Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.     

J1/tenascin is a repulsive substrate for central nervous system neurons.

J1/tenascin mediates neuron-astrocyte interactions in vitro and is transiently expressed during CNS development in vivo. It is detectable in discrete zones, for example on astrocytes delineating "barrels" in the rodent somatosensory cortex. To investigate the effects of J1/tenascin on neural cell behavior in vitro, we have generated two monoclonal antibodies specific for protein epitopes on J1/tenascin and used them for immunoaffinity isolation of the molecule from postnatal mouse brain. The purified ECM molecule alone did not support attachment and growth of cerebral astrocytes or E14 mesencephalic, E18 hippocampal, and P6 cerebellar neurons. When various ECM constituents were adsorbed to polyornithine-conditioned glass, a favorable substrate for neural cells, the neurons avoided J1/tenascin-, but not laminin- or fibronectin-coated surfaces, while they grew on J1/tenascin-free, polyornithine-containing areas of the coverslip. In contrast, astrocytes formed uniform monolayers on all of these substrates. We conclude that J1/tenascin could serve to define repulsive territories for CNS neurons from different stages of neural development.     

SMAD4 is essential for generating subtypes of neurons during cerebellar development

Cerebellum development involves the coordinated production of multiple neuronal cell types. The cerebellar primordium contains two germinative zones, the rhombic lip (RL) and the ventricular zone (VZ), which generate different types of glutamatergic and GABAergic neurons, respectively. What regulates the specification and production of glutamatergic and GABAergic neurons as well as the subtypes for each of these two broad classes remains largely unknown. Here we demonstrate with conditional genetic approaches in mice that SMAD4, a major mediator of BMP and TGFβ signaling, is required early in cerebellar development for maintaining the RL and generating subsets of RL-derived glutamatergic neurons, namely neurons of the deep cerebellar nuclei, unipolar brush cells, and the late cohort of granule cell precursors (GCPs). The early cohort of GCPs, despite being deficient for SMAD4, is still generated. In addition, the numbers of GABAergic neurons are reduced in the mutant and the distribution of Purkinje cells becomes abnormal. These studies demonstrate a temporally and spatially restricted requirement for SMAD4 in generating subtypes of cerebellar neurons.     

Changes in neurotrophin responsiveness during the development of cerebellar granule neurons.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.     

Slit antagonizes netrin-1 attractive effects during the migration of inferior olivary neurons

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. We have looked for a possible involvement of slit-1/2 during ION migration. We report that: (1) slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development; (2) robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate; (3) using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei; (4) slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but (5) when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. Our results indicate that slit silences the attractive effects of netrin-1 and could participate in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate.     

SRPS associated protein WDR60 regulates the multipolar-to-bipolar transition of migrating neurons during cortical development

Mutations of WD40 repeat domain 60 (WDR60) have been identified in short-rib polydactyly syndromes (SRPS I–V), a group of lethal congenital disorders characterized by short ribs, polydactyly, and a range of extraskeletal phenotypes. However, the underlying mechanism is still unclear. Here, we report that WDR60 is essential for embryonic development and plays a critical role in the multipolar-bipolar transition and migration of newborn neurons during brain development. Mechanically, we found that WDR60 was located at the microtubule-organizing center to control microtubule organization and possibly, the trafficking of cellular components. Importantly, the migration defect caused by Wdr60 knockdown could be rescued by the stable form of α-Tubulin, α-Tubulin^K40Q (an acetylation-mimicking mutant). These findings identified a non-cilia function of WDR60 and provided insight into its biological function, as well as the pathogenesis of WDR60 deficiency associated with SRPS.Subject terms: Cell migration, Embryogenesis     

The novel ankyrin-repeat containing kinase ARCK-1 acts as a suppressor of the Spalten signaling pathway during Dictyostelium development

Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasB^GTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.     

Survivin acts as anti-apoptotic factor during the development of bovine pre-implantation embryos

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos.     

Expression of fascin-1, an actin-bundling protein,in migrating hepatoblasts during rat liver development

Fascin-1 is an actin-bundling protein localized at the core actin bundles within microvillar projections and filopodial extensions in migrating cells. It is expressed at a low level in normal epithelial cells, but at a high level in tumor cells, indicating its importance in the invasion and motility of tumor cells. In addition, fascin-1 is expressed in human and murine embryos, occurring at high levels especially in developing nervous tissues. In this study, we have investigated the expression patterns of fascin-1 immunohistochemically during the early stages of rat hepatogenesis. A high expression of fascin-1 was detected in the liver bud and hepatoblasts at embryonic day (ED) 10.5, ED11.5, and ED12.5. Expression fell by ED13.5 and was not detectable at ED14.5. These observations demonstrate that the expression of fascin-1 is correlated with the migration activity of hepatoblasts during the early stages of liver development in rats. This study was supported in part by grant-in-aid for scientific research from the Japan Society for the Promotion of Science and by a grant from the Smoking Research Foundation, Japan.     

Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development.

Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1

ADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression.     

CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Eya 1 acts as a critical regulator for specifying the metanephric mesenchyme

Although it is well established that the Gdnf-Ret signal transduction pathway initiates metanephric induction, no single regulator has yet been identified to specify the metanephric mesenchyme or blastema within the intermediate mesoderm, the earliest step of metanephric kidney development and the molecular mechanisms controlling Gdnf expression are essentially unknown. Previous studies have shown that a loss of Eya 1 function leads to renal agenesis that is a likely result of failure of metanephric induction. The studies presented here demonstrate that Eya 1 specifies the metanephric blastema within the intermediate mesoderm at the caudal end of the nephrogenic cord. In contrast to its specific roles in metanephric development, Eya 1 appears dispensable for the formation of nephric duct and mesonephric tubules. Using a combination of null and hypomorphic Eya 1 mutants, we now demonstrated that approximately 20% of normal Eya 1 protein level is sufficient for establishing the metanephric blastema and inducing the ureteric bud formation but not for its normal branching. Using Eya 1, Gdnf, Six 1 and Pax 2 mutant mice, we show that Eya 1 probably functions at the top of the genetic hierarchy controlling kidney organogenesis and it acts in combination with Six 1 and Pax 2 to regulate Gdnf expression during UB outgrowth and branching. These findings uncover an essential function for Eya 1 as a critical determination factor in acquiring metanephric fate within the intermediate mesoderm and as a key regulator of Gdnf expression during ureteric induction and branching morphogenesis.