FGF/FGFR2 signaling regulates the generation and correct positioning of Bergmann glia cells in the developing mouse cerebellum.

The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse.

Combination of in silico and in situ hybridisation approaches to identify potential Dll1 associated miRNAs during mouse embryogenesis.

MicroRNAs (miRNAs) have regulatory functions during vertebrate embryogenesis. They are short approximately 21bp long endogenously expressed single-stranded RNAs, which preferentially bind to complementary sequences in the 3' untranslated regions (UTR) of mRNAs and typically down-regulate the respective target mRNAs by translational repression or enhanced mRNA degradation. The Notch ligand Delta-like 1 (Dll1) is expressed in a highly dynamic pattern and has pleiotropic functions during embryogenesis and in adult tissues. Here, we report an interspecies in silico analysis to identify 16 miRNAs, which potentially bind to the mouse, human and chicken Dll1 3'UTRs. To analyze whether these miRNAs could regulate Dll1 gene expression during somitogenesis and neurogenesis, we performed a systematic whole mount in situ hybridisation screen, followed by radioactive in situ hybridisation on sections, using LNA modified DNA probes in mouse embryos. We find that 7 miRNAs (miR-34a, miR-103, miR-107, miR-130a, miR-130b, miR-449a and miR-449c) are expressed in developing somites, limbs, restricted regions of the brain and neural tube between 9.5 dpc and 12.5 dpc. This suggests that these miRNAs could possibly target the Dll1 3'UTR in these regions. The other miRNAs are not expressed or below the detection limit and thus are unlikely to regulate Dll1 at the analyzed embryonic stages.

Periphilin is strongly expressed in the murine nervous system and is indispensable for murine development.

Periphilin is involved in multiple processes in vivo. To explore its physiological role from an organismic perspective, we generated mice with a gene trap insertion in the periphilin-1 gene. Based on beta-gal reporter activity, a widespread periphilin expression was evident, especially in the developing somites and limbs, the embryonic nervous system, and the adult brain. In accordance with this broad expression, homozygous deficiency of periphilin was lethal in early embryogenesis. Mice with a heterozygous deficiency did not show any abnormalities of brain morphology and function, neither histologically nor regarding the transcriptome. Interestingly, the reduction of the periphilin-1 gene dosage was compensated by an increased expression of the remaining wild-type allele in the brain. These results point to an indispensable function of periphilin during murine development and an important role in the nervous system, reflected by a strong and tightly regulated expression in the murine brain.

Activation of ERK/MAPK in the lateral amygdala of the mouse is required for acquisition of a fear-potentiated startle response.

There is considerable interest in examining the genes that may contribute to anxiety. We examined the function of ERK/MAPK in the acquisition of conditioned fear, as measured by fear-potentiated startle (FPS) in mice as a model for anticipatory anxiety in humans. We characterized the following for the first time in the mouse: (1) the expression of the ERK/MAPK signaling pathway components at the protein level in the lateral amygdala (LA); (2) the time course of activation of phospho-activated MAPK in the LA after fear conditioning; (3) if pharmacological inhibition of pMAPK could modulate the acquisition of FPS; (4) the cell-type specificity of pMAPK in the LA after fear conditioning. Using western blot and immunohistochemistry techniques and injecting the MEK inhibitor U0126 in the LA, we showed the following: (1) both MEK1/MEK2 and ERK1/ERK2 were co-expressed in the LA of the adult mouse brain; (2) there is a peak of pMAPK at 60 min after fear conditioning; (3) the ERK/MAPK signaling pathway activation is essential for the acquisition of an FPS response; (4) at 60 min, the pMAPK are exclusively neuronal and not glial. These results emphasize the importance of this signaling pathway in the acquisition of conditioned fear in the mouse. Given the widely held view that conditioned fear models the essential aspects of anxiety disorders, the results confirm the ERK/MAPK signaling pathway as a molecular target for the treatment of anxiety disorders in the clinic.

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo.

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.

Expression of Fgf receptors 1, 2, and 3 in the developing mid- and hindbrain of the mouse.

Fibroblast growth factor 8 (FGF8) mediates the function of the midbrain-hindbrain organizer (MHO). FGF signals are transmitted by means of four known FGF receptors (FGFRs). Studies of Fgfr expression in early vertebrate development have shown that Fgfr1 is expressed along the entire neural tube, whereas Fgfr2 and Fgfr3 expression has been shown to spare the tissue adjacent to the MHO. The FGF8 signal from the MHO, therefore, was believed to be transmitted by FGFR1 exclusively. However, incongruent results from conditional mutants of Fgf8 and Fgfr1 in the midbrain-hindbrain (MHB) region contradict this hypothesis. Therefore, we reexamined the expression of the Fgfrs in this region. Fgfr1 is expressed all over the neural tube. Strikingly, Fgfr2 is expressed throughout the floor plate of the MHB region. In the basal plate, Fgfr2 directly abuts the Fgf8 expression domain at the MHO, anteriorly and posteriorly. Fgfr3 expression is in contact with the Fgf8 expression domain only in the rostroventral hindbrain. Based on these findings, we postulate a role for FGFR2 and FGFR3 in FGF signaling in the ventral midbrain and hindbrain.

Fgfr1-dependent boundary cells between developing mid- and hindbrain.

Signaling molecules regulating development of the midbrain and anterior hindbrain are expressed in distinct bands of cells around the midbrain-hindbrain boundary. Very little is known about the mechanisms responsible for the coherence of this signaling center. One of the fibroblast growth factor (FGF) receptors, Fgfr1, is required for establishment of a straight border between developing mid- and hindbrain. Here we show that the cells close to the border have unique features. Unlike the cells further away, these cells express Fgfr1 but not the other FGF receptors. The cells next to the midbrain-hindbrain boundary express distinct cell cycle regulators and proliferate less rapidly than the surrounding cells. In Fgfr1 mutants, these cells fail to form a coherent band at the boundary. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

Location and size of dopaminergic and serotonergic cell populations are controlled by the position of the midbrain-hindbrain organizer.

Midbrain dopaminergic and hindbrain serotonergic neurons play an important role in the modulation of behavior and are involved in a series of neuropsychiatric disorders. Despite the importance of these cells, little is known about the molecular mechanisms governing their development. During embryogenesis, midbrain dopaminergic neurons are specified rostral to the midbrain-hindbrain organizer (MHO), and hindbrain serotonergic neurons are specified caudal to it. We report that in transgenic mice in which Otx2 and accordingly the MHO are shifted caudally, the midbrain dopaminergic neuronal population expands to the ectopically positioned MHO and is enlarged. Complementary, the extension of the hindbrain serotonergic cell group is decreased. These changes are preserved in adulthood, and the additional, ectopic dopaminergic neurons project to the striatum, which is a proper dopaminergic target area. In addition, in mutants in which Otx2 and the MHO are shifted rostrally, dopaminergic and serotonergic neurons are relocated at the newly positioned MHO. However, in these mice, the size ratio between these two cell populations is changed in favor of the serotonergic cell population. To investigate whether the position of the MHO during embryogenesis is also of functional relevance for adult behavior, we tested mice with a caudally shifted MHO and report that these mutants show a higher locomotor activity. Together, we provide evidence that the position of the MHO determines the location and size of midbrain dopaminergic and hindbrain serotonergic cell populations in vivo. In addition, our data suggest that the position of the MHO during embryogenesis can modulate adult locomotor activity.