Marjo Salminen

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.

Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated.

Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with ∼65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518–525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 μm) than ATP-G-actin (KD = 1.96 μm) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.

Gata3 is required for early morphogenesis and Fgf10 expression during otic development

Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.

GATA2 is required for lymphatic vessel valve development and maintenance

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Partially overlapping expression of Gata2 and Gata3 during inner ear development.

Gata2 and Gata3 belong to the Gata family of transcription factors in vertebrates that bind to a consensus “GATA” DNA sequence. The Gata3 gene is one of the earliest markers for the developing mouse inner ear. Ear morphogenesis is blocked in Gata3‐deficient embryos, whereas nothing was known of the role of Gata2 in mouse inner ear. Here, we have compared the expression patterns of Gata2 and Gata3 during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 has been inactivated. The expression of the two Gata genes is highly overlapping at embryonic day (E)10.5 but becomes increasingly distinct later. Whereas Gata2 is predominantly expressed in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. No phenotypic abnormalities were observed in the inner ear of Gata2−/− embryos before lethality at E10.5 and Gata3 expression was unchanged. In contrast, a delay and strong reduction of Gata2 expression was detected in Gata3−/− otic epithelium. Developmental Dynamics 231:775–781, 2004.

Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures.

Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR– and F-BAR–domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR–domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension

Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P2-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.

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.

Transcriptional regulatory mechanisms underlying the GABAergic neuron fate in different diencephalic prosomeres

Diverse mechanisms regulate development of GABAergic neurons in different regions of the central nervous system. We have addressed the roles of a proneural gene, Ascl1, and a postmitotic selector gene, Gata2, in the differentiation of GABAergic neuron subpopulations in three diencephalic prosomeres: prethalamus (P3), thalamus (P2) and pretectum (P1). Although the different proliferative progenitor populations of GABAergic neurons commonly express Ascl1, they have distinct requirements for it in promotion of cell-cycle exit and GABAergic neuron identity. Subsequently, Gata2 is activated as postmitotic GABAergic precursors are born. In P1, Gata2 regulates the neurotransmitter identity by promoting GABAergic and inhibiting glutamatergic neuron differentiation. Interestingly, Gata2 defines instead the subtype of GABAergic neurons in the rostral thalamus (pTh-R), which is a subpopulation of P2. Without Gata2, the GABAergic precursors born in the pTh-R fail to activate subtype-specific markers, but start to express genes typical of GABAergic precursors in the neighbouring P3 domain. Thus, our results demonstrate diverse mechanisms regulating differentiation of GABAergic neuron subpopulations and suggest a role for Gata2 as a selector gene of both GABAergic neuron neurotransmitter and prosomere subtype identities in the developing diencephalon. Our results demonstrate for the first time that neuronal identities between distinct prosomeres can still be transformed in postmitotic neuronal precursors.