direction of the leading process, with colours representing a 180 degree arc. c, Coronal maps at birth and 3 months of age segmented into sectors (dotted lines) with rose frequency histograms. d, Individual DCX+ neurons at birth, 7 months, and 3 years of age in the EC and their relationship to vimentin+ processes. e, Sagittal maps at 10 days postnatal from medial (i) to lateral (v) segmented into sectors (dotted lines). Within each sector, the orientations of the leading process of all individual DCX+ cells are plotted on a 360-degree (rose) frequency histogram. The magenta bar indicates the percentage of cells that fall into the most frequently occupied orientation within that sector. f–i, Macaque temporal lobe immunostaining between birth and 17 months. f, Tilescan of the macaque medial temporal lobe at birth showing location of g in adjacent section and dispersed DCX⁺ cell labelling in the EC stream region (insets). g, DCX⁺PSA-NCAM⁺ cell clusters are present in the lateral wall of the macaque temporal lobe, closer to the CGE region at birth. h, Macaque EC stream region at 3 months stained for DCX and DLX2, with no evidence of DCX-immunolabelled neurons (inset) in sharp contrast to the robustly DCX⁺DLX2⁺ RMS (right). i, DCX immunostaining uncovers no labelled cells in the EC stream region in the macaque at 6 months and 17 months. Scale bars: 2 mm (c, h top left), 1 mm (a, b, d), 500 µm (g overview, h bottom and right, i), 100 µm (f, g inset), 10 µm (e). Abbreviations: BLA basolateral amygdala; EC: entorhinal cortex; HP: hippocampus; LPL: lateral paralaminar amygdala; MPL medial paralaminar amygdala; PL: paralaminar amygdala; RMS: rostral migratory stream; tLV: temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 3. The EC stream forms next to a fused ventricle in humans during gestation.

a, EC stream region at 22 GW stained for Ki-67⁺ and HOPX⁺ cells showing location of insets in b–d. b,c, Ki-67⁺, HOPX⁺, and DCX⁺ cells in the VZ (b) and  outer subventricular zone (c) along the closely opposed walls of the ventricle in the EC stream anlage. d, Staining of an immediately adjacent section for FOXJ1 and vimentin in the region indicated in a shows FOXJ1⁺ cells not contacting the ventricle. e, At 29 GW (top) and birth (bottom), the ventricle has closed in this region which contains a field of DCX⁺ cells at 29 GW and multilayered lamina of coalesced streams at birth between the EC and the V-SVZ. In the wall of the ventricle the ependymal layer contains vimentin⁺ cells in the VZ and Ki-67⁺ cells in the V-SVZ (insets). f, Semithin section stained with toluidine blue showing the EC stream at birth (left), and TEM of cells displaying motile cilia (square inset) and an elongated neuron with ultrastructural features of a migrating cell in the EC stream at birth (right). g, Macaque temporal lobe at 3 and 17 months stained for DCX and vimentin showing open ventricle within the same region where a closed ventricle and the EC stream is found in humans (arrows). Scale bars: 1 mm (g top row), 500 µm (a, e left overviews, f left, g bottom row), 100 µm (d left, e top middle bottom right), 50 µm (b, c, d middle and right), 20 µm (e top right), 2 µm (f right panels). Abbreviations: CGE: caudal ganglionic eminence; EC: entorhinal cortex; HP: hippocampus; tLV: temporal lobe lateral ventricle.

Extended Data Fig. 4. EC stream neurons express COUPTFII between 38 GW and 11 months.

a, Map at 38 GW of DCX⁺COUPTFII⁺ cell clusters in a coronal section at the level of the anterior tip of the temporal lobe lateral ventricle (tLV) as shown in Fig. 1d. DCX⁺COUPTFII⁺ cells are located within and emanating from dense streams (arrow) at this age alongside individual DCX⁺ cells (arrowheads). b, At 38 GW, DCX⁺SP8⁺ cells individually migrating (arrowheads) and within the EC stream (arrow) are infrequent (~2%). c, Coronal map at 38 GW of the cell clusters shown in Fig. 1b from a level caudal to the map in (a). These DCX⁺ cells are COUPTFII⁺ and the stream directed to the EC breaks away from the ventricle at its closest point to the EC. d, Sagittal maps at 10 days postnatal spaced by 1 mm showing the whole tissue sections that are magnified in Fig. 1e. Dense DCX⁺COUPTFII⁺ cell clusters are present between the ventricle and EC and processes and individual cells are visible extending from the clusters (arrows). e, DCX⁺PSA-NCAM⁺ COUPTFII⁺ clusters in the EC stream at 7 months and 11 months and low magnification views of the magnified region shown (inset). f, Immunostaining of DCX⁺ cells in the EC stream at birth shows a small subset co-expressing PROX1 or LHX6. g, The percentage of DCX⁺ nuclei in the human EC between birth and 2 years; n = 3 individuals across 2 independent experiments, data points are individual images, large dots and bars are mean values +/− SD for each individual. h, COUPTFII^– DCX⁺ cells in the human RMS at 39 GW. i, At birth in the macaque, immunostaining in the temporal lobe V-SVZ and EC stream region shows dense DCX⁺PSA-NCAM⁺ cell clusters that do not co-express PAX6. Scale bars: 2 mm (d maps), 1 mm (a and c maps), 500 µm (a insets 1 and 2 left, c inset 2 left), 100 µm (a inset 1 middle panels inset 2 right panels, b top row, c inset 1, e inset), 50 µm (c inset 2 right panels, d bottom row, f, h, i), 20 µm (a inset 1 right, b bottom row, e high magnification view). Abbreviations: BLA basolateral amygdala; EC: entorhinal cortex; HP: hippocampus; PL: paralaminar amygdala; RMS: rostral migratory stream; tLV: temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 5. The EC stream supplies interneurons to the temporal lobe.

a, Coronal section at birth showing DCX⁺DLX2⁺TBR1⁻ cells with processes emanating at a branch-point (arrow) from the EC stream (1) and a stream cluster branch-point (arrow) away from the ventricle (2). b, DCX⁺ cells in the EC stream clusters at birth are DLX2⁺COUPTFII⁺ at distal, middle, and proximal distances from the tLV. Cells can be observed sending processes out of the dense cell clusters toward the EC (arrow). c, In the same section as b, the lateral wall of the tLV contains large collections of DCX⁺DLX2⁺COUPTFII⁺ cells. d, At 7 months of age, individual DCX⁺DLX2⁺ cells are found ventrally to the tLV (arrows). e, At 7 months the EC stream contains DCX⁺DLX2⁺TBR1^– cells (arrows). f, At birth, the macaque temporal lobe V-SVZ contains cell clusters with DCX⁺DLX2⁺ cells that are COUPTFII^– (arrowheads), and COUPTFII⁺ (arrows). g, At 7 months the EC contains DCX⁺DLX2⁺TBR1^– cells (arrows). h, In the EC at 38 GW, DCX⁺ cells can be observed that co-express the cortical excitatory neuron transcription factor TBR1. These cells typically had a rounded morphology (arrows) and lacked migratory features. i,j, Quantifications of marker co-expression by cells in the EC stream and in the EC between birth and 11 months for DCX, PSA-NCAM, and COUPTFII (i) or in the EC stream for DCX, PSA-NCAM, and DLX2 (j). Scale bars: 1 mm (a), 100 µm (a insets 1 and 2, b top row, c top, e top and middle left, f left overview and inset overviews 1 and 2, g left), 50 µm (b bottom row, c bottom, d top, h), 20 µm (f insets), 10 µm (a bottom right insets, d bottom, e right, bottom, and bottom right insets, g insets i and ii). Abbreviations: BLA basolateral amygdala; EC: entorhinal cortex; HP: hippocampus; tLV: temporal lobe lateral ventricle.

Extended Data Fig. 6. The EC stream forms between 22 and 27 GW.

a–d, Anatomically matched coronal sections of the medial temporal lobe at the rostral tip of the uncus of the hippocampus across ages. At 18 GW (a), Ki-67⁺SOX2⁺ cells line the walls of the temporal lobe ventricle (same section level shown in (ii) in Extended Data Fig. 8c), and the medial wall facing the EC and hippocampus has fewer of these cells and no anatomical features separating the hippocampus and EC. At this age, there is a slight tissue protrusion of the hippocampus in the lateral direction towards the ventricle, a feature that becomes rapidly more prominent in the next weeks. At 22 GW (b), the hippocampus and surrounding tissue have all grown larger and a seam has begun to form between the hippocampus and EC. At this age, there is no heightened accumulation of Ki-67⁺ SOX2⁺ or DCX⁺ cells within this region. At 27 GW (c), this region has formed a higher concentration of Ki-67⁺SOX2⁺ cells as well as DCX⁺ cell clusters indicating that the EC stream has formed by 27 GW. At 38 GW (d), the density of both the Ki-67⁺ SOX2⁺ cells and the DCX⁺ cell clusters is increased, with many of both populations present within the EC stream extending from the medioventral tLV to the EC. e, Map of the location of Ki-67⁺SOX2^– (yellow) and Ki-67⁺SOX2⁺ (magenta) cells surrounding the tLV at 38 GW. Most Ki-67⁺ cells at this age are SOX2⁺, and are found in the paralaminar nucleus of the amygdala (PL) (1), the lateral V-SVZ (2), and in the EC stream (3). Scale bars: 1 mm (a–e all left panel overviews), 100 µm (a–d right panel overviews, e 1–3), 20 µm (a right insets). Abbreviations: BLA basolateral amygdala; EC: entorhinal cortex; HP: hippocampus; oSVZ: outer subventricular zone; PL: paralaminar nucleus; tLV: temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 7. Clusters of dividing progenitors and immature neurons persist along the walls of the temporal lobe at birth.

a, Maps of coronal sections of the temporal lobe lateral ventricle (tLV) indicating Ki-67⁺ cells (green dots), the V-SVZ region (grey), blood vessels (BV, red), and DCX⁺ cell clusters (cyan). Sections are spaced by 4 mm along the rostral-caudal axis beginning at the anterior/ventral uncus of the hippocampus (i), extending across the rostral dentate gyrus (DG) (v), and more caudally across the DG (ix, xiii). The three inset regions are (1) remnants of the CGE along the dorsolateral wall of the tLV, (2), the most lateral extension of the tLV, and (3) the EC stream. b, Dividing Ki-67⁺ cells expressing MCM2 are present along the lateral extension of the tLV in the temporal lobe in the V-SVZ. Sections are spaced by 1 mm. c, A stream of DCX⁺ neurons wraps around the lateral extension of the tLV (1) and is present near Ki-67⁺COUPTFII⁺ cells (arrows) (2) which are a subset of the Ki-67⁺ population in the lateral wall at birth. d, A subset of Ki-67⁺ cells in the EC stream express COUPTFII (arrows) shown at level (i). e, Sections stained for Ki-67 and MCM2 in the CGE-remnant region in sections adjacent to one in main Fig. 5e showing cell clusters within the V-SVZ (arrows). Scale bars: 1 mm (a top row, d maps, e map), 500 µm (a insets 1,2 and 3), 100 µm (b i–xiii overviews, d top right i and v overviews, e top right ix and x overviews and bottom insets), 50 µm (b i–xiii insets, c, d i, v top right insets and i insets 1–3). Abbreviations: BLA: basolateral amygdala; CGE: caudal ganglionic eminence; DG: dentate gyrus; EC: entorhinal cortex; HP: hippocampus; ProS: prosubiculum; Sub: subiculum; tCd: tail of the caudate nucleus; tLV: temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 8. At 18 GW, the temporal lobe ventricle is surrounded by dividing progenitors in the CGE and DCX⁺DLX2⁺COUPTFII⁺ neurons extending toward the EC.

a,b, Diagrams of coronal (a) and horizontal (b) sections of the anterior temporal lobe at 18 GW showing the location of the CGE relative to the ventricle and EC. c,d, Immunostaining for Ki-67⁺SOX2⁺ progenitors and DCX⁺ young neurons in sections corresponding to those diagrammed in a,b. (Note: level ii corresponds to Extended Data Fig. 6a and level iii corresponds to Fig. 3a.) e,f, Immunostaining of sections immediately adjacent to those in c,d for SP8, COUPTFII and PROX1. g, On the ventral wall of the temporal lobe ventricle, many Ki-67⁺ cells expressing vimentin and HOPX are present in the V-SVZ and oSVZ. Similar cells are observed more dorsally along the lateral wall, in the CGE. h,i, The same region in g facing the EC contains Ki-67⁺SOX2⁺ cells mixed with DCX⁺ neurons (h), the vast majority of which are DLX2⁺ (i). j, Immunostaining of a section adjacent to the middle section in the coronal series (a) reveals a mixed population of COUPTFII⁺ and COUPTFII^– cells expressing DLX2 along the ventricle facing the EC (1) and within the EC (2). Scale bars: 2 mm (a, b), 1 mm (c–f, j left panel), 100 µm (g left overview, right CGE overview, h left panel, i left panel, j 1 and 2 left panels), 50 µm (g 1, 2, CGE right panel, h 1, 2, i right panel), 10 µm (j 1, two right panels, 2 right panel). Abbreviations: BLA: basolateral amygdala; CGE: caudal ganglionic eminence; DG: dentate gyrus; EC: entorhinal cortex; HP: hippocampus; oSVZ: outer subventricular zone; tLV: temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 9. At 22 GW, the CGE contains Ki-67⁺SOX2⁺ progenitors and many SP8⁺, COUPTFII⁺ and PROX1⁺ cells.

a, Diagrams of coronal sections of one hemisphere of the human brain at 22 GW at one anterior and one posterior level of the CGE. Red boxes indicate inset locations of immunostaining for Ki-67⁺SOX2⁺ cells. Immature DCX⁺ neurons are present in clusters between the Ki-67⁺SOX2⁺ cells (i-vi). In the ventral V-SVZ, fewer Ki-67⁺SOX2⁺ cells are visible (vii-ix). b, Diagrams of coronal sections at three cross-sections across the temporal lobe and immunostains for SP8, COUPTFII, and PROX1 at each level showing that each marker is highly expressed throughout the CGE. c, Ultrastructure of a migratory young neuron at 22 GW in the temporal lobe CGE. This cell has a classical localization of the Golgi (green arrow) and centrosome (magenta arrowhead) in the leading process filled with microtubules and displays an adherens junction (magenta arrow). Immunogold labelling for DCX reveals processes filled with microtubules at this age. d, Immunostaining of the 22-GW human temporal lobe at the level of the anterior hippocampus. Insets show higher magnification of SCGN+ cells densely clustered near the ventral extension of the temporal lobe lateral ventricle and extending into the EC. Scale bars: 2 mm (a maps, b all panels), 500 µm (a left immunostaining overviews, d), 100 µm (d inset), 20 µm (a right panels i-ix), 1 µm (c top left), 200 nm (c top right, bottom panels). Abbreviations: BLA: basolateral amygdala; CGE: caudal ganglionic eminence; EC: entorhinal cortex; HP: hippocampus; oSVZ: outer subventricular zone; tLV temporal lobe lateral ventricle; V-SVZ: ventricular–subventricular zone.

Extended Data Fig. 10. Composition of the entire snRNA-seq dataset.

a, All cells from the merged dataset, coloured by the main cell types. b–e, UMAP plots showing (b) donor age, (c) individual samples, (d) regions of origin, and (e) nuclear fraction (fraction of reads containing intronic regions). f, Heat map of the top upregulated protein-coding DE genes for each cell type in b. Clusters are ordered by the results of hierarchical clustering.

Extended Data Fig. 11. Composition of the snRNA-seq interneuron maturation dataset.

a–e, All cells from the interneuron maturation dataset. UMAP plots showing (a) results of unsupervised clustering, (b) their sample identifiers, (c) the distribution of cells from the EC stream, (d) distribution of cells from the embryonic EC at 23 GW, and (e) the calculated pseudotime. f, Heat map of top DE genes that are broadly expressed (>70% of cells) in each of the clusters identified in (a). g–j, Feature plots highlighting expression of key genes. Expression of genes associated with (g) dividing cells (TOP2A) and immature neurons (DCX, SOX4, SOX11, and SCGN); (h) interneuron origins (PROX1 and NR2F2 highly co-expressed in CGE-derived interneurons, LHX6 in MGE-derived neurons); (i) common CGE-derived interneuron subpopulations; and (j) mature neuron synaptic communication.

Extended Data Fig. 12. WGCNA analysis of inhibitory-neuron maturation.

a, WGCNA revealed five modules of genes that are highly co-expressed. The most connected genes for each module are listed and their eigengene values, a proxy for the combined gene expression within each module is shown in UMAP plots. b,c, Eigengene for each module plotted against pseudotime, with individual cells coloured by (b) donor age and (c) region of origin. d, Violin plots showing the distribution of eigengene across different regions and ages.

Extended Data Fig. 13. Spatial transcriptomic label transfer reveals superficial- and deep-layer LAMP5⁺ neurons.

a–g, A subset of the interneuron maturation dataset consisting of (a) only maturing CGE-derived LAMP5+ cells (Im. CGE-2 and LAMP5⁺). UMAP plots showing the (b) region of origin, (c) donor age, (d) cluster number, (e) spatial transcriptomic label transfer result and (f) inferred layer identities of LAMP5⁺ cells in our dataset. g, Module scores for superficial- and deep-layer LAMP5⁺ cells based on the top 10 DE genes between the two subpopulations. h, Module scores for each of the cells in a–g, colored by their inferred layer identities as in f. i, Heat map showing the top 25 most DE genes between superficial- (top panel) and deep-layer (bottom panel) cells. Cells were split by sample origin (first annotation row on top of heat map) and layer identity (second row). In each column, cells were ordered by pseudotime (third row) and donor age information is also shown (fourth row). Among immature LAMP5⁺ neurons derived from the EC stream (leftmost column), cells had a mixed expression of genes associated with superficial and deep layers, with some cells starting to express some markers such as NCAM2, PLD5, NR2F2-AS1 and ROBO2.

Extended Data Fig. 14. The EC stream supplies CR⁺ and RELN⁺ interneurons to the EC.

a–c, At birth, only a few individual DCX⁺RELN⁺ neurons (arrow) are present in the EC stream (a), whereas the ventral wall of the tLV contains multiple chains of DCX⁺RELN⁺COUPTFII⁺ neurons (b), location of inset shown in Fig. 6m. Some DCX+RELN+COUPTFII+ neurons are observed in the DCX⁺ chains wrapping around the lateral edge of the tLV (c). d, At 7 months of age, DCX⁺RELN⁺ cells are present in some dense clusters within the EC stream. e, Examples of DCX⁺RELN⁺COUPTFII⁺ cells in the EC at 11 months of age (arrows). f, Examples of of RELN⁺COUPTFII⁺ cells in the upper layers of the EC at 2 years of age. g, 38 GW coronal section with high magnification insets of DCX⁺CR⁺TBR1^– cells (arrows) in the EC stream clusters and EC (1–4). These cells are detectable in the EC stream at distal (1), middle (2), and proximal (3) levels relative to the medioventral tLV where the stream breaks away from the ventricle. Many additional DCX⁺CR⁺TBR1^– cells (arrows) are present in the EC (4) at birth, at the putative termini of the stream. h, At birth the EC contains DCX⁺CR⁺COUPTFII⁺ cells. i, At 2 years of age the vast majority of CR⁺ neurons in the EC co-express COUPTFII (inset). These cells display multiple morphologies including unipolar, bipolar, and multipolar. Scale bars: 1 mm (g left), 100 µm (g 1–3 top, h left, i left, a left, b left, c top left, d left, e left), 50 µm (g 4 top, a right insets, b right insets, c bottom and right insets, d right insets, e right, f), 10 µm (g 1–4 bottom, h right, i right). Abbreviations: EC: entorhinal cortex; HP: hippocampus; tLV temporal lobe lateral ventricle.