Daniela M. Vogt Weisenhorn

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 Parkinsons disease.

FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals.

Fibroblast growth factors (FGFs) are signaling molecules of the isthmic organizer, which regulates development of the midbrain and cerebellum. Tissue‐specific inactivation of one of the FGF receptor (FGFR) genes, Fgfr1, in the midbrain and rhombomere 1 of the hindbrain of mouse embryos results in deletion of the inferior colliculi in the posterior midbrain and vermis of the cerebellum. Analyses of both midbrain–hindbrain and midbrain‐specific Fgfr1 mutants suggest that after establishment of the isthmic organizer, FGFR1 is needed for continued response to the isthmic signals, and that it has direct functions on both sides of the organizer. In addition, FGFR1 appears to modify cell adhesion properties critical for maintaining a coherent organizing center. This may be achieved by regulating expression of specific cell‐adhesion molecules at the midbrain–hindbrain border.

Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region

The secreted glycoprotein WNT1 is expressed in the caudal midbrain and is essential for proper development of the entire mid-/hindbrain region. To get better insights into Wnt1 function in the mid-/hindbrain region, we ectopically expressed Wnt1 under the control of the endogenous En1 promoter, thereby extending Wnt1 expression rostrally into the anterior midbrain and caudally into rhombomere 1. In these transgenic mice, the position of the mid-/hindbrain organizer is not altered and pattern formation is not changed. During midgestation, ectopic Wnt1 induced strong overproliferation of precursor cells only in the caudal midbrain in a gene dosage-dependent manner. Enhanced proliferation is at least in part mediated by shortening of the cell cycle length. In adults, Wnt1 exhibited a cell size promoting effect specifically on neurons. We suggest that Wnt1 acts as a regulator of proliferation of specific precursor populations in the developing mid-/hindbrain region and is only secondarily involved in maintenance of the mid-/hindbrain organizer.

Fibroblast growth factor receptors cooperate to regulate neural progenitor properties in the developing midbrain and hindbrain.

Fibroblast growth factors (FGFs) secreted from the midbrain–rhombomere 1 (r1) boundary instruct cell behavior in the surrounding neuroectoderm. For example, a combination of FGF and sonic hedgehog (SHH) can induce the development of the midbrain dopaminergic neurons, but the mechanisms behind the action and integration of these signals are unclear. We studied how FGF receptors (FGFRs) regulate cellular responses by analyzing midbrain–r1 development in mouse embryos, which carry different combinations of mutant Fgfr1, Fgfr2, and Fgfr3 alleles. Our results show that the FGFRs act redundantly to support cell survival in the dorsal neuroectoderm, promote r1 tissue identity, and regulate the production of ventral neuronal populations, including midbrain dopaminergic neurons. The compound Fgfr mutants have apparently normal WNT/SHH signaling and neurogenic gene expression in the ventral midbrain, but the number of proliferative neural progenitors is reduced as a result of precocious neuronal differentiation. Our results suggest a SoxB1 family member, Sox3, as a potential FGF-induced transcription factor promoting progenitor renewal. We propose a model for regulation of progenitor cell self-renewal and neuronal differentiation by combinatorial intercellular signals in the ventral midbrain.

Megane/Heslike is required for normal GABAergic differentiation in the mouse superior colliculus

The mouse Mgn protein (Helt) is structurally related to the neurogenic Drosophila hairy and Enhancer of split [h/E(spl)] proteins, but its unique structural properties distinguish it from other members of the family. Mgn expression shows a spatiotemporal correlation with GABAergic markers in several brain regions. We report here that homozygous Mgn-null mice die between the second and the fifth postnatal week of age, and show a complete depletion of Gad65 and Gad67 expression in the superior colliculus and a reduction in the inferior colliculus. Other brain regions, as well as other neural systems, are not affected. The progenitor GABAergic cells appear to be generated in right numbers but fail to become GABAergic neurons. The phenotype of the mice is consistent with reduced GABAergic activity. Thus, our in vivo study provides evidence that Mgn is the key regulator of GABAergic neurons, controlling their specification in the dorsal midbrain. Another conclusion from our results is that the function of Mgn shows a previously unrecognized role for h/E(spl)-related transcription factors in the dorsal midbrain GABAergic cell differentiation. Vertebrate h/E(spl)-related genes can no longer be regarded solely as a factors that confer generic neurogenic properties, but as key components for the subtype-neuronal identity in the mammalian CNS.

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

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. Developmental Dynamics 233:1023–1030, 2005.

Differential mRNA distribution of components of the ERK/MAPK signalling cascade in the adult mouse brain

The mitogen‐activated protein kinases (MAPKs), also called extracellular signal‐regulated kinases (ERKs), are a group of serine/threonine terminal protein kinases activated downstream of a pleiotrophy of transmembrane receptors. Main intracellular components of the MAPK signalling pathway are the RAF, MEK, and ERK proteins, which work in a cascade of activator and effector proteins. They regulate many fundamental cellular functions, including cell proliferation, cell survival, and cell differentiation by transducing extracellular signals to cytoplasmic and nuclear effectors. To reveal more details about possible activation cascades in this pathway, the present study gives a complete description of the differential expression of Braf, Mek1, Mek2, Mek5, Erk1, Erk2, Erk3, and Erk5 in the adult murine brain by way of in situ hybridization analysis. In this study, we found that each gene is widely expressed in the whole brain, except for Mek2, but each displays a very distinct expression pattern, leading to distinct interactions of the MAPK components within different regions. Most notably we found that 1) Braf and Erk3 are coexpressed in the hippocampus proper, confirming a possible functional interaction; 2) in most forebrain areas, Mek5 and Erk5 are coexpressed; and 3) in the neurogenic regions of the brain, namely, the olfactory bulb and the dentate gyrus, Braf is absent, indicating that other activator proteins have to take over its function. Despite these differences, our results show widespread coexpression of the pathway components, thereby confirming the hypothesis of redundant functions among several MEK and ERK proteins in some regions of the brain. J. Comp. Neurol. 500:542–556, 2007.

Pink1-deficiency in mice impairs gait, olfaction and serotonergic innervation of the olfactory bulb

Parkinsons Disease (PD) is the most common neurodegenerative movement disorder. Autosomal-recessive mutations in the mitochondrial protein kinase PINK1 (PTEN-induced kinase 1) account for 1-2% of the hereditary early-onset cases. To study the mechanisms underlying disease development, we generated Pink1-deficient mice. In analogy to other genetic loss-of-function mouse models, Pink1(-/-) mice did not show morphological alterations in the dopaminergic system. As a consequence, no gross motor dysfunctions were observed indicating that these mice do not develop the cardinal symptoms of PD. Nonetheless, symptoms which develop mainly before bradykinesia, rigidity and resting tremor were clearly evident in Pink1-deficient mice. These symptoms were gait alterations and olfactory dysfunctions. Remarkably in the glomerular layer of the olfactory bulb the density of serotonergic fibers was significantly reduced. Concerning mitochondrial morphology, neurons in Pink1(-/-) mice had less fragmented mitochondria. In contrast, upon acute knock-down of Pink1 increased mitochondrial fragmentation was observed in neuronal cultures. This fragmentation was, however, evened out within days. Taken together, we demonstrate that Pink1-deficient mice exhibit behavioral symptoms of early phases of PD and present systematic experimental evidence for compensation of Pink1-deficiency at the cellular level. Thus, Pink1-deficient mice represent a model for the early phases of PD in which compensation may still impede the onset of neurodegeneration. Consequently, these mice are a valuable tool for studying Pink1-related PD development, as well as for searching for reliable PD biomarkers.

Novel allele of crybb2 in the mouse and its expression in the brain.

PURPOSE O377 was identified as a new dominant cataract mutation in mice after radiation experiments. The purpose of this study was to genetically characterize the mutation and to analyze its biological consequences. METHODS Linkage analysis of the O377 mouse mutant was performed; candidate genes including Crybb2 were sequenced. The authors analyzed eyes and brains of the mutants by histology and the expression domains of Crybb2 by in situ hybridization and immunohistochemistry. RNA was isolated from whole brains of heterozygous and homozygous O377 mutants, and differential expression arrays were performed. All studies were compared with age- and strain-matched wild-type mice. RESULTS The mutation was mapped to chromosome 5 and characterized as an A-->T substitution at the end of intron 5 of the Crybb2 gene. It led to alternative splicing with a 57-bp insertion in the mRNA and to 19 additional amino acids in the protein. In the brain, betaB2-crystallin was expressed in the cerebellum, olfactory bulb, cerebral cortex, and hippocampus. The only morphologic difference in the brain is the increased number of Purkinje cells in the cerebellum of homozygous strain-matched mutants. Differential expression analysis revealed the upregulation of calpain-3 in the brain of homozygous mutants, which was confirmed by quantitative real-time PCR. CONCLUSIONS These results confirm the third allele of Crybb2 in the mouse that also affected exon 6 and the fourth Greek key motif. Moreover, expression analysis of Crybb2 identified for the first time distinct regions of expression in the brain, and the differential expression analysis points to the participation of Ca2+ in the corresponding pathologic processes.