Molecular Regionalization of the Diencephalon

The anatomic complexity of the diencephalon depends on precise molecular and cellular regulative mechanisms orchestrated by regional morphogenetic organizers at the neural tube stage. In the diencephalon, like in other neural tube regions, dorsal and ventral signals codify positional information to specify ventro-dorsal regionalization. Retinoic acid, Fgf8, BMPs, and Wnts signals are the molecular factors acting upon the diencephalic epithelium to specify dorsal structures, while Shh is the main ventralizing signal. A central diencephalic organizer, the zona limitans intrathalamica (ZLI), appears after neurulation in the central diencephalic alar plate, establishing additional antero-posterior positional information inside diencephalic alar plate. Based on Shh expression, the ZLI acts as a morphogenetic center, which cooperates with other signals in thalamic specification and pattering in the alar plate of diencephalon. Indeed, Shh is expressed first in the basal plate extending dorsally through the ZLI epithelium as the development proceeds. Despite the importance of ZLI in diencephalic morphogenesis the mechanisms that regulate its development remain incompletely understood. Actually, controversial interpretations in different experimental models have been proposed. That is, experimental results have suggested that (i) the juxtaposition of the molecularly heterogeneous neuroepithelial areas, (ii) cell reorganization in the epithelium, and/or (iii) planar and vertical inductions in the neural epithelium, are required for ZLI specification and development. We will review some experimental data to approach the study of the molecular regulation of diencephalic regionalization, with special interest in the cellular mechanisms underlying planar inductions

FGF8 activates proliferation and migration in mouse post-natal oligodendrocyte progenitor cells.

Fibroblast growth factor 8 (FGF8) is a key molecular signal that is necessary for early embryonic development of the central nervous system, quickly disappearing past this point. It is known to be one of the primary morphogenetic signals required for cell fate and survival processes in structures such as the cerebellum, telencephalic and isthmic organizers, while its absence causes severe abnormalities in the nervous system and the embryo usually dies in early stages of development. In this work, we have observed a new possible therapeutic role for this factor in demyelinating disorders, such as leukodystrophy or multiple sclerosis. In vitro, oligodendrocyte progenitor cells were cultured with differentiating medium and in the presence of FGF8. Differentiation and proliferation studies were performed by immunocytochemistry and PCR. Also, migration studies were performed in matrigel cultures, where oligodendrocyte progenitor cells were placed at a certain distance of a FGF8-soaked heparin bead. The results showed that both migration and proliferation was induced by FGF8. Furthermore, a similar effect was observed in an in vivo demyelinating mouse model, where oligodendrocyte progenitor cells were observed migrating towards the FGF8-soaked heparin beads where they were grafted. In conclusion, the results shown here demonstrate that FGF8 is a novel factor to induce oligodendrocyte progenitor cell activation, migration and proliferation in vitro, which can be extrapolated in vivo in demyelinated animal models

Culture and Differentiation of Oligodendrocyte Progenitor Cells (OPCs).

<p>A) OPCs cultured for 3 days after extraction on poly-L-lysine coated culture dishes. Numerous astrocytes are present. B) OPCs after passaging and expanding in the presence of FGF2 and PDGF-AA, to induce proliferation and maintenance of the OPCs. C) OPCs cultured in differentiation inducing medium for 7 days. Mature oligodendrocytes appear in the culture. D) OPCs cultured for 7 days in the differentiating medium and FGF8. All images taken at 100X magnification. E–G) Immunocytochemistry of OPCs in undifferentiating medium (E), differentiation inducing medium (F), and in the presence of FGF8 (G), staining for Olig2 (in green) and DAPI for nuclear staining (in blue). H) Histogram presenting the percentage of Olig2+ cells in the various cultures observed in E–G. Percentages are with respect to the total number of cells, calculated by DAPI staining. I–K) Immunocytochemistry of NG2+/Brdu+ cell in undifferentiating medium (I), differentiation inducing medium (J), and in the presence of FGF8 (K). L) Histogram presenting the percentage of NG2+/BrdU+ cells in the different cultures, with respect to the total number of cells. *p<0.05 (paired t-test), n = 4. Scale Bar indicates 150 µm for A–D and 100 µm for E–G and I–K.</p

Matrigel cultures of OPCs.

<p>A) Oligodendrocyte progenitor cell matrigel culture where a PBS-soaked bead was placed 0.5 mm away from the cell cluster. Image taken at 40X magnification. B–C) OPCs where a cluster of cells migrating towards a FGF8-soaked bead can be observed. D) Histogram depicting the oligodendrocyte progenitor cell proximal/distal rate in control cultures and where FGF8 was included. *p<0.01 (paired t-test, n = 4). (E–G) Matrigel cultures where on one side a FGF8-soaked bead was placed and on the other side a FGF8 inhibitor. (F, G) Close-up images of the areas near the inhibitor-soaked bead (F) or near the FGF8-soaked bead (G). H) OPCs where a FGF8-soaked bead was placed and on the other side a PBS-soaked bead, as control of the inhibitor. Scale bar indicates 500 µm for (A–C, D, H) and 200 µm for (F, G). All images were taken 48 hours after culture.</p

Oligodendrocyte progenitor cell differentiation with and without FGF8.

<p>A) Immunocytochemistry of NG2+/Olig2+, PDGFR-A+/Olig2+ and O4+ cells in the undifferentiated, differentiating and FGF8 culture media. In all images, blue staining corresponds to the nuclei (DAPI). Scale bar indicates 50 µm. B) Histogram depicting the percentage of cells in the different cell cultures staining for markers analyzed in A. Percentages are with respect to the total number of cells. C) Histogram depicting the results from the quantitative PCR analysis of the different cell cultures. *p<0.05, **p<0.01, ***p<0.001 (paired t-test in B, one way-ANOVA in C), n = 4.</p

In Vivo Transplantation of FGF8-soaked beads in Chronically Demyelinated Mice.

<p>A) FGF8-soaked heparin beads (seen in green) surrounded by NG2-positive cells (in red. B) Control experiment where PBS-soaked beads were transplanted. C) Histogram depicting the percentage of NG2 immunoreactivity in the area injected with the heparin beads. The red bar indicates the percentage in the case FGF8-soaked beads was used, while the blue bar indicates the sham control. D) NG2 immunoreactivity in the corpus callsoum and cortex in the area near the PBS-soaked beads. E) NG2 immunoreactivity in the corpus callsoum and cortex in the area near the FGF8-soaked beads. F, G) MBP immunoreactivity in the area near the PBS or FGF8-soaked beads, respectively. H) Histogram depicting the percentage of MBP immunoreactivity in the area injected with the PBS- (blue bar) or FGF8- (red bar) soaked heparin beads. Scale bar measures 50 µm. *p<0.01 (paired t-test), n = 4.</p

Fgf15 regulates thalamic development by controlling the expression of proneural genes

The establishment of the brain structural complexity requires a precisely orchestrated interplay between extrinsic and intrinsic signals modulating cellular mechanisms to guide neuronal differentiation. However, little is known about the nature of these signals in the diencephalon, a complex brain region that processes and relays sensory and motor information to and from the cerebral cortex and subcortical structures. Morphogenetic signals from brain organizers regulate histogenetic processes such as cellular proliferation, migration, and differentiation. Sonic hedgehog (Shh) in the key signal of the ZLI, identified as the diencephalic organizer. Fgf15, the mouse gene orthologous of human, chick, and zebrafish Fgf19, is induced by Shh signal and expressed in the diencephalic alar plate progenitors during histogenetic developmental stages. This work investigates the role of Fgf15 signal in diencephalic development. In the absence of Fgf15, the complementary expression pattern of proneural genes: Ascl1 and Nng2, is disrupted and the GABAergic thalamic cells do not differentiate; in addition dorsal thalamic progenitors failed to exit from the mitotic cycle and to differentiate into neurons. Therefore, our findings indicate that Fgf15 is the Shh downstream signal to control thalamic regionalization, neurogenesis, and neuronal differentiation by regulating the expression and mutual segregation of neurogenic and proneural regulatory genes.This work was supported by the following Grants: European consortium EUCOMMTOOLS (Contract HEALTH-FA-2010-261492), Spanish Ministry of Science and Innovation Grant BFU-2011-27326, Consolider Grant CSD2007-00023, Institute of Health Carlos III, Spanish Cell Therapy Network and Research Center of Mental Health, and General Council of Valencia (Prometeo 2009/028 and 11/2011/042).Peer reviewe

FGF8 Activates Proliferation and Migration in Mouse Post-Natal Oligodendrocyte Progenitor Cells

Fibroblast growth factor 8 (FGF8) is a key molecular signal that is necessary for early embryonic development of the central nervous system, quickly disappearing past this point. It is known to be one of the primary morphogenetic signals required for cell fate and survival processes in structures such as the cerebellum, telencephalic and isthmic organizers, while its absence causes severe abnormalities in the nervous system and the embryo usually dies in early stages of development. In this work, we have observed a new possible therapeutic role for this factor in demyelinating disorders, such as leukodystrophy or multiple sclerosis. In vitro, oligodendrocyte progenitor cells were cultured with differentiating medium and in the presence of FGF8. Differentiation and proliferation studies were performed by immunocytochemistry and PCR. Also, migration studies were performed in matrigel cultures, where oligodendrocyte progenitor cells were placed at a certain distance of a FGF8-soaked heparin bead. The results showed that both migration and proliferation was induced by FGF8. Furthermore, a similar effect was observed in an in vivo demyelinating mouse model, where oligodendrocyte progenitor cells were observed migrating towards the FGF8-soaked heparin beads where they were grafted. In conclusion, the results shown here demonstrate that FGF8 is a novel factor to induce oligodendrocyte progenitor cell activation, migration and proliferation in vitro, which can be extrapolated in vivo in demyelinated animal models

A High-Resolution Spatiotemporal Atlas of Gene Expression of the Developing Mouse Brain

To provide a temporal framework for the genoarchitecture of brain development, we generated in situ hybridization data for embryonic and postnatal mouse brain at seven developmental stages for ∼2,100 genes, which were processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, seven reference atlases, an ontogenetic ontology, and tools to explore coexpression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org)