Maarja Haugas

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.

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.

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.

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development.

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory–neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken. Developmental Dynamics 236:306–313, 2007.

Differentiation and molecular heterogeneity of inhibitory and excitatory neurons associated with midbrain dopaminergic nuclei.

Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits. Summary: Tal1 and Gata transcription factors select between inhibitory GABAergic and excitatory glutamatergic fates of neurons regulating the dopaminergic system.

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. We show that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3, which are activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3-expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. Our study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation. Summary: Dorsal raphe neurons in mice exhibit functionally significant heterogeneity that is established early during their differentiation via the actions of Gata2 and Gata3.

The role of Gata2 in the midbrain GABAergic neuron development

Emergence of the hilar ectopic granule cells is a well-known pathological aberration found in the dentate gyrus of temporal lobe epilepsy patients. However, the cellular mechanisms underlying the phenomena remain to be elucidated. In this study, we asked if early-life febrile seizures affect the normal migration of the granule cells in the postnatal hippocampus because the development of the granule cell layer is almost completed in the postnatal periods when most of the patients experience febrile seizures. To investigate themechanisms that regulate the granule cell migration, we developed a novel slice coculture system. We prepared the hilar micro-slices from early-postnatal transgenic rats expressing green fluorescent protein and inserted them to the hippocampal slices from wild-type littermates. Using confocal microscopy, we succeeded in the real-time imaging of the migrating granule cells. The immature granule cells had long leading processes and performed the radial migration to the granule cell layer. We found that the granule cells migrate reversely in the hilus when the hilar slices were derived from rats that underwent experimental febrile seizures. The reverse migration was blocked by a bath application of the GABAA receptor antagonist bicuculline. We also found that muscimol, a GABAA receptor agonist, slows down or reverses the migration of the granule cells which originate from the wild-type hilar slices. These results suggest that GABAA receptor activation interrupts the normal migration of the immature granule cells in the postnatal hippocampus undergoing febrile seizures. Considering that the development of the dentate gyrus is mostly completed postnatally, the slowly or reverselymigrating granule cells during the periods likely give rise to the hilar ectopic granule cells later in life.

A complex genomic locus drives mtDNA replicase POLG expression to its disease-related nervous system regions

DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG, containing three functional CNS‐specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925‐RNA and MIR9‐3, which are coexpressed with POLG. The MIR9‐3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non‐coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS‐specific manifestations in POLG disease.

Melanoblast development coincides with the late emerging cells from the dorsal neural tube in turtle Trachemys scripta

Ectothermal reptiles have internal pigmentation, which is not seen in endothermal birds and mammals. Here we show that the development of the dorsal neural tube-derived melanoblasts in turtle Trachemys scripta is regulated by similar mechanisms as in other amniotes, but significantly later in development, during the second phase of turtle trunk neural crest emigration. The development of melanoblasts coincided with a morphological change in the dorsal neural tube between stages mature G15 and G16. The melanoblasts delaminated and gathered in the carapacial staging area above the neural tube at G16, and differentiated into pigment-forming melanocytes during in vitro culture. The Mitf-positive melanoblasts were not restricted to the dorsolateral pathway as in birds and mammals but were also present medially through the somites similarly to ectothermal anamniotes. This matched a lack of environmental barrier dorsal and lateral to neural tube and the somites that is normally formed by PNA-binding proteins that block entry to medial pathways. PNA-binding proteins may also participate in the patterning of the carapacial pigmentation as both the migratory neural crest cells and pigment localized only to PNA-free areas.

12-P003 Gata2 is a postmitotic selector gene for GABAergic neurons in the midbrain

Vertebrate neurotrophins (NTs: NGF, BDNF, NT3, NT4) form a family of signalling molecules with key functions in nervous system development and function. They regulate cell survival, axon guidance and targeting, synaptic formation and function, learning and memory. Deficient NGF function occurs in Alzheimer’s disease and alterations in BDNF function underlie psychiatric and cognitive disorders such as epilepsy, autism, anxiety and depression. NTs are therapeutic targets in pain control (e.g. chronic pain in cancer). We recently reported that a family of Drosophila neurotrophins (DNTs) formed by DNT1, DNT2 and Spz regulate neuronal survival and targeting in fruit-flies (Zhu et al., 2008 PLoS Biology 6, e284). We are now investigating the roles that DNTs play in neuronal function, using both loss and gain of function conditions for each of the DNTs. Here, we report the involvement of DNTs in synapse formation and in adult locomotion. With the help of mathematical algorithms that we have developed, we quantify automatically and objectively phenotypic changes in: (i) the number of boutons and active zones at the larval neuromuscular junction (NMJ) synapse; (ii) axon terminal length and branch number at the NMJ; (iii) and speed and trajectory of adult flies in a locomotion paradigm. Using genetic rescue experiments, we investigate functional redundancy and whether the DNTs are required in muscle or neurons. Our findings suggest that neuronal functions of NTs are evolutionarily conserved, supporting the notion that NTs underlie multiple aspects of nervous system development and function in all animals with a CNS and brain.