Telophase correction refines division orientation in stratified epithelia.

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs.

Impaired αVß8 and TGFß signaling lead to microglial dysmaturation and neuromotor dysfunction.

Microglia play a pivotal role in the coordination of brain development and have emerged as a critical determinant in the progression of neurodegenerative diseases; however, the role of microglia in the onset and progression of neurodevelopmental disorders is less clear. Here we show that conditional deletion of αVß8 from the central nervous system (Itgb8ΔCNS mice) blocks microglia in their normal stepwise development from immature precursors to mature microglia. These "dysmature" microglia appear to result from reduced TGFß signaling during a critical perinatal window, are distinct from microglia with induced reduction in TGFß signaling during adulthood, and directly cause a unique neurodevelopmental syndrome characterized by oligodendrocyte maturational arrest, interneuron loss, and spastic neuromotor dysfunction. Consistent with this, early (but not late) microglia depletion completely reverses this phenotype. Together, these data identify novel roles for αVß8 and TGFß signaling in coordinating microgliogenesis with brain development and implicate abnormally programmed microglia or their products in human neurodevelopmental disorders that share this neuropathology.

Afadin controls cell polarization and mitotic spindle orientation in developing cortical radial glia.

BACKGROUND: In developing tissues, cell polarity and tissue architecture play essential roles in the regulation of proliferation and differentiation. During cerebral cortical development, adherens junctions link highly polarized radial glial cells in a neurogenic niche that controls their behavior. How adherens junctions regulate radial glial cell polarity and/or differentiation in mammalian cortical development is poorly understood. RESULTS: Conditional deletion of Afadin, a protein required for formation and maintenance of epithelial tissues, leads to abnormalities in radial glial cell polarity and subsequent loss of adherens junctions. We observed increased numbers of obliquely-oriented progenitor cell divisions, increased exit from the ventricular zone neuroepithelium, and increased production of intermediate progenitors. CONCLUSIONS: Together, these findings indicate that Afadin plays an essential role in regulating apical-basal polarity and adherens junction integrity of radial glial cells, and suggest that epithelial architecture plays an important role in radial glial identity by regulating mitotic orientation and preventing premature exit from the neurogenic niche.

Glutamatergic axon-derived BDNF controls GABAergic synaptic differentiation in the cerebellum.

To study mechanisms that regulate the construction of inhibitory circuits, we examined the role of brain-derived neurotrophic factor (BDNF) in the assembly of GABAergic inhibitory synapses in the mouse cerebellar cortex. We show that within the cerebellum, BDNF-expressing cells are restricted to the internal granular layer (IGL), but that the BDNF protein is present within mossy fibers which originate from cells located outside of the cerebellum. In contrast to deletion of TrkB, the cognate receptor for BDNF, deletion of Bdnf from cerebellar cell bodies alone did not perturb the localization of pre- or postsynaptic constituents at the GABAergic synapses formed by Golgi cell axons on granule cell dendrites within the IGL. Instead, we found that BDNF derived from excitatory mossy fiber endings controls their differentiation. Our findings thus indicate that cerebellar BDNF is derived primarily from excitatory neurons--precerebellar nuclei/spinal cord neurons that give rise to mossy fibers--and promotes GABAergic synapse formation as a result of release from axons. Thus, within the cerebellum the preferential localization of BDNF to axons enhances the specificity through which BDNF promotes GABAergic synaptic differentiation.

Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking αVß8-TGFß signaling in the brain.

Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVß8 activates angiosuppressive TGFß gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVß8 in the brain or downstream TGFß1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfb(ret/ret) mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage.

Selective targeting of TGF-ß activation to treat fibroinflammatory airway disease.

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-ß (TGF-ß) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-ß is expressed in a latent form that requires activation. The integrin αvß8 (encoded by the itgb8 gene) is a receptor for latent TGF-ß and is essential for its activation. Expression of integrin αvß8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvß8 (B5) inhibited TGF-ß activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-ß activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvß8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvß8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-ß pathway to treat fibroinflammatory airway diseases.

Transcriptional corepressors HIPK1 and HIPK2 control angiogenesis via TGF-ß-TAK1-dependent mechanism.

Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-ß (TGF-ß) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-ß remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-ß-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-ß type II receptor (TßRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-ß in a TAK1-dependent manner. Remarkably, TGF-ß-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-ß signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.

De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity.

A fundamental process in biology is the de novo formation and morphogenesis of polarized tubules. Although these processes are essential for the formation of multiple metazoan organ systems, little is known about the molecular mechanisms that regulate them. In this study, we have characterized several steps in tubule formation and morphogenesis using the mouse kidney as a model system. We report that kidney mesenchymal cells contain discrete Par3-expressing membrane microdomains that become restricted to an apical domain, coinciding with lumen formation. Once lumen formation has been initiated, elongation occurs by simultaneous extension and additional de novo lumen generation. We demonstrate that lumen formation and elongation require afadin, a nectin adaptor protein implicated in adherens junction formation. Mice that lack afadin in nephron precursors show evidence of Par3-expressing membrane microdomains, but fail to develop normal apical-basal polarity and generate a continuous lumen. Absence of afadin led to delayed and diminished integration of nectin complexes and failure to recruit R-cadherin. Furthermore, we demonstrate that afadin is required for Par complex formation. Together, these results suggest that afadin acts upstream of the Par complex to regulate the integration and/or coalescence of membrane microdomains, thereby establishing apical-basal polarity and lumen formation/elongation during kidney tubulogenesis.

A novel method that improves sensitivity of protein detection in PAGE and Western blot.

We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available.

Rare missense neuronal cadherin gene (CDH2) variants in specific obsessive-compulsive disorder and Tourette disorder phenotypes.

The recent finding that the neuronal cadherin gene CDH2 confers a highly significant risk for canine compulsive disorder led us to investigate whether missense variants within the human ortholog CDH2 are associated with altered susceptibility to obsessive-compulsive disorder (OCD), Tourette disorder (TD) and related disorders. Exon resequencing of CDH2 in 320 individuals identified four non-synonymous single-nucleotide variants, which were subsequently genotyped in OCD probands, Tourette disorder probands and relatives, and healthy controls (total N=1161). None of the four variants was significantly associated with either OCD or TD. One variant, N706S, was found only in the OCD/TD groups, but not in controls. By examining clinical data, we found there were significant TD-related phenotype differences between those OCD probands with and without the N845S variant with regard to the co-occurrence of TD (Fisher's exact test P=0.014, OR=6.03). Both N706S and N845S variants conferred reduced CDH2 protein expression in transfected cells. Although our data provide no overall support for association of CDH2 rare variants in these disorders considered as single entities, the clinical features and severity of probands carrying the uncommon non-synonymous variants suggest that CDH2, along with other cadherin and cell adhesion genes, is an interesting gene to pursue as a plausible contributor to OCD, TD and related disorders with repetitive behaviors, including autism spectrum disorders.

Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is critical for formation of α-smooth muscle actin filaments during myofibroblast differentiation.

Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and in myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-ß1 (TGF-ß1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-ß1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and maturation.

TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry.

Dysfunction of basal forebrain cholinergic neurons (BFCNs) is an early pathological hallmark of Alzheimer's disease (AD). Numerous studies have indicated that nerve growth factor (NGF) supports survival and phenotypic differentiation of BFCNs. Consistent with a potential link to AD pathogenesis, TrkA, a NGF receptor, is expressed in cholinergic forebrain neuronal populations including those in BF and striatum, and is markedly reduced in individuals with mild cognitive impairment (MCI) without dementia and early-stage AD. To investigate the role of TrkA in the development, connectivity, and function of the BF cholinergic system and its contribution to AD pathology, we have generated a forebrain-specific conditional TrkA knock-out mouse line. Our findings show a key role for TrkA signaling in establishing the BF cholinergic circuitry through the ERK pathway, and demonstrate that the normal developmental increase of choline acetyltransferase expression becomes critically dependent on TrkA signaling before neuronal connections are established. Moreover, the anatomical and physiological deficits caused by lack of TrkA signaling in BFCNs have selective impact on cognitive activity. These data demonstrate that TrkA loss results in cholinergic BF dysfunction and cognitive decline that is reminiscent of MCI and early AD.

Integrin ß1 signals through Arg to regulate postnatal dendritic arborization, synapse density, and behavior.

Integrins are heterodimeric extracellular matrix receptors that are essential for the proper development of the vertebrate nervous system. We report here that selective loss of integrin ß1 in excitatory neurons leads to reductions in the size and complexity of hippocampal dendritic arbors, hippocampal synapse loss, impaired hippocampus-dependent learning, and exaggerated psychomotor sensitivity to cocaine in mice. Our biochemical and genetic experiments demonstrate that the intracellular tail of integrin ß1 binds directly to Arg kinase and that this interaction stimulates activity of the Arg substrate p190RhoGAP, an inactivator of the RhoA GTPase. Moreover, genetic manipulations that reduce integrin ß1 signaling through Arg recapitulate the integrin ß1 knock-out phenotype in a gene dose-sensitive manner. Together, these results describe a novel integrin ß1-Arg-p190RhoGAP pathway that regulates dendritic arbor size, promotes synapse maintenance, supports proper hippocampal function, and mitigates the behavioral consequences of cocaine exposure.

Afadin, a Ras/Rap effector that controls cadherin function, promotes spine and excitatory synapse density in the hippocampus.

Many molecules regulate synaptogenesis, but intracellular signaling pathways required for their functions are poorly understood. Afadin is a Rap-regulated, actin-binding protein that promotes cadherin complex assembly as well as binding many other cell adhesion molecules and receptors. To examine its role in mediating synaptogenesis, we deleted afadin (mllt1), using a conditional allele, in postmitotic hippocampal neurons. Consistent with its role in promoting cadherin recruitment, afadin deletion resulted in 70% fewer and less intense N-cadherin puncta with similar reductions of ß-catenin and αN-catenin puncta densities and 35% reduction in EphB2 puncta density. Its absence also resulted in 40% decreases in spine and excitatory synapse densities in the stratum radiatum of CA1, as determined by morphology, apposition of presynaptic and postsynaptic markers, and synaptic transmission. The remaining synapses appeared to function normally. Thus, afadin is a key intracellular signaling molecule for cadherin recruitment and is necessary for spine and synapse formation in vivo.

Defective retinal vascular endothelial cell development as a consequence of impaired integrin αVß8-mediated activation of transforming growth factor-ß.

Deletions of the genes encoding the integrin αVß8 (Itgav, Itgb8) have been shown to result in abnormal vascular development in the CNS, including prenatal and perinatal hemorrhage. Other work has indicated that a major function of this integrin in vivo is to promote TGFß activation. In this paper, we show that Itgb8 mRNA is strongly expressed in murine Müller glia and retinal ganglion cells, but not astrocytes. We further show that Itgb8 deletion in the entire retina severely perturbs development of the murine retinal vasculature, elevating vascular branch point density and vascular coverage in the superficial vascular plexus, while severely impairing formation of the deep vascular plexus. The stability of the mutant vasculature is also impaired as assessed by the presence of hemorrhage and vascular basal lamina sleeves lacking endothelial cells. Specific deletion of Itgb8 in Müller glia and neurons, but not deletion in astrocytes, recapitulates the phenotype observed following Itgb8 in the entire retina. Consistent with αVß8's role in TGFß1 activation, we show that retinal deletion of Tgfb1 results in very similar retinal vascular abnormalities. The vascular deficits appear to reflect impaired TGFß signaling in vascular endothelial cells because retinal deletion of Itgb8 reduces phospho-SMAD3 in endothelial cells and endothelial cell-specific deletion of the TGFßRII gene recapitulates the major deficits observed in the Itgb8 and TGFß1 mutants. Of special interest, the retinal vascular phenotypes observed in each mutant are remarkably similar to those of others following inhibition of neuropilin-1, a receptor previously implicated in TGFß activation and signaling.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis.

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

TrkB (tropomyosin-related kinase B) controls the assembly and maintenance of GABAergic synapses in the cerebellar cortex.

Inhibitory interneurons play a critical role in coordinating the activity of neural circuits. To explore the mechanisms that direct the organization of inhibitory circuits, we analyzed the involvement of tropomyosin-related kinase B (TrkB) in the assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex. We show that TrkB acts directly within each cell-type to regulate synaptic differentiation. TrkB is required not only for assembly, but also maintenance of these synapses and acts, primarily, by regulating the localization of synaptic constituents. Postsynaptically, TrkB controls the localization of a scaffolding protein, gephyrin, but acts at a step subsequent to the localization of a cell adhesion molecule, Neuroligin-2. Importantly, TrkB is required for the localization of an Ig superfamily cell adhesion molecule, Contactin-1, in Golgi and granule cells and the absence of Contactin-1 also results in deficits in inhibitory synaptic development. Thus, our findings demonstrate that TrkB controls the assembly and maintenance of GABAergic synapses and suggest that TrkB functions, in part, through promoting synaptic adhesion.

Mesangial cell integrin αvß8 provides glomerular endothelial cell cytoprotection by sequestering TGF-ß and regulating PECAM-1.

Integrins are heterodimeric receptors that regulate cell adhesion, migration, and apoptosis. Integrin αvß8 is most abundantly expressed in kidney and brain, and its major ligand is latent transforming growth factor-ß (TGF-ß). Kidney αvß8 localizes to mesangial cells, which appose glomerular endothelial cells and maintain glomerular capillary structure by mechanical and poorly understood paracrine mechanisms. To establish kidney αvß8 function, mice with homozygous Itgb8 deletion (Itgb8(-/-)) were generated on outbred and C57BL/6 congenic backgrounds. Most Itgb8(-/-) mice died in utero, and surviving Itgb8(-/-) mice failed to gain weight, and rarely survived beyond 6 weeks. A renal glomerular phenotype included azotemia and albuminuria, as well as increased platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which was surprisingly not associated with conventional functions, such as endothelial cell hyperplasia, hypertrophy, or perivascular inflammation. Itgb8(-/-) mesangial cells demonstrated reduced latent TGF-ß binding, resulting in bioactive TGF-ß release, which stimulated glomerular endothelial cell apoptosis. Using PECAM-1 gain and loss of function strategies, we show that PECAM-1 provides endothelial cytoprotection against mesangial cell TGF-ß. These results clarify a singular mechanism of mesangial-to-endothelial cell cross-talk, whereby mesangial cell αvß8 homeostatically arbitrates glomerular microvascular integrity by sequestering TGF-ß in its latent conformation. Under pathological conditions associated with decreased mesangial cell αvß8 expression and TGF-ß secretion, compensatory PECAM-1 modulation facilitates glomerular endothelial cell survival.