PMC

At 15 gw, relative density of CalR⁺ cells is still the highest in the GE with much lower density calculated in the cortical VZ/SVZ (). This distribution suggests that at 15 gw the GE is still the main source of cortical CalR⁺ cells. At 15 gw GE is divided into LGE and MGE, with CGE at more caudal telencephalic levels. From the immunolabeling studies it is not clear where exactly in the GE do CalR⁺ or CB⁺ cells originate, since they are seen as postmitotic, probably migrating cells.

Numerous CalR⁺ and CB⁺ cells were present in a well developed GE positioned caudally and inferiorly, around the temporal extension of the lateral ventricle (). A number of Ki67⁺ cells in the inferior GE indicates cell proliferation in this region at mid-term. This part of GE would correspond by its position to the caudal GE (CGE) described in mice (; ).

During the next two months, from 8 gw to 15 gw, the number of CalR⁺ cells increases throughout the developing cerebral cortex. In sagittally cut sections through the 15 gw brain, the density of CalR⁺ cells is higher in occipital than in frontal cortical regions (), demonstrating a caudo-rostral gradient of CalR expression. A similar caudo-rostral developmental gradient has been reported for calcium-binding protein expression in embryonic () and fetal human cortex (Bayatti et al., 2007), as well as for general cellular and functional maturation of the CNS (e.g., Yan et al., 1995; ).

At 8 gw (CS 20), the cortical plate (CP), emerges as initially only 1–2 cells rows thick layer dividing the PPL into upper and lower parts. From the very beginning GABA⁺ or CalR⁺ neurons are present in the newly formed CP (). These early immunolabeled cells will eventually become deep layers V–VI interneurons, as the inside-out formation of the CP proceeds. On coronal sections from an 8-week-old embryo the width of the CP is seven times larger in the lateral (140 µm) than in the medial (20 µm) regions of the telencephalic wall. CalR⁺ cells are observed to follow the inside-out developmental gradient, as described for projection neurons ().

At 6 gw (CS 17) neurons in the PPL and the GE express two calcium-binding proteins, CalR and CB, but not PV or Sst (, ). The density of immunolabeled cells with either of these two markers is the greatest in the matrix of the GE, which at this stage is not yet divided into LGE and MGE. There is a clear ventro-dorsal gradient of immunoreactivity for both CalR and CB from the GE towards the emerging neocortex (, ). Importantly, all immunolabeled cells were demonstrated outside of the proliferative ventricular zone, consistent with labeling of young interneurons, and not their progenitors. Immunoreactive cells had the appearance suggestive of tangentional migration to the neocortex, based on the orientation of their processes and their ventro-dorsal gradient (). Notably, a stream of migrating cells appeared to come into close contact with ventral brain surface, before “bouncing off” to more dorsally located cortical primordium (). At a similar embryonic age (7gw, CS 19), among tangentially migrating cells, occasional radially oriented cells within the telencephalic wall could be double-labeled with neuronal markers (β-III tubulin or GABA) and ventral transcription factors, Nkx2.1 or Dlx2 (, ). The early presence of these double-labeled cells extending from the VZ to the pia, is suggestive of their local, cortical origin.

At 6 gw (CS 17) neurons in the PPL and the GE express two calcium-binding proteins, CalR and CB, but not PV or Sst (, ). The density of immunolabeled cells with either of these two markers is the greatest in the matrix of the GE, which at this stage is not yet divided into LGE and MGE. There is a clear ventro-dorsal gradient of immunoreactivity for both CalR and CB from the GE towards the emerging neocortex (, ). Importantly, all immunolabeled cells were demonstrated outside of the proliferative ventricular zone, consistent with labeling of young interneurons, and not their progenitors. Immunoreactive cells had the appearance suggestive of tangentional migration to the neocortex, based on the orientation of their processes and their ventro-dorsal gradient (). Notably, a stream of migrating cells appeared to come into close contact with ventral brain surface, before “bouncing off” to more dorsally located cortical primordium (). At a similar embryonic age (7gw, CS 19), among tangentially migrating cells, occasional radially oriented cells within the telencephalic wall could be double-labeled with neuronal markers (β-III tubulin or GABA) and ventral transcription factors, Nkx2.1 or Dlx2 (, ). The early presence of these double-labeled cells extending from the VZ to the pia, is suggestive of their local, cortical origin.

At 6 gw (CS 17) neurons in the PPL and the GE express two calcium-binding proteins, CalR and CB, but not PV or Sst (, ). The density of immunolabeled cells with either of these two markers is the greatest in the matrix of the GE, which at this stage is not yet divided into LGE and MGE. There is a clear ventro-dorsal gradient of immunoreactivity for both CalR and CB from the GE towards the emerging neocortex (, ). Importantly, all immunolabeled cells were demonstrated outside of the proliferative ventricular zone, consistent with labeling of young interneurons, and not their progenitors. Immunoreactive cells had the appearance suggestive of tangentional migration to the neocortex, based on the orientation of their processes and their ventro-dorsal gradient (). Notably, a stream of migrating cells appeared to come into close contact with ventral brain surface, before “bouncing off” to more dorsally located cortical primordium (). At a similar embryonic age (7gw, CS 19), among tangentially migrating cells, occasional radially oriented cells within the telencephalic wall could be double-labeled with neuronal markers (β-III tubulin or GABA) and ventral transcription factors, Nkx2.1 or Dlx2 (, ). The early presence of these double-labeled cells extending from the VZ to the pia, is suggestive of their local, cortical origin.

In the neocortical VZ/SVZ the percentage of CalR⁺ cells from all cells increases approximately six times from only 1% at 15 gw to 5.9% at 20gw (). In layer I, both small cells and much larger Cajal-Retzius cells are strongly reactive for CalR at this stage (, ), similar to earlier fetal stages (, ). Relative density of CalR⁺ cells in different forebrain regions also changes and for the first time at mid-term becomes higher in the cortical VZ/SVZ relative to the GE (). In respect to the ratio of two Ca-binding proteins, CalR⁺ cells at mid-term outnumber CB⁺ cells in all cortical regions except in deep layers V–VI (). Some of the deep CB⁺ cells may represent a subpopulation of pyramidal cells. This observation is corroborated by the strong labeling of axonal tracts, including the internal capsule which contains corticospinal axons (). Additionally, in the upper cortical layers (layers II–III) single CB⁺ cells with large round cell bodies were labeled, as well as numerous radially spreading processes (). CalR⁺ cells are closely apposed to these CB⁺ processes, as if migrating along them. CB did not label CalR+ Cajal-Retzius cells, but in layer I, 30% of small CB neurons co-expressed CalR+; single labeled cells for CalR and CB were also present (). It is interesting to note that in all cortical regions studied at mid-term, layer I was filled with CalR⁺ puncta, reminiscent of axonal terminals and/or dendrites (). In contrast to numerous CalR⁺ cells in the cortical VZ/SVZ only rare CB⁺ cells could be detected in that region (, ), in contrast to the GE, where both single CalR⁺, CB⁺, and double-labeled cells were present (not shown).

In the neocortical VZ/SVZ the percentage of CalR⁺ cells from all cells increases approximately six times from only 1% at 15 gw to 5.9% at 20gw (). In layer I, both small cells and much larger Cajal-Retzius cells are strongly reactive for CalR at this stage (, ), similar to earlier fetal stages (, ). Relative density of CalR⁺ cells in different forebrain regions also changes and for the first time at mid-term becomes higher in the cortical VZ/SVZ relative to the GE (). In respect to the ratio of two Ca-binding proteins, CalR⁺ cells at mid-term outnumber CB⁺ cells in all cortical regions except in deep layers V–VI (). Some of the deep CB⁺ cells may represent a subpopulation of pyramidal cells. This observation is corroborated by the strong labeling of axonal tracts, including the internal capsule which contains corticospinal axons (). Additionally, in the upper cortical layers (layers II–III) single CB⁺ cells with large round cell bodies were labeled, as well as numerous radially spreading processes (). CalR⁺ cells are closely apposed to these CB⁺ processes, as if migrating along them. CB did not label CalR+ Cajal-Retzius cells, but in layer I, 30% of small CB neurons co-expressed CalR+; single labeled cells for CalR and CB were also present (). It is interesting to note that in all cortical regions studied at mid-term, layer I was filled with CalR⁺ puncta, reminiscent of axonal terminals and/or dendrites (). In contrast to numerous CalR⁺ cells in the cortical VZ/SVZ only rare CB⁺ cells could be detected in that region (, ), in contrast to the GE, where both single CalR⁺, CB⁺, and double-labeled cells were present (not shown).