Fig 2: Immunofluorescent staining of HOPX and BLBP in coronal sections of occipital (a), temporal (b), parietal (c) and frontal (d) neocortex from 21 wpc (a) and 19 wpc (b–d) human fetal brains. The parietal cortex shown here has been sectioned close to the frontal lobe, which may explain their similar appearance. The outer fibrous layer (OFL) is visible in (a). The inner fibrous layer (IFL)/outer subventricular zone (OSVZ) seem to increase in size in parietal and frontal cortex with a higher density of horizontal fibers interdigitating with radially oriented radial glial cells and their extensions. The dotted line in (a–d) illustrates a proposed outer demarcation of the OSVZ. FC, frontal cortex; OC, occipital cortex; PC, parietal cortex; TC, temporal cortex. Scale bars: 500 μm.

Fig 3: Distribution of toluidine blue (TB) (a), vimentin (VIM) (b), BLBP (c) and HOPX (d) immunoreactivity in consecutive coronal sections of occipital cortex from a 21 wpc fetus (CRL: 200 mm). HOPX, BLBP and vimentin are all present in the endfeet layer (EFL) (arrows in (d)) lining the pial surface and in a large proportion of cells in the outer subventricular zone (OSVZ), separating the inner‐ (IFL) and outer fibrous layer (OFL). The zonal configuration of OSVZ as depicted with VIM, BLBP and HOPX is attenuated towards the calcarine sulcus (CS) and a narrower possible OSVZ emerges below CS where the majority of HOPX‐positive cells is stained with less intensity and oriented tangentially as opposed to the radial orientation of HOPX‐positive cells in the adjacent OSVZ (not shown). The cortical plate as defined by TB is divided into two sections by BLBP. Vimentin stains the ventricular zone (VZ), which is almost devoid of HOPX and contains few BLBP‐positive cells. The framed area in (a) is shown in higher magnification in Figure 2. CS, calcarine sulcus; EFL, endfeet layer; IFL, inner fibrous layer; LW, lateral wall; MW, medial wall; OFL, outer fibrous layer; OSVZ, outer subventricular zone; SVZ, subventricular zone; TB, toluidine blue; VIM, vimentin; VZ, ventricular zone. All figures are of same magnification. Scale bar: 5000 μm.

Fig 4: (a) Each heatmap shows log2 signal‐to‐noise ratios of different proteins in 22 regions of interest (ROI) (rows) in selected areas/regions (columns) from 13 wpc (frontal), 19 wpc (temporal, parietal) and 21 wpc (occipital, brain stem and cerebellum) human fetal brain samples profiled using Nanostring GeoMx ® . Values below 1 or missing are depicted in grey. Proteins are grouped according to cell types in which they are known to be prevalent. Apart from indusium griseum (b) (green, HOPX; red, BLBP), examples of selected areas/regions are illustrated on Figure 1, 2 (neocortical zones), Figure 6 (olfactory bulb), Figure 7 (hippocampus), Figure 8 (cerebellar cortex, center of dentate nucleus, axonal fibers and corpus pontobulbare). All regions of interest are depicted in Figure S1–S5. The screening reveals differences in protein abundance between regions/areas and/or ages in fetal developing brain. Scale bar: 500 μm.

Fig 5: Distribution of HOPX and BLBP in cerebellum and brain stem from a 21 wpc human fetal brain. (b–e) are higher magnifications of boxed areas in (a). HOPX is most prevalent in corpus pontobulbare (CPB) depicted in (a–d). HOPX (b) and BLBP (c) seem to occupy different regions (d) of this cellular aggregate dominated by strong HOPX immunoreactivity. HOPX is also scattered in the brain stem and in the dentate nucleus (a) as well as in blood vessel walls (e). In the cerebellar cortex HOPX is mostly confined to the Purkinje cell layer (e). CB, cerebellum; CP, choroid plexus; CPB, corpus pontobulbare; D, dentate nucleus; EFL, endfeet layer; EGL: external granular/germinal layer; IGL: internal granular layer; P, pons; PCL: Purkinje cell layer. Scale bars: (a): 2000 μm; (b–e) 1000 μm.