Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells.

BAP31 is a transmembrane protein that associates with nascent membrane proteins in transit between endoplasmic reticulum (ER) and cis-Golgi. Its C-terminal dilysine (KKEE) motif, mediating return to the ER, is consistent with a role in early sorting of membrane proteins. An initiator caspase-binding site in the C-terminal domain of BAP31 is implicated in cytoplasmic membrane fragmentation events of apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization has not been determined. To gain further insight into its possible functions, we localized BAP31 in primate tissues using monoclonal antibodies. Immunoreactivity was prominent in T- and B-lymphocytes in blood and in thymus, in cerebellar Purkinje neuron bodies and dendrites, in gonadotrophs of the anterior pituitary, ovarian thecal and follicular cells, active but not quiescent thyroid epithelium, adrenal cortex more than medulla, and proximal more than distal renal tubules. Blood vessels and skeletal muscle were nonreactive. The anatomic distribution of BAP31 and the nature of proteins identified thus far as its cargo exiting the ER, suggest an interaction with proteins assembling in macromolecular complexes en route to selected sites of exocytotic and signaling activities. Apoptotic associations in mature tissues could be physiological (lymphocytes, endocrine cells) or pathological (Purkinje neurons, renal tubules).     

Synaptotagmin 8 is expressed both as a calcium-insensitive soluble and membrane protein in neurons, neuroendocrine and endocrine cells

Synaptotagmins (syt) form a large family of transmembrane proteins and some of its isoforms are known to regulate calcium-induced membrane fusion during vesicular traffic. In view of the reported implication of the isoform syt8 in exocytosis we investigated the expression, localisation and calcium-sensitivity of syt8 in secretory cells. An immunopurified antipeptide antibody was generated which is directed against a C-terminal sequence and devoid of crossreactivity towards syt1 to 12. Subcellular fractionation and immunocytochemistry revealed two forms of synaptotagmin 8 (50 and 40 kDa). Whereas the 40-kDa was present in the cytosol in brain, in PC12 and in clonal beta-cells, the 50-kDa form was localised in very typical clusters and partially colocalised with the SNARE protein Vti1a. Moreover, in primary hippocampal neurons syt8 was only found within the soma. Amplification of syt8 by RT-PCR indicated that the observed protein variants were not generated by alternative splicing of the 6th exon and are most likely linked to variations in the N-terminal region. In contrast to the established calcium sensor syt2, endogenous cytosolic syt8 and transiently expressed syt8-C2AB-eGFP did not translocate upon a raise in cytosolic calcium in living cells. Syt8 is therefore not a calcium sensor in exocytotic membrane fusion in endocrine cells.     

The LIM domain protein Lmo4 is highly expressed in proliferating mouse epithelial tissues.

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.     

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8–22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9 wga and reached its maximum expression levels between 14 and 18 wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.     

The protein product of the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and oligodendrocytes.

von Recklinghausen's neurofibromatosis (NF1) is a common inherited human disease. The events leading to patient symptoms from inheritance of a defective NF1 gene are unknown. Since knowledge of the distribution of the normal NF1 gene product should improve understanding of the pathogenesis of the disease, we raised antibodies against peptides coded by portions of the recently cloned human NF1 cDNA. These antibodies specifically recognize a 220 kd protein (neurofibromin) in both human and rat spinal cord. Neurofibromin is most abundant in the nervous system. Immunostaining of tissue sections indicates that neurons, oligodendrocytes, and nonmyelinating Schwann cells contain neurofibromin while astrocytes and myelinating Schwann cells do not. These results suggest a function for neurofibromin in the normal nervous system. Some NF1 disease manifestations, such as Schwann cell tumors and learning disabilities, may result from abnormalities in the cells that express neurofibromin.     

Krit1/cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult

Cavernous malformations are capillaro-venous lesions mostly located within the central nervous system (CCM/OMIM#116860) and occasionally within the skin and/or retina. They occur as a sporadic or hereditary condition. Three CCM loci have been mapped, and the sole gene identified so far, CCM1, has been shown to encode KRIT1, a protein of unknown function. In an attempt to get some insight on the relationship between KRIT1 mutations and CCM lesions, we investigated Krit1 mRNA expression during mouse development from E7.5 to E20.5 and in adult tissues, of both mouse and human origin. A ubiquitous Krit1 mRNA expression was detected from E7.5 up to E9.5. Then, it became progressively restricted from E10.5 to E12.5, to become detectable later essentially in the nervous system and various epithelia. Strong labelling was observed in neurons in the brain, cerebellum, spinal cord, retina and dorsal root ganglia. In epithelia, Krit1 mRNA expression was detected in differentiating epidermal, digestive, respiratory, uterine and urinary epithelia. A similar pattern of expression persisted in mouse and man adult nervous system and epithelia. Unexpectedly, in vascular tissues, expression of Krit1 was detected only in large blood vessels of the embryo.     

The Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.     

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.     

LIM kinase 2 is widely expressed in all tissues.

The LIM kinase family includes two proteins: LIMK1 and LIMK2. These proteins have identical genomic structure and overall amino acid identity of 50%. Both proteins regulate actin polymerization via phosphorylation and inactivation of the actin depolymerizing factors ADF/cofilin. Although the function of endogenous LIMK1 is well established, little is known about the function of the endogenous LIMK2 protein. To understand the specific role of endogenous LIMK2 protein, we examined its expression in embryonic and adult mice using a rat monoclonal antibody, which recognizes specifically the PDZ domain of LIMK2 but not that of LIMK1. Immunoblotting and immunoprecipitation analyses of mouse tissues and human and mouse cell lines revealed widespread expression of the 75-kDa LIMK2 protein. Immunofluorescence analysis demonstrated that the cellular localization of LIMK2 is different from that of LIMK1. LIMK2 protein is found in the cytoplasm localized to punctae and is not enriched within focal adhesions like LIMK1. Immunohistochemical studies revealed that LIMK2 is widely expressed in embryonic and adult mouse tissues and that its expression pattern is similar to that of LIMK1 except in the testes. We have also demonstrated that endogenous LIMK1 and LIMK2 form heterodimers, and that LIMK2 does not always interact with the same proteins as LIMK1.     

DLK1 is a somato-dendritic protein expressed in hypothalamic arginine-vasopressin and oxytocin neurons

Delta-Like 1 Homolog, Dlk1, is a paternally imprinted gene encoding a transmembrane protein involved in the differentiation of several cell types. After birth, Dlk1 expression decreases substantially in all tissues except endocrine glands. Dlk1 deletion in mice results in pre-natal and post-natal growth deficiency, mild obesity, facial abnormalities, and abnormal skeletal development, suggesting involvement of Dlk1 in perinatal survival, normal growth and homeostasis of fat deposition. A neuroendocrine function has also been suggested for DLK1 but never characterised. To evaluate the neuroendocrine function of DLK1, we first characterised Dlk1 expression in mouse hypothalamus and then studied post-natal variations of the hypothalamic expression. Western Blot analysis of adult mouse hypothalamus protein extracts showed that Dlk1 was expressed almost exclusively as a soluble protein produced by cleavage of the extracellular domain. Immunohistochemistry showed neuronal DLK1 expression in the suprachiasmatic (SCN), supraoptic (SON), paraventricular (PVN), arcuate (ARC), dorsomedial (DMN) and lateral hypothalamic (LH) nuclei. DLK1 was expressed in the dendrites and perikarya of arginine-vasopressin neurons in PVN, SCN and SON and in oxytocin neurons in PVN and SON. These findings suggest a role for DLK1 in the post-natal development of hypothalamic functions, most notably those regulated by the arginine-vasopressin and oxytocin systems.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.