Evolutionary origin of Tbr2-expressing precursor cells and the subventricular zone in the developing cortex.
ABSTRACT: The subventricular zone (SVZ) is greatly expanded in primates with gyrencephalic cortices and is thought to be absent from vertebrates with three-layered, lissencephalic cortices, such as the turtle. Recent work in rodents has shown that Tbr2-expressing neural precursor cells in the SVZ produce excitatory neurons for each cortical layer in the neocortex. Many excitatory neurons are generated through a two-step process in which Pax6-expressing radial glial cells divide in the VZ to produce Tbr2-expressing intermediate progenitor cells, which divide in the SVZ to produce cortical neurons. We investigated the evolutionary origin of SVZ neural precursor cells in the prenatal cerebral cortex by testing for the presence and distribution of Tbr2-expressing cells in the prenatal cortex of reptilian and avian species. We found that mitotic Tbr2(+) cells are present in the prenatal cortex of lizard, turtle, chicken, and dove. Furthermore, Tbr2(+) cells are organized into a distinct SVZ in the dorsal ventricular ridge (DVR) of turtle forebrain and in the cortices of chicken and dove. Our results are consistent with the concept that Tbr2(+) neural precursor cells were present in the common ancestor of mammals and reptiles. Our data also suggest that the organizing principle guiding the assembly of Tbr2(+) cells into an anatomically distinct SVZ, both developmentally and evolutionarily, may be shared across vertebrates. Finally, our results indicate that Tbr2 expression can be used to test for the presence of a distinct SVZ and to define the boundaries of the SVZ in developing cortices.
Project description:The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.
Project description:Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. Here, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices.
Project description:While several major classes of neocortical neural precursor cells have been identified, the lineal relationships and molecular profiles of these cells are still largely unknown. Furthermore, the individual contribution of each cell class to neocortical growth during normal development and in neurodevelopmental disorders has not been determined. Using a novel fate-mapping approach, we demonstrate that precursors in the embryonic ventricular (VZ) and subventricular zones (SVZ), which give rise to excitatory neurons, are divided into distinct subtypes based on lineage profile, morphology, and transcription factor expression in vivo. Using this technique, we show that short neural precursors are a unique class of VZ intermediate progenitors derived from radial glial cells and are distinct from the multipolar Tbr2((+)) intermediate progenitors, which divide in the SVZ. To test whether these multiple groups of intermediate progenitors are redundant or whether they are necessary for proper neocortical growth, we measured precursor cell diversity in the Ts65Dn mouse model of Down syndrome (DS), which exhibits reduced neurogenesis and postnatal microcephaly. We report that SNP generation is markedly reduced in the Ts65Dn VZ during mid-neurogenesis, indicating that faulty specification of this progenitor pool is a central component of the neocortical abnormality in DS. Together, these findings demonstrate that neocortical neurons are produced via multiple indirect routes during embryonic development and that these parallel streams of neurogenesis collectively contribute to the proper growth and development of the neocortex.
Project description:The ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (Sufu) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B1 cells. In contrast, we found a significant decrease in Gsx2+ and a more dramatic decrease in Tbr2+ transit amplifying cells (TACs) indicating that innate differences between dorsal and ventral forebrain derived Type B1 cells influence Sufu function. However, many precursors accumulated in the dorsal V-SVZ or failed to survive, demonstrating that despite the over-proliferation of Type B1 cells, they are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression and surprisingly, a significant downregulation of Sonic hedgehog (Shh) signaling. Therefore, these findings indicate a potential role of the Sufu-Gli3 regulatory axis in the neonatal dorsal V-SVZ independent of Shh signaling in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ.
Project description:Cerebral cortical neurons arise from radial glia (direct neurogenesis) or from intermediate progenitors (indirect neurogenesis); intriguingly, the sizes of intermediate progenitor populations and the cortices they generate correlate across species. The generation of intermediate progenitors is regulated by the transcription factor Tbr2, whose expression marks these cells. We investigated how this mechanism might be controlled. We found that acute blockade of mature microRNA biosynthesis in murine cortical progenitors caused a rapid cell autonomous increase in numbers of Tbr2-expressing cells. Acute microRNA-92b (miR-92b) gain of function caused rapid reductions in numbers of Tbr2-expressing cells and proliferating intermediate progenitors. Acute miR-92b loss of function had opposite effects. These findings indicate that miR-92b limits the production of intermediate cortical progenitors.
Project description:The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.
Project description:Because folding of the cerebral cortex in the mammalian brain is believed to be crucial for higher brain functions, the mechanisms underlying its formation during development and evolution are of great interest. Although it has been proposed that increased neural progenitors in the subventricular zone (SVZ) are responsible for making cortical folds, their roles in cortical folding are still largely unclear, mainly because genetic methods for gyrencephalic mammals had been poorly available. Here, by taking an advantage of our newly developed in utero electroporation technique for the gyrencephalic brain of ferrets, we investigated the role of SVZ progenitors in cortical folding. We found regional differences in the abundance of SVZ progenitors in the developing ferret brain even before cortical folds began to be formed. When Tbr2 transcription factor was inhibited, intermediate progenitor cells were markedly reduced in the ferret cerebral cortex. Interestingly, outer radial glial cells were also reduced by inhibiting Tbr2. We uncovered that reduced numbers of SVZ progenitors resulted in impaired cortical folding. When Tbr2 was inhibited, upper cortical layers were preferentially reduced in gyri compared to those in sulci. Our findings indicate the biological importance of SVZ progenitors in cortical folding in the gyrencephalic brain.
Project description:Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.
Project description:Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.
Project description: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.