Ghrelin is dispensable for embryonic pancreatic islet development and differentiation

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.     

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.     

Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling

Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1-4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4-deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling.     

Trb2, a mouse homolog of tribbles, is dispensable for kidney and mouse development

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.     

Gene expression and cell growth are modified by silencing SUMO2 and SUMO3 expression

Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. Here we report that blocking SUMO2 and SUMO3 conjugation by silencing their expression markedly modifies gene expression. A microRNA-based RNAi system was used to specifically silence SUMO2 and SUMO3 expression simultaneously and stably transfected neuroblastoma B35 cells expressing dual SUMO2/3 microRNA were created. In cells stably expressing SUMO2/3 microRNA, mRNA levels of 105 and 58 known genes were significantly up- and down-regulated, respectively. About 20% of differentially regulated genes were associated with pathways involved in cell growth and differentiation. Cell division was significantly suppressed in SUMO2/3 miRNA expressing cells. Elucidating what effect the silencing of SUMO2/3 expression has on gene expression will help to identify the impact of SUMO2/3 conjugation on the various cellular pathways.     

RBP-Jkappa-dependent notch signaling is dispensable for mouse early embryonic development

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.     

Mint3 is dispensable for pancreatic and kidney functions in mice

Munc-18 interacting protein 3 (Mint3) is an activator of hypoxia-inducible factor-1 in cancer cells, macrophages, and cancer-associated fibroblasts under pathological conditions. However, exactly which cells highly express Mint3 in vivo and whether Mint3 depletion affects their physiological functions remain unclear. Here, we surveyed mouse tissues for specific expression of Mint3 by comparing Mint3 expression in wild-type and Mint3-knockout mice. Interestingly, immunohistochemical analyses revealed that Mint3 was highly expressed in islet cells of the pancreas, distal tubular epithelia of the kidney, choroid plexus ependymal cells of the cerebrum, medullary cells of the adrenal gland, and epithelial cells of the seminal gland. We also studied whether Mint3 depletion affects the physiological functions of the islets and kidneys. Mint3-knockout mice did not show any abnormalities in glucose-tolerance and urine-biochemical tests, indicating that Mint3 depletion was compensated for in these organs. Thus, loss of Mint3 might be compensated in the islets and kidneys under physiological conditions in mice.     

Golga5 is dispensable for mouse embryonic development and postnatal survival

Golgins are a family of coiled‐coil proteins located at the cytoplasmic surface of the Golgi apparatus and have been implicated in maintaining Golgi structural integrity through acting as tethering factors for retrograde vesicle transport. Whereas knockdown of several individual golgins in cultured cells caused Golgi fragmentation and disruption of vesicle trafficking, analysis of mutant mouse models lacking individual golgins have discovered tissue‐specific developmental functions. Recently, homozygous loss of function of GOLGA2, of which previous in vitro studies suggested an essential role in maintenance of Golgi structure and in mitosis, has been associated with a neuromuscular disorder in human patients, which highlights the need for understanding the developmental roles of the golgins in vivo. We report here generation of Golga5‐deficient mice using CRISPR/Cas9‐mediated genome editing. Although knockdown studies in cultured cells have implicated Golga5 in maintenance of Golgi organization, we show that Golga5 is not required for mouse embryonic development, postnatal survival, or fertility. Moreover, whereas Golga5 is structurally closely related to Golgb1, we show that inactivation of Golga5 does not enhance the severity of developmental defects in Golgb1‐deficient mice. The Golga5‐deficient mice enable further investigation of the roles and functional specificity of golgins in development and diseases.     

CCN2 is necessary for the function of mouse embryonic fibroblasts

CCN2 is expressed by mesenchymal cells undergoing active tissue remodeling, and is characteristically overexpressed in connective tissue pathologies such as fibrosis and cancer. However, the physiological roles and mechanism of action of CCN2 are largely unknown. Here, we probe the contribution of CCN2 to the biology of mouse embryonic fibroblasts (MEFs) using genome-wide mRNA expression profiling, proteomic and functional bioassay analyses. We show that ccn2-/- mouse embryonic fibroblasts (MEFs) have significantly reduced the expression of pro-adhesive, pro-inflammatory and pro-angiogenic genes such as interleukin-6 (IL-6), ceruloplasmin, thrombospondin-1, lipocalin-2 and syndecan 4. Anti-syndecan 4 antibody reduced ERK phosphorylation in ccn2+/+ MEFs. In ccn2+/+ MEFs, the MEK inhibitor U0126 and dominant negative ras reduced expression of IL-6 and lipocalin-2. Overexpressing syndecan 4 in ccn2-/- MEFs restored IL-6 and lipocalin-2 mRNA expression. Syndecan 4 has been shown to mediate cell migration. We found that ccn2+/+ MEFs migrated significantly faster than ccn2-/- MEFs; anti-syndecan 4 antibody and U0126 reduced the migration of ccn2+/+ MEFs to that of ccn2-/- MEFs. These results collectively support the notion that syndecan 4 acts downstream of CCN2 in MEFs, and that reduced syndecan 4 expression contributes to at least part of the ccn2-/- phenotype. Further, these results suggest that CCN2 is required for MEFs to contribute to aspects of tissue remodeling. Consistent with this notion, whereas ccn2+/+ MEFs displayed actin stress fibers and focal adhesions at the cell periphery consistent with a migratory phenotype, ccn2-/- MEFs displayed reduced focal adhesions and actin stress fibers, and a reduced ability to transduce forces across a collagen gel matrix. Collectively, these results suggest that CCN2 supplies essential, non-redundant functions required for fibroblasts to properly participate in features of embryogenesis, and further suggest that CCN2 may play essential roles in adult wound healing, tissue repair and fibrogenesis.     

A functional Jak2 tyrosine kinase domain is essential for mouse development

Jak2 is a member of the Janus family of tyrosine kinases and is involved in cytokine signaling. As a part of a study to determine biological functions of Jak2, we used molecular modeling to identify W1038 as a residue that is critical for tyrosine kinase function. Mutation of W1038, in tandem with E1046, generates a dominant-negative form of the Jak2 protein. Mice that were engineered to express two copies of this dominant-negative Jak2 protein died in utero. Additionally, heterozygous mice expressing Jak2 with kinase activity that is moderately reduced when compared to wild-type activity appear phenotypically normal. Collectively, these data suggest that Jak2 kinase activity is essential for normal mammalian development.     

Twinfilin-2a is dispensable for mouse development

Twinfilins are evolutionarily conserved regulators of cytoskeletal dynamics. They inhibit actin polymerization by binding both actin monomers and filament barbed ends. Inactivation of the single twinfilin gene from budding yeast and fruit fly results in defects in endocytosis, cell migration, and organization of the cortical actin filament structures. Mammals express three twinfilin isoforms, of which twinfilin-1 and twinfilin-2a display largely overlapping expression patterns in non-muscle tissues of developing and adult mice. The expression of twinfilin-2b, which is generated through alternative promoter usage of the twinfilin-2 gene, is restricted to heart and skeletal muscles. However, the physiological functions of mammalian twinfilins have not been reported. As a first step towards understanding the function of twinfilin in vertebrates, we generated twinfilin-2a deficient mice by deleting exon 1 of the twinfilin-2 gene. Twinfilin-2a knockout mice developed normally to adulthood, were fertile, and did not display obvious morphological or behavioural abnormalities. Tissue anatomy and morphology in twinfilin-2a deficient mice was similar to that of wild-type littermates. These data suggest that twinfilin-2a plays a redundant role in cytoskeletal dynamics with the biochemically similar twinfilin-1, which is typically co-expressed in same tissues with twinfilin-2a.     

Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.     

The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451 isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro. Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo, ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line.     

Arp3 is required during preimplantation development of the mouse embryo

The role of Arp3 in mouse development was investigated utilizing a gene trap mutation in the Arp3 gene. Heterozygous Arp3(WT/GT) mice are normal, however, homozygous Arp3(GT/GT) embryos die at blastocyst stage. Earlier embryonic stages appear unaffected by the mutation, probably due to maternal Arp3 protein. Mutant blastocysts isolated at E3.5 fail to continue development in vitro, lack outgrowth of trophoblast-like cells in culture and express reduced levels of the trophoblast marker Cdx2, while markers for inner cell mass continue to be present. The recessive embryonic lethal phenotype indicates that Arp3 plays a vital role for early mouse development, possibly when trophoblast cells become critical for implantation.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.