Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM-sleep regulatory neurons.

The midbrain reticular formation (MRF) is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including sleep regulation. However, the molecular characteristics and development of MRF neurons are poorly understood. As the transcription factor, Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is required for the expression of several GABAergic lineage-specific transcription factors, including Nkx2-2 and Skor2, which are co-expressed in a restricted group of post-mitotic GABAergic precursors in the MRF. Both Gata2 and Nkx2-2 function is required for Skor2 expression in GABAergic precursors. In the adult mouse and rat midbrain, Nkx2-2-and Skor2-expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the MRF, an area containing REM-off neurons regulating REM sleep. In addition to the characteristic localization, Skor2+ cells increase their activity upon REM-sleep inhibition, send projections to the dorsolateral pons, a region associated with sleep control, and are responsive to orexins, consistent with the known properties of midbrain REM-off neurons.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone.

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.

A network including TGFß/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells.

Transforming growth factor ß (TGFß) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFß-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFß response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFß signaling effector molecule p57 was found to be upregulated as a secondary response to TGFß. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFß-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.

Gata2 Is a Rheostat for Mesenchymal Stem Cell Fate in Male Mice.

Gata2 is a zinc finger transcription factor that is important in hematopoiesis and neuronal development. However, the roles of Gata2 in the mesenchymal lineages are poorly understood. In vitro studies suggest that Gata2 modulates adipocyte differentiation and mesenchymal stem cell (MSC) proliferation. To systematically determine the in vivo functions of Gata2 in the MSC lineage commitment and development, we have generated three mouse models in which Gata2 is specifically deleted in MSCs, adipocytes, or osteoblasts. During the MSC expansion stage, Gata2 promotes proliferation and attenuates differentiation; thereby Gata2 loss in MSCs results in enhanced differentiation of both adipocytes and osteoblasts. During the differentiation stage, Gata2 also plays MSC-independent roles to impede lineage commitment; hence, Gata2 loss in adipocyte or osteoblast lineages also augments adipogenesis and osteoblastogenesis, respectively. These findings reveal Gata2 as a crucial rheostat of MSC fate to control osteoblast and adipocyte lineage 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.

Human bocaviruses are commonly found in stools of hospitalized children without causal association to acute gastroenteritis.

UNLABELLED: Human bocaviruses (HBoVs) may be grouped into respiratory (HBoV1) and enteric (HBoV2-4) types. We examined this association of HBoV types and clinical symptoms in 955 children who had acute gastroenteritis (AGE, n = 172), acute respiratory tract infection (ARTI, n = 545) or symptoms of both (n = 238). Both nasal swab and stool specimens were studied for such patients. HBoV1 DNA was detected in 6.2 % of patients with ARTI and 9.2 % of patients with symptoms of both ARTI and AGE, but in only 1.7 % of patients with AGE alone. In about one half of the cases, HBoV1 was detected concomitantly in nasal swab and stool samples. HBoV2 was found in stool samples of patients with AGE (5.8 %), ARTI (5.1 %) and symptoms of both (5.5 %) but only rarely in nasal swabs. HBoV3 was found in the stools, but not in nasal swabs, in 0.6, 1.1 and 0.8 % of patients with, respectively, AGE, ARTI and both. HBoV4 was not found. All but one HBoV-positive stool sample of AGE patients contained a known gastroenteritis virus (rotavirus, norovirus, sapovirus, astrovirus or enteric adenovirus) that was probably responsible for the symptoms of the respective case. Sera of 30 HBoV-positive patients were available, and IgM antibodies for HBoVs were found in ten cases and HBoV DNA in eight of these. CONCLUSIONS: HBoV2 and HBoV3 were more commonly found in stool than in nasal swab samples, but the findings could not be causally linked with AGE. HBoV1 was commonly found in stool samples during ARTI, with or without gastrointestinal symptoms.

Mechanisms regulating GABAergic neuron development.

Neurons using gamma-aminobutyric acid (GABA) as their neurotransmitter are the main inhibitory neurons in the mature central nervous system (CNS) and show great variation in their form and function. GABAergic neurons are produced in all of the main domains of the CNS, where they develop from discrete regions of the neuroepithelium. Here, we review the gene expression and regulatory mechanisms controlling the main steps of GABAergic neuron development: early patterning of the proliferative neuroepithelium, production of postmitotic neural precursors, establishment of their identity and migration. By comparing the molecular regulation of these events across CNS, we broadly identify three regions utilizing distinct molecular toolkits for GABAergic fate determination: telencephalon-anterior diencephalon (DLX2 type), posterior diencephalon-midbrain (GATA2 type) and hindbrain-spinal cord (PTF1A and TAL1 types). Similarities and differences in the molecular regulatory mechanisms reveal the core determinants of a GABAergic neuron as well as provide insights into generation of the vast diversity of these neurons.

Distinct developmental origins and regulatory mechanisms for GABAergic neurons associated with dopaminergic nuclei in the ventral mesodiencephalic region.

GABAergic neurons in the ventral mesodiencephalic region are highly important for the function of dopaminergic pathways that regulate multiple aspects of behavior. However, development of these neurons is poorly understood. We recently showed that molecular regulation of differentiation of the GABAergic neurons associated with the dopaminergic nuclei in the ventral midbrain (VTA and SNpr) is distinct from the rest of midbrain, but the reason for this difference remained elusive. Here, we have analyzed the developmental origin of the VTA and SNpr GABAergic neurons by genetic fate mapping. We demonstrate that the majority of these GABAergic neurons originate outside the midbrain, from rhombomere 1, and move into the ventral midbrain only as postmitotic neuronal precursors. We further show that Gata2, Gata3 and Tal1 define a subpopulation of GABAergic precursors in ventral rhombomere 1. A failure in GABAergic neuron differentiation in this region correlates with loss of VTA and SNpr GABAergic neurons in Tal1 mutant mice. In contrast to midbrain, GABAergic neurons of the anterior SNpr in the diencephalon are not derived from the rhombomere 1. These results suggest unique migratory pathways for the precursors of important GABAergic neuron subpopulations, and provide the basis for understanding diversity within midbrain GABAergic neurons.

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.

Netrin1 is required for neural and glial precursor migrations into the olfactory bulb.

Netrin1 (NTN1) deficiency in mouse brain causes defects in axon guidance and cell migration during embryonic development. Here we show that NTN1 is required for olfactory bulb (OB) development at late embryogenesis and at early postnatal stages to facilitate the accumulation of proper numbers of granular and glomerular neuron subtypes and oligodendrocytes into the OB. In addition to the analysis of Ntn1-/- mice we made tissue and neurosphere cultures to clarify the role of NTN1 in the anterior forebrain. We propose that a subset of neural progenitors/precursors requires NTN1 to efficiently enter the rostral migratory stream to migrate into the OB. The analysis of postnatal Ntn1-/- OBs revealed a reduction of specific types of interneurons which have been shown to originate from particular subregions of the lateral ventricle walls. Based on Ntn1 expression in ventral parts of the ventricle walls, we observed a decrease in the mainly ventrally derived type II interneurons that express calcium-binding proteins calretinin and calbindin. Instead, no change in the numbers of dorsally derived tyrosine hydroxylase expressing interneurons was detected. In addition to the specific reduction of type II interneurons, our results indicate that NTN1 is required for oligodendroglial migration into the OB. Furthermore, we characterised the Ntn1 expressing subpopulation of neurosphere-forming cells from embryonic and adult brain as multipotent and self-renewing. However, NTN1 is dispensable for the proliferation of neurosphere forming progenitor cells and for their differentiation.

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.

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)P(2)-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.

Analysis of Netrin 1 receptors during inner ear development.

Netrin 1 plays key roles in axon guidance and neuronal migration during central nervous system (CNS) development. Outside the CNS, Netrin 1 has been shown to be involved in epithelial morphogenesis of various organs. We have shown that Netrin 1 is essential for inner ear semicircular duct formation, but the involvement of Netrin 1 receptors in this process has remained unknown. Netrin 1 receptors include members of the Deleted in colorectal cancer (Dcc), Unc5-homologue and integrin families. Here we have analysed the expression of these receptor genes during inner ear development and verified the inner ear phenotypes of several receptor mutant mice. Special interest was directed to receptors that could cooperate with Netrin 1 during semicircular duct formation. We show that Neogenin (Neo1), Unc5c as well as integrin b1 (Itgb1) are expressed in periotic mesenchyme, while Dcc, Unc5b, Unc5c, Itga3, Itga6 and Itgb1 are expressed in different parts of the otic epithelium. In spite of the broad and strong expression of several receptors in ear region, none of the analysed receptor mutant embryos showed any defects in inner ear development.

Apaf1-dependent programmed cell death is required for inner ear morphogenesis and growth.

During inner ear development programmed cell death occurs in specific areas of the otic epithelium but the significance of it and the molecules involved have remained unclear. We undertook an analysis of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene led to a dramatic decrease in apoptosis in the inner ear epithelium, severe morphogenetic defects and a significant size reduction of the membranous labyrinth, demonstrating that an Apaf1-dependent apoptotic pathway is necessary for normal inner ear development. This pathway most probably operates through the apoptosome complex because caspase 9 mutant mice suffered similar defects. Inactivation of the Bcl2-like (Bcl2l) gene led to an overall increase in the number of cells undergoing apoptosis but did not cause any major morphogenetic defects. In contrast, decreased apoptosis was observed in specific locations that suffered from developmental deficits, indicating that proapoptotic isoform(s) produced from Bcl2l might have roles in inner ear development. In Apaf1(-/-)/Bcl2l(-/-) double mutant embryos, no cell death could be detected in the otic epithelium, demonstrating that the cell death regulated by the anti-apoptotic Bcl2l isoform, Bcl-X(L) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle failed to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development.

Developmentally regulated expression of Netrin-1 and -3 in the embryonic mouse molar tooth germ.

The Netrins form a small, conserved family of laminin-related signaling proteins regulating axon guidance in the developing nervous system. Here, we analyzed the roles of Netrin-1 and -3 in trigeminal axon guidance to the first lower molar of the embryonic mouse. Netrin-1 showed a restricted epithelial expression domain buccal to the tooth germ, toward which the pioneer tooth axons initially appear to navigate. Later, before birth, transcripts were colocalized with nerve fibers around the bell stage tooth germ. Analysis of Netrin-1-deficient mice, however, did not reveal any obvious disturbances in the axon growth or pattern of tooth innervation. In contrast, Netrin-3 showed a prominent, distinct expression in the axon pathway and target field mesenchyme around the tooth. Hence, it is possible that Netrin-3 may regulate pioneer axon growth toward and within the embryonic tooth target field.

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 approximately 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 microM) than ATP-G-actin (KD = 1.96 microM) 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.

Mouse MIM, a tissue-specific regulator of cytoskeletal dynamics, interacts with ATP-actin monomers through its C-terminal WH2 domain.

The WH2 (WASP homology domain-2) is a small actin monomer-binding motif and is found in many proteins that regulate the actin cytoskeleton, including the beta-thymosins, ciboulot, WASP, and verprolin/WIP (WASP-interacting protein). In sequence database searches we identified a novel mouse protein containing a WH2 domain in its C-terminal region. This mouse gene also shows strong sequence homology to human MIM (Missing in Metastasis), a cDNA fragment that is present in non-metastatic but absent in metastatic bladder cancer cell lines. Northern blot and in situ hybridizations show that MIM is strongly expressed in the developing neurons and skeletal and cardiac muscles in mouse embryos. In adult mice, the strongest expression of MIM mRNA is in liver, outer layers of the kidney, and in the Purkinje cells of the brain. Recombinant MIM protein interacts with actin monomers and inhibits actin filament nucleation in vitro. However, the MIM/ATP-G-actin complex can participate in actin filament assembly at the barbed end. MIM binds ATP-G-actin with a higher affinity (K(D) = 0.06 microm) than ADP-G-actin (K(D) = 0.3 microm) and inhibits the nucleotide exchange on actin monomers. Site-directed mutagenesis demonstrates that the actin monomer-binding site resides in the C-terminal WH2 domain of MIM. Overexpression of mouse MIM in NIH 3T3 cells results in the disappearance of actin stress fibers and appearance of abnormal actin filament structures. These data show that MIM is an ATP-G-actin binding protein that regulates cytoskeletal dynamics in specialized mammalian cell-types.