TGFß-Signaling and FOXG1-Expression Are a Hallmark of Astrocyte Lineage Diversity in the Murine Ventral and Dorsal Forebrain.

Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. To investigate the impact of TGF (transforming growth factor) ß-signaling on astrocyte development in the telencephalon we deleted the TGFBR2 (transforming growth factor beta receptor 2) in early neural progenitor cells in mice using a FOXG1 (forkhead box G1)-driven CRE-recombinase. We used quantitative proteomics to characterize TGFBR2-deficient cells derived from the mouse telencephalon and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFß-signaling. Impaired TGFß-signaling affected numbers of GFAP astrocytes in the ventral telencephalon. In contrast, TGFß reduced MFGE8-expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells.

The FOXG1/FOXO/SMAD network balances proliferation and differentiation of cortical progenitors and activates Kcnh3 expression in mature neurons.

Transforming growth factor ß (TGFß)-mediated anti-proliferative and differentiating effects promote neuronal differentiation during embryonic central nervous system development. TGFß downstream signals, composed of activated SMAD2/3, SMAD4 and a FOXO family member, promote the expression of cyclin-dependent kinase inhibitor Cdkn1a. In early CNS development, IGF1/PI3K signaling and the transcription factor FOXG1 inhibit FOXO- and TGFß-mediated Cdkn1a transcription. FOXG1 prevents cell cycle exit by binding to the SMAD/FOXO-protein complex. In this study we provide further details on the FOXG1/FOXO/SMAD transcription factor network. We identified ligands of the TGFß- and IGF-family, Foxo1, Foxo3 and Kcnh3 as novel FOXG1-target genes during telencephalic development and showed that FOXG1 interferes with Foxo1 and Tgfß transcription. Our data specify that FOXO1 activates Cdkn1a transcription. This process is under control of the IGF1-pathway, as Cdkn1a transcription increases when IGF1-signaling is pharmacologically inhibited. However, overexpression of CDKN1A and knockdown of Foxo1 and Foxo3 is not sufficient for neuronal differentiation, which is probably instructed by TGFß-signaling. In mature neurons, FOXG1 activates transcription of the seizure-related Kcnh3, which might be a FOXG1-target gene involved in the FOXG1 syndrome pathology.

PI3K-p110-alpha-subtype signalling mediates survival, proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2.

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFß) and insulin-like growth factor 1 (IGF1) have a central role. The TGFß- and IGF1-pathways cross-talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down-stream molecules of TGFß and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5-derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5-derived cells. TGFß has little effect at E13.5. It does not activate the PI3K- and mTOR-signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5-derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K-p110-alpha signalling. PI3K-p110-beta signalling activates mTORC2 in E16.5-derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms. Within developing cortical cells TGFß- and IGF-signalling activities are timely separated. TGFß dominates in E16.5-derived cells and drives neuronal differentiation. IGF influences survival, proliferation and neuronal differentiation in E13.5-derived cells. mTORC2-signalling in E16.5-derived cells influences survival, proliferation and differentiation, activated through PI3K-p110-alpha. PI3K-p110-beta-signalling activates a different mTORC2. Both PI3K/mTORC2-signalling pathways are required but not directly activated in TGFß-mediated neuronal differentiation.