Progressive loss of PAX6, TBR2, NEUROD and TBR1 mRNA gradients correlates with translocation of EMX2 to the cortical plate during human cortical development

Abstract

The transcription factors Emx2 and Pax6 are expressed in the proliferating zones of the developing rodent neocortex, and gradients of expression interact in specifying caudal and rostral identities. Pax6 is also involved in corticoneurogenesis, being expressed by radial glial progenitors that give rise to cells that also sequentially express Tbr2, NeuroD and Tbr1, genes temporally downstream of Pax6. In this study, using in situ hybridization, we analysed the expression of EMX2, PAX6, TBR2, NEUROD and TBR1 mRNA in the developing human cortex between 8 and 12 postconceptional weeks (PCW). EMX2 mRNA was expressed in the ventricular (VZ) and subventricular zones (SVZ), but also in the cortical plate, unlike in the rodent. However, gradients of expression were similar to that of the rodent at all ages studied. PAX6 mRNA expression was limited to the VZ and SVZ. At 8 PCW, PAX6 was highly expressed rostrally but less so caudally, as has been seen in the rodent, however this gradient disappeared early in corticogenesis, by 9 PCW. There was less restricted compartment-specific expression of TBR2, NEUROD and TBR1 mRNA than in the rodent, where the gradients of expression were similar to that of PAX6 prior to 9 PCW. The gradient disappeared for TBR2 by 10 PCW, and for NEUROD and TBR1 by 12 PCW. These data support recent reports that EMX2 but not PAX6 is more directly involved in arealization, highlighting that analysis of human development allows better spatio-temporal resolution than studies in rodents.

Fig. 1

Laminar localization of EMX2 and PAX6 during early foetal development of the human neocortex. ISH revealed a changing laminar distribution of EMX2 mRNA between 8 and 12 postconceptional weeks (PCW). Expression was observed only in the subventricular and ventricular zones (SVZ/VZ) at 8 PCW (A). However, by 9 PCW expression was also noted in the cortical plate (CP) (C). Subsequently, expression of EMX2 intensified in the CP at 10 PCW (E) and 12 PCW (G), while still present in the SVZ and VZ at lower intensities. At 12 PCW, the highest level of EMX2 mRNA expression was found in the CP most proximal to the marginal zone (MZ). PAX6 was observed predominantly in the proliferative zones (SVZ/VZ) of the developing cortex (B, D, F, H). PAX6 mRNA was observed most intensely at 8 PCW in the VZ (A), after which a decrease was observed at 9 PCW (C). Staining intensity of PAX6 decreased at 10 PCW and 12 PCW (F and H, respectively) due to the decrease in the relative thickness of the VZ. Sections for EMX2 and PAX6 are taken from the caudal and rostral poles respectively. Scale bars: 100 μm (A, C); 200 μm (E, G). IZ, intermediate zone; SP, subplate.

Fig. 2

EMX2 and PAX6gradients in the developing human cortex. ISH analysis of sagittal sections at 8 postconceptional weeks (PCW; A, B) revealed that EMX2 and PAX6 mRNA were expressed in reciprocal rostro-caudal gradients. While EMX2 was expressed high caudally and low rostrally, PAX6 showed an opposite gradient of expression. In sagittal sections at 9 PCW (C, D), EMX2 mRNA maintained a similar caudal-rostral gradient to that seen at 8 PCW. The previously observed PAX6 expression gradient, however, disappeared. Similarly, in horizontal sections at 8 PCW, EMX2 and PAX6 exhibited reciprocal opposing medial-lateral gradients (E, F). EMX2 expression was observed high medially and low laterally with the opposite for PAX6. While the EMX2 gradient still persisted at 9 PCW (G), the PAX6 medial-gradient disappeared (H). Scale bars: 500 μm. Ca, caudal; Lt, lateral; Md, medial; Ro, rostral.

Fig. 4

TBR2, NEUROD, and TBR1 gradients in the developing human cortex. Expression analysis of TBR2, NEUROD and TBR1 transcription factors temporally downstream of PAX6 during neurogenesis demonstrates similar gradients of expression. At 8 postconceptional weeks (PCW), all three genes exhibited high rostral-low caudal (A–C), high lateral-low medial (D–F) expression. These gradients persisted at 9 PCW (G–I and J–L, respectively); however, the TBR2 gradient was not as pronounced (G and J). By 10 PCW the TBR2 gradient was absent (M and P), the NEUROD and TBR1 gradients were becoming less pronounced (N, O, Q and R). By 12 PCW, there were no gradients of expression of any of these three transcription factors (data not shown). Higher magnification images of respective boxed areas are indicated by single and double asterisks. Sections shown are in the sagittal plane (A–C, G–I and M–O), horizontal plane (D–F) or coronal plane (J–L and P–R). Scale bars: 200 μm. Ca, caudal; Lt, lateral; Md, medial; Ro, rostral.

Fig. 3

Laminar localization of TBR2, NEUROD and TBR1 mRNA during early foetal development of the human neocortex. ISH analysis of TBR2, NEUROD and TBR1 mRNA in the developing human cortex between 8 and 12 postconceptional weeks (PCW). At 8 PCW, TBR2 and NEUROD exhibit restricted expression in what is probably the subventricular zone (SVZ) of the developing cortex (A, B), while TBR1 expression additionally includes the cortical plate (CP; C). By 9 PCW, the SVZ is more distinctly identified at the border with the ventricular zone (VZ). All three genes are absent from the majority VZ but possibly present at the SVZ border, TBR2 being restricted to the SVZ (D) while NEUROD and TBR1 are additionally present in the CP (E, F). The expression patterns of these genes are similar at 10 PCW (G–I); however, TBR2 exhibits a more restricted expression close to the SVZ/VZ border. By 12 PCW, all three show high expression in the ISVZ, while NEUROD and TBR1 show more widespread expression in the SVZ and CP. Scale bars: 100 μm (C, F); 200 μm (I, L). ISVZ, inner SVZ; IZ, intermediate zone; MZ, marginal zone; OSVZ, outer SVZ; SP, subplate.

Fig. 5

Summary of changes in expression patterns of EMX2, PAX6, TBR2, NEUROD and TBR1 during early development of the human cerebral cortex. At 8 postconceptional weeks (PCW) EMX2 and PAX6 are localized within the proliferative zones of the developing cortex in opposing rostro-lateral/caudo-medial gradients. From 9 to 12 PCW onwards the majority of EMX2 expression is found in the cortical plate (CP), where a similar gradient exists to that observed in the subventricular zone (SVZ)/ventricular zone (VZ). By this time the PAX6 gradient has disappeared and expression is widespread throughout the SVZ/VZ only. However, the mRNA of transcription factors downstream of PAX6 in neurogenesis form PAX6-like gradients in different compartments during this time period. TBR2 exhibits a PAX6-like gradient within the SVZ until 10 PCW. NEUROD, which initially is not expressed in the CP, also forms a gradient within the SVZ at 8 PCW, which extends to the SP/intermediate zone (IZ) and CP at 9 PCW. The TBR1 gradient is also observed from 8 PCW and encompasses all compartments outside of the VZ, most prominently within the CP, but disappears before 12 PCW. Thus, as EMX2-expressing cells migrate from the VZ to the CP, they pass through compartments expressing genes downstream of PAX6 that exhibit PAX6-like gradients.