Kersti Lilleväli

Gata2 is a tissue-specific post-mitotic selector gene for midbrain GABAergic neurons

Midbrain GABAergic neurons control several aspects of behavior, but regulation of their development and diversity is poorly understood. Here, we further refine the midbrain regions active in GABAergic neurogenesis and show their correlation with the expression of the transcription factor Gata2. Using tissue-specific inactivation and ectopic expression, we show that Gata2 regulates GABAergic neuron development in the mouse midbrain, but not in rhombomere 1, where it is needed in the serotonergic lineage. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. These studies identify Gata2 as an essential post-mitotic selector gene of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of GABAergic neurons in the midbrain.

Comparative expression analysis of the genes encoding polypyrimidine tract binding protein (PTB) and its neural homologue (brPTB) in prenatal and postnatal mouse brain

The polypyrimidine tract binding protein (PTB) and its recently discovered homologue brain-enriched PTB (brPTB) are RNA binding proteins involved in the control of alternative splicing. We have characterized expression patterns of the PTB and brPTB in course of mouse brain development, using mRNA in situ hybridization. PTB is expressed in choroid plexi and ependyma at all the stages of development and temporarily in the mantle layer of migrating neuroblasts of fore-, mid- and hindbrain and in the external granular layer of cerebellum. In the neurons of adult mouse cerebrum and cerebellum expression of PTB is undetectable. In contrast to this, brPTB is expressed ubiquitously in neuroblasts of various parts of embryonic brain and in the differentiated neurons of postnatal cerebrum and cerebellum. brPTB mRNA is not observed in choroid plexi and ependymal layer. Thus, in the embryonic brain expression patterns of PTB and brPTB overlap, but in the course of brain development the patterns become complementary to each other.

Gata2 is required for the development of inner ear semicircular ducts and the surrounding perilymphatic space

Gata2 has essential roles in the development of many organs. During mouse inner ear morphogenesis, it is expressed in otic vesicle and the surrounding periotic mesenchyme from early on, but no defects in the ear development of Gata2 null mice have been observed before lethality at embryonic day (E) 10.5. Here, we used conditional gene targeting to reveal the role of Gata2 at later stages of inner ear development. We show that Gata2 is critically required from E14.5–E15.5 onward for vestibular morphogenesis. Without Gata2 the semicircular ducts fail to grow to their normal size and the surrounding mesenchymal cells are not removed properly to generate the perilymphatic space. Gata2 is the first factor known to control the clearing of the vestibular perilymphatic mesenchyme, but interestingly, it is not required for the formation of the cochlear perilymphatic areas, suggesting distinct molecular control for these processes. Developmental Dynamics 239:2452–2469, 2010.

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3’UTR targeting may constitute a useful tool in analyzing gene function.

Subdomain-mediated axon-axon signaling and chemoattraction cooperate to regulate afferent innervation of the lateral habenula

A dominant feature of neural circuitry is the organization of neuronal projections and synapses into specific brain nuclei or laminae. Lamina-specific connectivity is controlled by the selective expression of extracellular guidance and adhesion molecules in the target field. However, how (sub)nucleus-specific connections are established and whether axon-derived cues contribute to subdomain targeting are largely unknown. Here, we demonstrate that the lateral subnucleus of the habenula (lHb) determines its own afferent innervation by sending out efferent projections that express the cell adhesion molecule LAMP to reciprocally collect and guide dopaminergic afferents to the lHb-a phenomenon we term subdomain-mediated axon-axon signaling. This process of reciprocal axon-axon interactions cooperates with lHb-specific chemoattraction mediated by Netrin-1, which controls axon target entry, to ensure specific innervation of the lHb. We propose that cooperation between pretarget reciprocal axon-axon signaling and subdomain-restricted instructive cues provides a highly precise and general mechanism to establish subdomain-specific neural circuitry.

Defects in sensory organ morphogenesis and generation of cochlear hair cells in Gata3-deficient mouse embryos.

The development of the inner ear sensory epithelia involves a complex network of transcription factors and signaling pathways and the whole process is not yet entirely understood. GATA3 is a DNA-binding factor that is necessary for otic morphogenesis and without GATA3 variable defects have been observed already at early stages in mouse embryos. In the less severe phenotypes, one small oval shaped vesicle is formed whereas in the more severe cases, the otic epithelium becomes disrupted and the endolymphatic domain becomes separated from the rest of the otic epithelium. Despite these defects, the early sensory fate specification occurs in Gata3-/- otic epithelium. However, due to the early lethality of Gata3-deficient embryos, the later morphogenesis and sensory development have remained unclear. To gain information of these later processes we produced drug-rescued Gata3-/- embryos that survived up to late gestation. In these older Gata3-/- embryos, a similar variability was observed as earlier. In the more severely affected ears, the development of the separate endolymphatic domain arrested completely whereas the remaining vesicle formed an empty cavity with variable forms, but without any distinguishable otic compartments or morphologically distinct sensory organs. However, the dorsal part of this vesicle was able to adopt a sensory fate and to produce some hair cells. In the less severe cases of Gata3-/- ears, distinct utricular, saccular and cochlear compartments were present and hair cells could be detected in the vestibular sensory epithelia. Although clear cristae and maculae formed, the morphology and size of these sensory areas were abnormal and they remained often un-separated. In contrast to the vestibule, the cochlear sensory compartment remained more immature and no hair or supporting cells could be detected. Our results suggest that GATA3 is critical for normal vestibular and cochlear morphogenesis and that it is especially important for cochlear sensory differentiation.

The Embryonic Transcriptome of the Red-Eared Slider Turtle (Trachemys scripta).

The bony shell of the turtle is an evolutionary novelty not found in any other group of animals, however, research into its formation has suggested that it has evolved through modification of conserved developmental mechanisms. Although these mechanisms have been extensively characterized in model organisms, the tools for characterizing them in non-model organisms such as turtles have been limited by a lack of genomic resources. We have used a next generation sequencing approach to generate and assemble a transcriptome from stage 14 and 17 Trachemys scripta embryos, stages during which important events in shell development are known to take place. The transcriptome consists of 231,876 sequences with an N50 of 1,166 bp. GO terms and EC codes were assigned to the 61,643 unique predicted proteins identified in the transcriptome sequences. All major GO categories and metabolic pathways are represented in the transcriptome. Transcriptome sequences were used to amplify several cDNA fragments designed for use as RNA in situ probes. One of these, BMP5, was hybridized to a T. scripta embryo and exhibits both conserved and novel expression patterns. The transcriptome sequences should be of broad use for understanding the evolution and development of the turtle shell and for annotating any future T. scripta genome sequences.

Deletion of the Lsamp gene lowers sensitivity to stressful environmental manipulations in mice.

The Lsamp gene gives rise to limbic system-associated membrane protein (LAMP), which is expressed on the surface of somata and proximal dendrites of neurons. Lsamp-deficient mice have been shown to be slightly hyperactive in novel environments and less anxious, and they display alterations in swimming speed, fear reaction, fear conditioning and social behaviour. In human studies, links between the LSAMP gene and several psychiatric disorders have been found and LSAMP has been established as a tumour suppressor gene. To study the impact of environmental manipulations on the phenotype, we exposed male Lsamp-deficient mice to environmental enrichment (EE), a technique that has often been shown to abolish phenotypic deviations in knockout mice, and to social isolation, a stressful manipulation, after which all the mice were tested in a behavioural battery. EE abolished differences between the genotypes in body weight and anogenital sniffing, a behaviour related to aggressiveness, and amplified the anxiolytic-like phenotype of Lsamp-deficient mice both in the plus maze and motility box. Isolation abolished differences between the genotypes in body weight and anxiety and amplified the differences in swimming speed and anogenital sniffing. EE and isolation failed to modify the results as compared to standard housing in whisker trimming, locomotor activity, marble burying and corticosterone levels. In conclusion, Lsamp-deficient mice were less sensitive to isolation stress than their wild-type littermates. Lack of LAMP protein seemingly leads to a deterioration in the ability to adapt to novel stressful environments and stimuli.

Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain.

Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule involved in neurite formation and outgrowth. The purpose of the present study was to characterize the distribution of alternatively transcribed Lsamp isoforms in the mouse brain and its implications on the regulation of behavior. Limbic system-associated membrane protein 1b transcript was visualized by using a mouse strain expressing beta-galactosidase under the control of Lsamp 1b promoter. The distribution of Lsamp 1a transcript and summarized expression of the Lsamp transcripts was investigated by non-radioactive in situ RNA hybridization analysis. Cross-validation was performed by using radioactive in situ hybridization with oligonucleotide probes. Quantitative RT-PCR was used to study correlations between the expression of Lsamp isoforms and behavioral parameters. The expression pattern of two promoters differs remarkably from the developmental initiation at embryonic day 12.5. Limbic system-associated membrane protein 1a promoter is active in “classic” limbic structures where the hippocampus and amygdaloid area display the highest expression. Promoter 1b is mostly active in the thalamic sensory nuclei and cortical sensory areas, but also in areas that regulate stress and arousal. Higher levels of Lsamp 1a transcript had significant correlations with all of the measures indicating higher trait anxiety in the elevated plus-maze test. Limbic system-associated membrane protein transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social interaction test. The data are in line with decreased anxiety and alterations in social behavior in Lsamp-deficient mice. We propose that Lsamp is involved in emotional and social operating systems by complex regulation of two alternative promoters.

Characterization of MYG1 gene and protein: subcellular distribution and function.

Background information. MYG1 [Melanocyte proliferating gene 1, also known as Gamm1 (NM_021640)] is a recently described gene of unknown function. MYG1 orthologues are found in simple as well as complex eukaryotes. According to sequence homology, MYG1 is considered to have a metal‐dependent protein hydrolase (UPF0160) domain. The purpose of the present study was to determine the expression and subcellular localization of MYG1 protein and to identify physiological processes connected to MYG1 function.