Background: Hyaluronan is a crucial glycosaminoglycan of the vertebrate embryonic extracellular matrix able to influence cell behaviour, both by assembling the pericellular matrices and by activating signal transducing receptors such as CD44. Results: We showed that the hyaluronan synthases, Has1 and Has2, and CD44 display a dynamic expression pattern during cranial neural crest cells (NCC) development. By knocking down Has1 and Has2 gene functions, we revealed that hyaluronan synthesized by Has1 and Has2 is necessary for the proper development of the visceral skeleton. Conclusions: The data suggest that hyaluronan helps to maintain the active migratory behaviour of cranial NCC, and that its presence around pre-chondrogenic NCC is crucial for their survival. CD44 knock down also suggests that the role of hyaluronan in cranial NCC migration could be mediated, at least in part, by the activation of CD44. These findings contribute to the unveiling of the functional relation between NCC and their extracellular environment during craniofacial development. Developmental Dynamics 241:294-302, 2012. V C 2011 Wiley Periodicals, Inc.Key words: hyaluronan; neural crest cell; craniofacial development; Has1; Has2; CD44; extracellular matrix; Xenopus Key findings:Cranial NCC migrate throughout a hyaluronan rich extracellular matrix Hyaluronan synthases 1 and 2 activity is necessary to maintain the migratory behaviour of cranial NCC After NCC migration, the presence of Hyaluronan in the extracellular matrix is necessary for the survival of pre-chondrogenic cranial NCC. CD44, a Hyaluronan receptor, is required for cranial NCC migration but not for their survival. In Xenopus, the craniofacial skeleton formation is deeply influenced by the composition of the extracellular matrix during cranial NCC migration and differentiation.

No abstract

BACE1 (β-site amyloidogenic cleavage of precursor protein-cleaving enzyme 1) is a β-secretase protein that plays a central role in the production of the β-amyloid peptide in the brain and is thought to be involved in the Alzheimer's pathogenesis. In type 2 diabetes, amyloid deposition within the pancreatic islets is a pathophysiological hallmark, making crucial the study in the pancreas of BACE1 and its homologous BACE2 to understand the pathological mechanisms of this disease. The objectives of the present study were to characterize the localization of BACE proteins in human pancreas and determine their function. High levels of BACE enzymatic activity were detected in human pancreas. In normal human pancreas, BACE1 was observed in endocrine as well as in exocrine pancreas, whereas BACE2 expression was restricted to β-cells. Intracellular analysis using immunofluorescence showed colocalization of BACE1 with insulin and BACE2 with clathrin-coated vesicles of the plasma membrane in MIN6 cells. When BACE1 and -2 were pharmacologically inhibited, BACE1 localization was not altered, whereas BACE2 content in clathrin-coated vesicles was increased. Insulin internalization rate was reduced, insulin receptor β-subunit (IRβ) expression was decreased at the plasma membrane and increased in the Golgi apparatus, and a significant reduction in insulin gene expression was detected. Similar results were obtained after specific BACE2 silencing in MIN6 cells. All these data point to a role for BACE2 in the IRβ trafficking and insulin signaling. In conclusion, BACE2 is hereby presented as an important enzyme in β-cell function.

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.KEY WORDS: Has2, CD44, Hyaluronan, Somitogenesis, Myogenesis, Cell migration, Neural crest, ECM, Xenopus Development 133, 631-640 doi:10.1242/dev.02225 1 Laboratori di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e Biochimica, Università di Pisa, Via Carducci 13, Ghezzano, Pisa (PI) 56010, Italy. ). These findings have prompted us to investigate the possible role of XHas2 in somitogenesis, myogenic differentiation and trunk NCC migration, which, thus far, have not been possible to elucidate in Has2-null mice. To this end, we have also cloned the Xenopus orthologue of CD44, known to be the principal cell surface receptor of HA (Wheatley et al., 1993;Ponta et al., 2003) for which the precise role during vertebrate development still remains to be elucidated. MATERIALS AND METHODS Embryo manipulations and whole mount in situ hybridizationXenopus laevis embryos obtained by hormone induced laying, were in vitro fertilized, dejelled, washed and incubated in 0.1ϫMMR. Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967), fixed and stained for ␤-galactosidase (200-300 pg injected per embryo) as previously described (Sive et al., 2000) using alternatively a red or light-blue substrate for ...

BACE2 (b-site APP-cleaving enzyme 2) is a protease localized in the brain, where it appears to play a role in the development of Alzheimer disease (AD). It is also found in the pancreas, although its biologic function is not fully known. Amyloidogenic diseases, including AD and type 2 diabetes mellitus (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. Islet amyloid polypeptide (IAPP) deposits are a key pathogenic feature of T2D. Within this context, we found by global gene expression profiling that BACE2 was up-regulated in the rat pancreatic b-cell line INS1E stably transfected with human IAPP gene (hIAPP-INS1E). Glucose-stimulated insulin secretion (GSIS) in hIAPP-INS1E cells was 30% lower than in INS1E cells. Additionally, INS1E cells transfected with a transient overexpression of BACE2 showed a 60% decrease in proliferation, a 3-fold increase in reactive oxygen species production, and a 25% reduction in GSIS compared to control cells. Remarkably, silencing of endogenous BACE2 in hIAPP-INS1E cells resulted in a significant improvement in GSIS (3-fold increase vs. untransfected cells), revealing the significant role of BACE2 expression in b-cell function. Thus, BACE2 inhibition may be useful to recover insulin secretion in hIAPP-INS1E defective cells and may be proposed as a therapeutic target for T2D.-Alcarraz-Vizán, G., Casini, P., Cadavez, L., Visa, M., Montane, J., Servitja, J.-M., Novials, A. Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic b-cells. FASEB J. 29, 95-104 (2015). www.fasebj.org Key Words: amylin • BACE activity • secretase • type 2 diabetes INSULIN SECRETION DEFICIENCY plays a critical role in the onset of type 2 diabetes mellitus (T2D). It is well known that the progressive loss of b-cell mass and function are key features of the manifestation of the underlying disease (1). One of the main factors involved in the process of pancreatic b-cell deterioration is the accumulation of pancreatic amyloid deposits (2), which are considered a hallmark of T2D (3, 4). Because islet amyloid deposits colocalize with areas of b-cell degeneration, the process of amyloidosis has been strongly associated with a progressive loss of pancreatic b-cell mass and therefore with the pathology of T2D (5). Islet amyloid deposits are mainly composed by islet amyloid polypeptide (IAPP), a small peptide physiologically produced by pancreatic b-cells and cosecreted with insulin in response to different secretagogue stimuli (6, 7).The process of ordered aggregation into amyloid fibrils is a common event in many cell-degenerative diseases, such as Alzheimer disease (AD) and other local or systemic amyloidoses (8-10). A number of studies show that the toxicity of the amyloidogenic peptides lies in the oligomeric intermediates rather than in the mature fibrils. Thus, the conversion of a normally soluble protein into amyloid structures can allow for gained toxic function, providing a common mechanism by which amyloid contr...

The chondroitin sulfate proteoglycan (PG) PG-M/versican is known to be a primary component of the vertebrate embryonic extracellular matrix and, in the mouse, functional abrogation of the versican gene leads to severe cardiovascular malformations and embryonic lethality. In order to provide a means for approaching the study of the role of versican during embryogenesis, we have cloned the Xenopus versican cDNA and we have performed in situ hybridization on embryos at different stages of development. We showed maternal Xversican transcription, as well as a previously undocumented early expression of the PG during gastrulation and neurulation. At later stages of development, spatial transcription of Xversican correlates with the patterns of migrating neural crest cells (NCC) and it is expressed in embryonic regions representing the final sites of arrest of NCC. Xversican mRNA was also detected in a subpopulation of trunk NCC migrating into the fin, in tissues flanking the trunk NCC ventral migratory pathway and in post-migratory cranial skeletogenic NCC. Further embryonic sites expressing Xversican were the pronephros, pronephric ducts, heart anlage and branchial pouches. These findings constitute the basis for future studies aimed at clarifying unresolved aspects of versican function during embryogenesis. KEY WORDS: chondroitin sulfate proteoglycan, versican, embryo, neural crest, Xenopus laevisVersican is a chondroitin sulfate proteoglycan (PG) belonging to the hyalectan subgroup of PGs including aggrecan, neurocan, brevican and CD44 and having in common an N-terminal G1 domain embodying the binding site for hyaluronan. Aggrecan, neurocan and brevican show a rather restricted tissue distribution (e.g. aggrecan is most prominent in cartilage, whereas neurocan and brevican are predominantly expressed in the central nervous system), whereas versican is present in a variety of tissues and organs, implying that it may exert a diverse biological function (Iozzo, 2005). In addition to the hyaluronan-binding domain, hyalectans show common structural features such as a set of epidermal growth factor (EGF), lectin and complement regulatory protein (CRP) in the carboxy-terminal portion (G3 domain) of the molecule (Zimmermann and Ruoslahti, 1989;Shinomura et al., 1993). The glycosaminoglycan (GAG) attachment regions are located in the middle portion of the protein and are different in the Int. J. Dev. Biol. 52: 993-998 (2008) four extracellular matrix (ECM) PGs of the hyalectan subgroup. In versican, two different GAG attachment domains have thus far been identified in higher vertebrates and denoted GAGα and GAGβ. Both domains (exons) are present within the parental V0 isoform while they are differentially spliced out in isoforms V1 and V2 (Zimmermann and Ruoslahti, 1989;Shinomura et al., 1993).The complex structure of versican provides the basis for its multiple and sometime contrasting effects on cellular functions, such as cell adhesion, cell migration, cell proliferation and apoptosis, as well as on morphoge...

The thymus of 18-day-old normal-chick embryos, partially decerebrated chick embryos, and partially decerebrated embryos bearing hypophyseal allografts were analysed by light and transmission electron microscopy. The hypophyseal influence on the cytological differentiation of epithelial components has been studied. The thymus of partially decerebrated embryos showed a delayed differentiation of some types of epithelial cells and a marked decrease in number of lymphoid cells. Partially decerebrated embryos with hypophyseal implants showed a consistent recovery in the degree of differentiation of epithelial components. These findings indicate the influence of the hypophysis in establishing a correct environment for stromal cell differentiation.

No abstract