FIGURE 3. Expression of HAR1F and HAR1R in the developing neocortex.

Pattern of expression of HAR1F and reelin in the human and macaque developing brain, revealed by RNA in situ hybridization (HAR1F) and immunohistochemistry (reelin). a, In situ hybridization of HAR1F antisense (AS) and sense (S) probes on 9-GW human brain coronal sections, illustrating selective expression of HAR1F in the dorsal telencephalon (DT), whereas the ventral telencephalon (VT) and thalamus (Th) show no obvious expression. The HAR1 probes contain 112 bp of the region of overlap between HAR1F and HAR1R. Therefore, the HAR1F sense probe has the potential to detect HAR1R expression. Within the DT, HAR1F is selectively enriched in the most superficial part of the cortex, much like reelin at the same stage (arrows). Scale bars: 500 m on left panels; 250 m on right panels. Dorsal (D) is top; lateral (L) is right on left panels. b, At 11 GW, HAR1F expression is confined to the cerebral cortex, with expression in large cells situated at the border of the marginal zone (MZ) (arrows), corresponding to Cajal–Retzius neurons, and in the upper cortical plate (CP) (arrowheads). Scale bar: 125 m for the right panel. IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. c, At 17 GW, expression of HAR1F and reelin remain confined to Cajal–Retzius cells in the MZ (arrows), intermingled with other cells of the subpial granular layer (SGL). In addition, HAR1F is expressed in neurons of the upper CP (arrowheads). Scale bar: 1,000 m. d, At 19 GW, HAR1F and reelin show the same pattern in the MZ/SGL (arrows), and HAR1F remains expressed in the upper CP (arrowheads), whereas the HAR1F sense probe shows no signal. Lower panels centred on the MZ illustrate double in situ hybridization/immunohistochemistry experiments. Lower left and middle panels show that HAR1F (in dark blue) and reelin (in dark brown) are co-localized in most cells, corresponding to Cajal–Retzius neurons (black arrows), although some reelin-positive cells do not express HAR1F (brown arrows). The lower right panel illustrates that HAR1F cells (blue arrows) seem to be mainly distinct from interneurons (brown arrows) expressing vesicular GABA transporter (vGAT). Scale bar: 250 m. e, Expression of HAR1F in macaque developing cortex. In situ hybridization (HAR1F) and immunohistochemistry (reelin) on E79 macaque brain parasagittal sections. As in the human, HAR1F and reelin are expressed in Cajal–Retzius cells in the MZ (arrows), intermingled with other cells of the SGL. In addition, HAR1F is expressed in neurons of the upper CP (arrowheads). Scale bar: 1,000 m. The HAR1F sense probe does not reveal any obvious expression pattern.