Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex.

Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.

Integrin αvß3 and thyroid hormones promote expansion of progenitors in embryonic neocortex.

Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin αvß3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin αvß3 activation causes an increased cell cycle re-entry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin αvß3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin αvß3, completely abolishes the intermediate progenitor expansion observed upon targeted integrin αvß3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin αvß3. Convergence of ECM and thyroid hormones on integrin αvß3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.

Role of maternal thyroid hormones in the developing neocortex and during human evolution.

The importance of thyroid hormones during brain development has been appreciated for many decades. In humans, low levels of circulating maternal thyroid hormones, e.g., caused by maternal hypothyroidism or lack of iodine in diet, results in a wide spectrum of severe neurological defects, including neurological cretinism characterized by profound neurologic impairment and mental retardation, underlining the importance of the maternal thyroid hormone contribution. In fact, iodine intake, which is essential for thyroid hormone production in the thyroid gland, has been related to the expansion of the brain, associated with the increased cognitive capacities during human evolution. Because thyroid hormones regulate transcriptional activity of target genes via their nuclear thyroid hormone receptors (THRs), even mild and transient changes in maternal thyroid hormone levels can directly affect and alter the gene expression profile, and thus disturb fetal brain development. Here we summarize how thyroid hormones may have influenced human brain evolution through the adaptation to new habitats, concomitant with changes in diet and, therefore, iodine intake. Further, we review the current picture we gained from experimental studies in rodents on the function of maternal thyroid hormones during developmental neurogenesis. We aim to evaluate the effects of maternal thyroid hormone deficiency as well as lack of THRs and transporters on brain development and function, shedding light on the cellular behavior conducted by thyroid hormones.

Abundant occurrence of basal radial glia in the subventricular zone of embryonic neocortex of a lissencephalic primate, the common marmoset Callithrix jacchus.

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.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin ß1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling.

A major cause of the cerebral cortex expansion that occurred during evolution is the increase in subventricular zone (SVZ) progenitors. We found that progenitors in the outer SVZ (OSVZ) of developing human neocortex retain features of radial glia, in contrast to rodent SVZ progenitors, which have limited proliferation potential. Although delaminating from apical adherens junctions, OSVZ progenitors maintained a basal process contacting the basal lamina, a canonical epithelial property. OSVZ progenitor divisions resulted in asymmetric inheritance of their basal process. Notably, OSVZ progenitors are also found in the ferret, a gyrencephalic nonprimate. Functional disruption of integrins, expressed on the basal process of ferret OSVZ progenitors, markedly decreased the OSVZ progenitor population size, but not that of other, process-lacking SVZ progenitors, in slice cultures of ferret neocortex. Our findings suggest that maintenance of this epithelial property allows integrin-mediated, repeated asymmetric divisions of OSVZ progenitors, providing a basis for neocortical expansion.

Peripheral mural cell recruitment requires cell-autonomous heparan sulfate.

Blood vessel maturation and stability require recruitment of mural cells (MCs) to the nascent vessel. Loss or detachment of MCs causes vascular dysfunction in diseases. N-sulfation of heparan sulfate (HS) is required for platelet-derived growth factor B (PDGF-B) retention and platelet-derived growth factor receptor-beta (PDGFR-beta) signaling during MC recruitment. To analyze the specific role of MC-derived HS in this process, we inactivated HS synthesis in MCs. MC-specific loss of HS causes embryonic lethality associated with vascular patterning defects, edema, and hemorrhages during late gestation. MC recruitment in the skin is impaired, correlating with defective PDGFR-beta and transforming growth factor-beta (TGF-beta)-SMAD signaling. Accumulation of rounded cells positive for MC markers close to the vessels indicates defective polarization and migration of local MC progenitors. In contrast, MC recruitment and signaling in the central nervous system (CNS) are unaffected by MC HS loss. Our results suggest that HS is selectively required in a cell-autonomous manner, acting in cis with PDGFR-beta and TGF-beta receptors during induction/polarization and migration of local progenitor cells to the nascent vessel. Once MCs are in contact with the vessel, as during CNS vascularization, endothelial HS appears sufficient to facilitate PDGFR-beta activation in trans.

Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development.

During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor beta (PDGFRbeta) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.