NF1 is expressed in different cell lineages of the murine forebrain

We first verified the performance and specificity of our NF1 antibody through comprehensive control and absorption studies (Supplementary Fig. S1). Our controls demonstrated antibody reliability, allowing accurate spatiotemporal tracking throughout the developing brain. After this validation, we traced the expression of NF1 using IHC and observed broad expression in different parts of the mouse forebrain, such as the neocortex, hippocampus, choroid plexus, thalamus, and hypothalamus (Fig. 1A–P). In the postnatal day (P)35 hypothalamus, NF1 strongly marked the paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), and supraoptic nucleus (SON), involved in regulating circadian rhythm, neurohormone release and fluid balance. Similar localization of Nf1 mRNA was also observed at P35 and P0 using in situ hybridization (Supplementary Fig. S2A–B”). In embryonic ages E17.5, E14.5, and E12.5, NF1 protein expression was observed along the lateral and third ventricular linings, including neocortical and ventral telencephalic ventricular zones, cortical hem, and hippocampal primordium (Fig. 1D–F). Specifically, NF1 showed perinuclear expression in the neocortical plate and white matter as well as CA fields and hilus of the hippocampus; comparatively low expression was observed in the dentate gyrus (DG) (Fig. 1G–I). By colabelling with different cell-type-specific telencephalic markers across development, we identified sets of cells that express the NF1 protein (Fig. 2). We observed strong perinuclear labelling of NF1 in excitatory pyramidal neocortical neurons from the peak neurogenic stage at E14.5 till developed cortical stage at P35 (Fig. 2A–H, K,L). This includes colabeling with markers specific to all mature pyramidal neurons (NeuN), deep layer-specific neurons (Ctip2), and upper layer neurons (Satb2). When checked for colocalization with the progenitors at the neocortical ventricular zone, NF1 was found perinuclearly in the Sox2+ apical progenitors at E17.5, E14.5, and E12.5, but not in the Tbr2+ intermediate progenitor cells (Fig. 2I, J,M–P). Further, NF1 was strongly expressed in the hippocampal CA1 and CA3 pyramidal neurons and hilar neurons and mildly in dentate granule cells, as observed at P35, P0 and E17.5 (Fig. 2Q–Y”). These were determined by colabelling NF1 with NeuN to mark all differentiated hippocampal neurons across ages, and Ctip2, which prominently marks CA1 and developing DG at E17.5. Even at E14.5, weak NF1 expression was observed in the ventricular zone of cortical hem and hippocampal primordium/anlage (Fig. 2Z–Z”). E12.5 dorsal midline showed mild NF1 expression mostly concentrated along the ventricular lining (Fig. 2AA–AA”).

We also observed strong NF1 expression in different subtypes of cortical interneurons at P35, colabelling with Parvalbumin and Somatostatin (Supplementary Fig. S3A-D”). Interestingly, NF1 was observed to be present only in certain subsets of Olig2+ oligodendrocyte populations in the major axonal tracts of P35 mouse forebrain, namely corpus callosum, hippocampal commissure, and anterior commissure (Fig. 3A-F’’). This observation may suggest differential function of molecularly distinct subtypes of oligodendrocytes when NF1 levels are disrupted in the brain.

Fig. 1

NF1 is expressed in mouse forebrain. A Schematics showing representative coronal sections of the mouse forebrain at adult and embryonic stages. NF1 protein expression in whole brain (B–E), hippocampus (E–I), and neocortex (J–M) across descending ages P35, P0, E17.5, E14.5, and E12.5. B–F NF1 was expressed in distinct zones of thalamus (t) and hypothalamus (hy) in the diencephalon. In the hypothalamus, NF1 clearly marked the paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), and supraoptic nucleus (SON) (B). Dotted boxes in B–E mark insets of parts of telencephalon, namely the neocortex (nctx) and hippocampus (h). G-I Differential NF1 expression was observed in the hippocampus at E17.5, P0, and P35 – strong expression in CA1, CA3, and hilus and comparatively milder expression in the dentate gyrus (DG). J–K NF1 was also expressed along the lateral and third ventricular linings, encompassing midline structures, namely choroid plexus (cp), cortical hem (ch), hippocampal primordium (hp), and ventral telencephalon (vtel) across ages (arrows, H-K). L–P NF1 is strongly expressed in all mature pyramidal neurons perinuclearly and in the ventricular/subventricular progenitor zones of neocortex, across development. P, posterior; A, anterior; hp, hippocampal primordium; cc, corpus callosum; wm, white matter; 3 V, third ventricle; lv, lateral ventricle; mz, marginal zone; cp., cortical plate; sp, subplate layer; iz, intermediate zone; vz, ventricular zone; svz, subventricular zone. Scalebars: 500 μm (B–F); 100 μm (G–K); 50 μm (L–P)

Fig. 2

NF1 colocalizes with mature neurons and apical progenitors of forebrain. A–D’’ NF1 colocalizes with mature neuronal marker NeuN in P35 (A–A’’) and P0 (C–C’’) neocortical plates (cp); insets show colocalizations indicated as yellow in both ages (B–B’’, D–D’’). E–H NF1 was expressed in both deep and upper neocortical layers. Perinuclear NF1 expression surrounded the nuclear expression of transcription factors Ctip2 and Satb2, respectively demarcating deep and upper layers. I, J NF1 is present perinuclearly in Sox2+ apical progenitors at the ventricular zone (vz) but not in Tbr2+ intermediate progenitor cells. K–N NF1 was expressed in both the developing cortical plate marked by Ctip2 and Sox2+ apical progenitor zone at E14.5, but not in the Tbr2+ subventricular zone (svz). O, P NF1 was expressed in the Sox2+ apical progenitor zone at E12.5. Arrows in J, N, P indicate perinuclear NF1 expression in the apical progenitors. Q-Y’’ NF1 was expressed predominantly in the CA fields (CA1, CA3) and hilus of P35, P0, and E17.5 hippocampi; relatively low expression was seen in the granule neurons of dentate gyrus (DG) across ages. Hippocampal neuron-dense regions were denoted by NeuN and Ctip2 expressions. (Z-AA’’) At E14.5 and E12.5, the dorsal midline of the telencephalon showed NF1 expression in choroid plexus (cp), ventricular lining, and a subset of mature neurons derived from the cortical hem (ch) and the hippocampal primordium (hp). nctx, neocortex; wm, white matter; 3V, third ventricle; LV, lateral ventricle; mz, marginal zone; cp, cortical plate; sp, subplate layer; iz, intermediate zone; vz, ventricular zone; h, hippocampus. Scalebars: 100µm (Q–Q’’, T-T’’, W–W’’); 50µm (A–A’’, R-S’’, U–V’’); 25µm (B–C”, E-I, K, M, O, X–AA’’); 12.5 μm (D–D’’, F, H, J, L, N, P)

Fig. 3

NF1 is expressed in subsets of cortical oligodendrocytes. A–A’’ NF1 was expressed in a subset of Olig2+ oligodendrocytes in the main three cortical commissures of the P35 mouse brain - corpus callosum (A–B’’), anterior commissure (C–D’’), and hippocampal commissure (E–F’’). In a magnified view, cream dashed circles mark NF1+Olig2+ double-positive oligodendrocytes, while red dotted circles mark Olig2+ oligodendrocytes that did not express NF1 (B–B”, D–D”, F–F”). Scalebars: 50 μm (A–A’’, C–C’’, E–E’’); 25 μm (B–B’’, D–D’’, F–F’’)

NF1 is expressed in different cell lineages of the murine cerebellum

Unlike the forebrain, a more restricted protein expression profile of NF1 was observed in the murine cerebellum across the developmental timeline (Fig. 4A–J”). Particularly, strong perinuclear NF1 expression was observed in mature and migrating Purkinje cells, colabelling with Calbindin (Fig. 4E’,F’,G’; Fig. 5A–D”). On the other hand, relatively low NF1 expression was observed at any stage of cerebellar granule cell development, when colabelled with NeuN (Fig. 5E–H”,M, N). Nf1 mRNA was also observed in distinct parts of the cerebellum at P35 and P0 using in situ hybridization (Supplementary Fig. S2C–D”). Intriguingly, NF1 expression was observed throughout the development of the cerebellar neurons, be it in the migratory state or transitory state at the embryonic nuclear transitory zone (NTZ) or later when forming the deep cerebellar nuclei (DCN) located within the white matter (Fig. 4E”,F”,G”H”, I, I’,J, J’). At P12, NF1 expression was strong in the developing white matter tracts within folia, mostly depicting NF1+ oligodendrocytes and interneurons. NF1 coexpressed with NeuN, marking neurons at the DCN, from P3 to P35 (Fig. 5I–K”); NeuN+ cells were not present in the prospective DCN area at P0 (Fig. 5L–L”).

At embryonic stages, the cerebellar anlage has distinct proliferative zones, namely rhombic lip and cerebellar ventricular zone (CVZ) (Leto et al. 2016). At early cerebellar development (E12.5), CVZ is further subdivided molecularly into the Purkinje cell-producing zone and the Pax2+ interneuron-producing zone. Later in development, the whole CVZ becomes potent to produce cerebellar interneurons. Perinuclear NF1 expression was observed around Sox2 + nuclei in the apical progenitors of the CVZ at E12.5 and E14.5 (Fig. 6A–B-B”,D, E–E”). However, no overlap was observed in rhombic lip-derived Pax6+ cells at E12.5 or E14.5 (Fig. 6C–C”, F–F”). We also found NF1 to mark differentiated Pax2+ cells that have migrated out of the interneuron-producing side of the CVZ at E14.5 (Fig. 4I, I”, Supplementary Fig. S4A–B”). However, NF1 was expressed in only a small subset of Tbr2 + rhombic lip-derived unipolar brush cells at E14.5 (Supplementary Fig. S4E–E”).

Finally, similar to the forebrain results, NF1 showed an interesting expression pattern in glial lineages of the cerebellum (Fig. 7A–N”). At both the cerebellar folium and the DCN region, NF1 showed perinuclear expression in a subset of Olig2+ oligodendrocytes, as observed at P35 and P12 (Fig. 7A–H”). The same trend was observed in the subpopulations of Bergmann glia, which are unipolar astrocytes of the cerebellum (Fig. 7I–N”).

Taken together, here we report differential expression of NF1 in the mouse brain, specifically in glial populations, along development. This suggests the possibility of alternate regulatory mechanisms that eventually govern brain function.

Fig. 4

NF1 is expressed in the mouse cerebellum. A–D Schematics showing sagittal sections of the mouse cerebellum at adult, early postnatal, and embryonic stages, demonstrating how a complex lobular laminated structure like the cerebellum develops from a simple fate-specified neuroepithelium. Boxes mark insets at P35, P12, P3 and P0 indicating magnified views of folium and cerebellar ventricular zone. E–F’’ P35 and P12 cerebella showed strong NF1 expression in the Purkinje cell layer (pcl), different cells in the white matter (wm), and in the deep cerebellar nuclei (DCN). G–H’’ NF1 was also expressed in migrating Purkinje cells and in developing DCN at perinatal ages, P3 and P0. I-J”” At E14.5 and E12.5, NF1 was expressed very strongly in immature cerebellar neurons of the nuclear transitory zone (NTZ), and in differentiating neurons near the cerebellar ventricular zone (cvz) and/or extra cerebellar ventricular zone. Weak NF1 expressions were observed in migrating rhombic lip-derived cells. NF1 was expressed in the hindbrain choroid plexus (cp) across the developmental stages, from E12.5 to the adult stage. Mes, mesencephalon; rl, rhombic lip; ml, molecular layer; gcl, granular cell layer; egl, external granular cell layer; igl, internal granular cell layer. Scalebars: 200 μm (E, F, G, H, I, J); 50 μm (E’–F”); 20 μm (G’–H”, I’–J’’)

Fig. 5

NF1 is expressed in cerebellar neuronal lineages across development. A–D” NF1 was expressed in Purkinje cells across development, and arrowheads show its colocalization with Calbindin (Calb). Insets show magnified views of one Calb+ Purkinje cell across development and its colocalization with NF1. E-H” Cerebellar granule cells showed low coexpression of NF1 and NeuN across development. I–L” NF1 expression was predominant in the mature neurons of deep cerebellar nuclei (DCN), across development, colocalizing with NeuN (cream, dashed circles). NeuN+ mature cells were not observed here at P0. M, N Schematics demonstrate morphology and differential lamination of developing (< P21) and developed (> P21) mouse cerebella. Mes mesencephalon, rl rhombic lip, ml molecular layer, gcl granular cell layer, egl external granular cell layer, igl internal granular cell layer, bv blood vessels; cp choroid plexus, bg Bergmann glia. Scalebars: 25 μm (A–B”, E–F”); 10 μm (C–D”, G–L”)

Fig. 6

NF1 is expressed in the embryonic cerebellar ventricular progenitors. A, D Schematics demarcate different germinal zones of cerebellar anlage at E14.5 and E12.5. At E12.5, the cerebellar ventricular zone is subdivided into the zone producing Purkinje cells (deep blue) and the interneuron-producing zone (light blue). (B–B”, E–E”) NF1 is expressed perinuclearly in Sox2+ progenitors in the cerebellar ventricular zone at E12.5 and E14.5. However, NF1 was not expressed in Pax6+ secondary progenitor cells derived from the rhombic lip. Mes mesencephalon, NTZ nuclear transitory zone, 4 V fourth ventricle, cvz cerebellar ventricular zone. Scalebars: 20 μm (B–F”)

Fig. 7

NF1 is expressed in cerebellar glial lineages. (A–H’’) NF1 was expressed in a subset of Olig2+ oligodendrocytes in both folium (A–D”) and deep cerebellar nuclei (E–H”) at mature (P35) and developing (P12) mouse cerebella (arrowheads). In magnified views (B-B”, D–D”, F–F”, H–H”), cream dashed circles mark NF1+Olig2+ double-positive oligodendrocytes, while red dotted circles mark Olig2+ oligodendrocytes that did not express NF1. I–N” NF1 was also expressed in the soma and projections of the Bergmann glia across development. Colocalization with Blbp is shown using arrowheads. ml molecular layer, gcl granular cell layer, egl external granular cell layer, igl internal granular cell layer, pcl Purkinje cell layer, wm white matter. Scalebars: 25 μm (A–A”, C-C”, E–E”, G–G”, I-I”, K-K”, M–M”); 10 μm (B–B”, D-D”, F-F”, H–H”, J-J”, L–L”, N–N”)

NF1 is expressed in distinct cell layers of the eye and olfactory bulb

Beyond regular brain regions, NF1 was also found to be expressed in accessory brain regions like the eye and olfactory bulb, both of which are connected to neurosensation (Fig. 8A–R”). Expression of NF1 in the developing eye was substantially strong in the lens, retinal pigmented epithelium (RPE), and neuroretina. In the neuroretina, NF1 expression was distinct in the photoreceptor cell layer (PRC) and in the retinal ganglionic cells and Müller glial cells present in the ganglionic cell layer. A subset of NF1+ cells in the retinal ganglion cell (RGC) layer colocalized with Tbr2, which is known to be a key regulator of the intrinsically photosensitive RGCs (Chen et al. 2021). Sox2+ zone of E14.5 and E17.5 neuroretina largely did not overlap with the NF1+ layers, apart from the PRCs (Fig. 8A, C-F”). With respect to the developing olfactory bulb, NF1 was observed specifically in the mitral cell layer (MCL) of the main olfactory bulb (MOB), which contains projection neurons critical for processing the sense of odours (Davison and Katz 2007). NF1 was also expressed in the accessory olfactory bulb (AOB) from P0 to P35, which is important for aggression and reproduction (Ennis and Holy 2015) (Fig. 8G–J). NF1 overlapped with NeuN in the MCL at P35 and P12 (Fig. 8K, L,O–P’’). On the other hand, it did not have much overlap with Pax6+ cells at P3 and P0, apart from posterior AOB (Fig. 8M, N,Q–R”).

Fig. 8

NF1 expression in murine optic cup and olfactory bulb. A, B Schematics showing zones and cell layers of neurosensory organs, eye and olfactory bulb. C, F” NF1 is expressed in the developing mouse eye, predominantly in the lens (L) and neuroretina (NR), which includes the photoreceptor layer (PRC), retinal ganglionic cells and Müller glial cells in the broad retinal ganglion cell layer (RGC) at E17.5 and E14.5. Magnified insets (D–D”, F–F”) show colocalization of NF1 and Tbr2 in the lens and a subset of intrinsically photosensitive RGCs. NF1 also showed wide coexpression with Sox2 in PRC and retinal pigmented epithelium (RPE), but not with the interneurons of neuroretina. NF1 colocalized with Tbr2 in the lens and a subset of (G–J) NF1 was expressed strongly across the postnatal development of the olfactory bulb. It showed strong expression in the mitral cell layer (MCL) of the main olfactory bulb (MOB) and also in the accessory olfactory bulb (AOB). K, L, O–P” Colabelling with NeuN at P35 and P12 identified subsets of mitral cells that were double positive for NF1 and NeuN (arrowheads). M, N, Q–R” Strong coexpression with Pax6 was seen in posterior AOB (pAOB) at P0 and P3, but not in the other parts of MOB. IN interneurons, aAOB anterior accessory olfactory bulb, EPL external plexiform layer, GCL granule cell layer, GL glomerular layer. Scalebars: 200 μm (G, H, K, L); 100 μm (C–C”, E–E”, I, J, M, N); 50 μm (O–O”, P–P”); 25 μm (Q–R”); 20 μm (D–D”, F–F”)

Reanalysis of single-nuclei RNA-sequencing (snRNA-seq) data reveals enriched Nf1 expression in diverse neural cell types

To determine the expression of Nf1 at higher resolution, we utilized publicly available snRNA-seq databases (Mortberg et al. 2023; Kozareva et al. 2021). In the adult mouse cortex, Nf1 expression was identified in almost all the marked cortical clusters (Fig. 9A–C). However, detailed analysis revealed specific enrichment of Nf1 transcripts in the lineages of cortical interneurons, oligodendrocytes, and excitatory neurons (Fig. 9D). Specifically, Nf1 expression was highest in the oligodendrocyte lineage. Moreover, among different oligodendrocyte clusters, Nf1 expression was found to be highest in differentiating oligodendrocytes (Fig. 9D). Based on our IHC data, all Olig2+ cells in the cortex were not positive for NF1. Hence, we looked at the percentage of cells positive for Nf1 expression across different cortical clusters (Fig. 9E). Interestingly, oligodendrocyte precursor cells (OPCs) and differentiating OPCs showed a very high frequency of Nf1-expressing cells than mature oligodendrocytes. This data strongly suggests the importance of NF1 in oligodendrocyte development. Besides oligodendrocytes, cortical interneurons and excitatory pyramidal neurons also exhibit higher frequency and expression, corroborating our IHC findings.

We performed a similar analysis on adult cerebellum and identified an enriched expression of Nf1 in CVZ-derivatives, such as cerebellar interneurons, Purkinje cells, Bergmann glia, and rhombic lip-derivative unipolar brush cells (Fig. 9F–H). Among these clusters, the highest expression was found in the Bergmann glial cells (Fig. 9I). Similarly, Nf1 showed enriched expression in the oligodendrocyte lineage of the adult neocortex, with a higher frequency of Nf1-expressing cells in the OPC cluster as compared to mature oligodendrocytes (Fig. 9H). The frequency plot also showed that almost all cells in the Purkinje cell cluster were positive for Nf1, thus aligning with our IHC expression data.

Fig. 9

Reanalysis of single-nuclei RNA-seq data showing expression of Nf1 in adult mouse brain. A, F UMAP plot for single-nuclei RNA-seq data from adult mouse cortex and cerebellum, respectively. Transcriptionally distinct clusters were coloured based on cell type. B, G UMAP plot illustrating the expression level of Nf1 (red) across different cortical and cerebellar clusters, respectively. C–E Nf1 expression is higher in cortical interneurons, differentiating OPCs, and excitatory neurons of adult murine cortex compared to other neural populations. H–J In the adult murine cerebellum, Nf1 expression is enriched primarily in Purkinje cells, Golgi Ins, Bergmann glia, and OPCs. MGE_INs medial ganglionic eminence interneurons, dMGE_INs dorsal medial ganglionic eminence interneurons, CGE_INs caudal ganglionic eminence interneurons, OPCs oligodendrocyte precursor cells, MLI molecular layer interneurons, PLI Purkinje layer interneurons, INs interneurons, UBC unipolar brush cells

Overall, these reanalysis results not only validated our IHC data but also helped identify Nf1 expression in some neural cell types, such as differentiating oligodendrocytes, which are difficult to capture using conventional IHCs. Our in-depth analysis also uncovered the graded expression pattern of Nf1 across diverse cell types in the murine adult brain. This may suggest differential role of Nf1 in these diverse subtypes.