Janne Hakanen

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.

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.

E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells.

Many if not most tissues need a controlled number of stem cells to maintain normal function. Cancer can be seen as a process of disturbed tissue homeostasis, in which too many cells have or acquire too primitive identity. Here we measured how oncogenes and tumour suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. Neural progenitor cells (NPCs) were exposed to human papilloma virus E6, E7 or E6/E7 oncogenes, which degrade tumour suppressors p53 and pRb family members, respectively. E6/E7-expressing or p53−/− NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family, and involved MEK–ERK signalling. Decreased amount of p53 increased self-renewal and proliferation, whereas pRb affected only proliferation. Our results suggest that the oncogenes increase whereas p53 and pRb family tumour suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumour suppressors control tissue homeostasis and highlight their importance in stem cell self- renewal, linked both to cancer and life-long tissue turnover.

Defects in neural guidepost structures and failure to remove leptomeningeal cells from the septal midline behind the interhemispheric fusion defects in Netrin1 deficient mice

Corpus callosum (CC) is the largest commissural tract in mammalian brain and it acts to coordinate information between the two cerebral hemispheres. During brain development CC forms at the boundary area between the cortex and the septum and special transient neural and glial guidepost structures in this area are thought to be critical for CC formation. In addition, it is thought that the fusion of the two hemispheres in the septum area is a prerequisite for CC formation. However, very little is known of the molecular mechanisms behind the fusion of the two hemispheres. Netrin1 (NTN1) acts as an axon guidance molecule in the developing central nervous system and Ntn1 deficiency leads to the agenesis of CC in mouse. Here we have analyzed Ntn1 deficient mice to better understand the reasons behind the observed lack of CC. We show that Ntn1 deficiency leads to defects in neural, but not in glial guidepost structures that may contribute to the agenesis of CC. In addition, Nnt1 was expressed by the leptomeningeal cells bordering the two septal walls prior to fusion. Normally these cells are removed when the septal fusion occurs. At the same time, the Laminin containing basal lamina produced by the leptomeningeal cells is disrupted in the midline area to allow the cells to mix and the callosal axons to cross. In Ntn1 deficient embryos however, the leptomeninges and the basal lamina were not removed properly from the midline area and the septal fusion did not occur. Thus, NTN1 contributes to the formation of the CC by promoting the preceding removal of the midline leptomeningeal cells and interhemispheric fusion.

14-P019 Netrin1 in forebrain development

Netrin1 is a member of a family of secreted molecules implicated in axon guidance, neuronal migration and apoptosis during development of central nervous system (CNS). Netrin1 signals in CNS through a group of transmembrane receptors belonging to the DCC (deleted in colorectal cancer) and C. elegans Unc5-related families. Netrin1 mutant mice die usually at birth with several CNS defects, but heterozygous mice are normal. We made Netrin1 expression analysis in developing and postnatal mouse brain. Netrin1 was strongly expressed in the proliferative ventricular zone (VZ) in embryonic brain and in the developmentally related ependymal layer of the postnatal brain. Isolated Netrin1 positive neurospheres from VZ/ependymal layer expressed several neural stem cell markers, were able to self-renew and differentiated into neurons, astrocytes and oligodendrocytes. Netrin1 expressing cells were also apparent in the rostral migratory stream (RMS), along which newly born neuroblasts migrate from lateral ventricles to the olfactory bulb contributing a constant renewal of the peripheral olfactory system. Netrin1 –/– mice had a smaller olfactory bulbs and immunohistological analysis revealed that Netrin1 expressing cells form a heterogenous subset of precursor cells contributing both neural and glial populations in olfactory bulb.

E6/E7 oncogenes increase the proportion of self-renewing neural progenitor cells

Neurofibromatosis type 1 (NF1) predisposes to benign, incurable peripheral nerve tumors. We hypothesized that expansion of a progenitor population in Nf1 mutant peripheral nerves contributes to tumorigenesis. To test this hypothesis, we studied progenitor cells from Nf1 mutant mice. We identified an embryonic dorsal root ganglia (DRG) derived cell population, which is expandable as EGF-dependent self-renewing spheres; loss of Nf1 is associated with an increase in sphere formation. Spheres from both wild type and Nf1 mutant DRG contained cells capable of glial differentiation. Spheres from Nf1 mutants contained cells with increased propensity to form neurons in vitro when compared with wild type cells. An EGFR+;p75+ cell population was previously isolated from Nf1 DRG cultures; here we show that these cells (Nf1−/−TXF) can be propagated as spheres at greatly (20×) increased efficiency. Consistent with a progenitor phenotype, Nf1−/−TXF cells showed migratory characteristics of neural crest stem cells in a chicken xenograft model and expressed markers (as assessed using Affymetrix GeneChips) of neural crest and at least three crest derivatives: neurons, Schwann cells, and melanocytes. Knockout of Nf1 at the embryonic Schwann cell stage of development, but not earlier or later, generated this progenitor like population, implying that loss of Nf1 at a critical stage in development causes an expansion of progenitor cells. Nf1 but not wild type perinatal mouse nerves contained cells expressing EGFR and p75, suggesting abnormal persistence of the progenitor population. We also prospectively identified EGFR+;p75+ cells by FACS analysis of human neurofibromas, and have derived spheres from primary neurofibroma cells in the presence of EGF. These data support the hypothesis that an EGFR+ neural progenitor is amplified in Nf1 mutants and is relevant to peripheral nerve tumorigenesis in NF1.