Roles of CUP-5, the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> orthologue of human TRPML1, in lysosome and gut granule biogenesis

Background  

CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells.     

Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans

Mutations in MCOLN1, which encodes the protein mucolipin 1, result in the lysosomal storage disease mucolipidosis Type IV. Studies on human mucolipin 1 and on CUP-5, the Caenorhabditis elegans ortholog of mucolipin 1, have shown that these proteins are required for lysosome biogenesis/function. Loss of CUP-5 results in a defect in lysosomal degradation, leading to embryonic lethality. We have identified a mutation in the ABC transporter MRP-4 that rescues the degradation defect and the corresponding lethality, owing to the absence of CUP-5. MRP-4 localizes to endocytic compartments and its levels are elevated in the absence of CUP-5. These results indicate that the lysosomal degradation defect is exacerbated in some cells because of the accumulation of MRP-4 in lysosomes rather than the loss of CUP-5 per se. We also show that under some conditions, loss of MRP-4 rescues the embryonic lethality caused by the loss of the cathepsin L protease, indicating that the accumulation of ABC transporters may be a more general mechanism whereby an initial lysosomal dysfunction is more severely compromised.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

A role for Xrcc2 in the early stages of mouse development

Xrcc2 is one of a family of five Rad51-like genes with important roles in the repair of DNA damage by homologous recombination (HR) in mammals. We have shown previously that loss of Xrcc2 in mice results in severe but variable developmental defects and embryonic lethality, potentially linked to excessive apoptosis. To look at the causes of lethality, and possibly to allow Xrcc2-/- mice to survive to birth, we have produced double knockout mice deficient in either the p53 oncoprotein or Ataxia telangiectasia mutated (Atm). Overall we show that the excessive apoptosis observed in Xrcc2-/- embryos is p53-dependent, and that loss of p53 can restore growth capacity to Xrcc2-/- fibroblasts in culture, but that it cannot rescue the embryonic lethality. Additionally, although the Xrcc2-/- Trp53-/- embryos show a near-normal morphology they remain relatively small in size. Loss of Atm in an Xrcc2-/- embryo has little effect, suggesting that response to loss of HR capacity is not mediated through the Atm kinase in the early stages of mouse development. Further, as seen by reduced expression of the early developmental marker, Delta-like1, the normal developmental programme is perturbed in Xrcc2-/- embryonic tissues, particularly during neurogenesis and somitogenesis. Taken together our data suggest that the accumulation of spontaneous damage in HR-deficient embryos has severe consequences for the development and survival of mammals due to the unregulated loss of cells important to the developmental programme.     

Lysosomal trafficking functions of mucolipin-1 in murine macrophages

Background  

Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes.     

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.     

Systematic Screens for Proteins That Interact with the Mucolipidosis Type IV Protein TRPML1

Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.     

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency-Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.     

A functional overlap of plasminogen and MMPs regulates vascularization during placental development

Both plasminogen activators and matrix metalloproteinases (MMPs) have been implicated in a variety of developmental processes in the mouse during embryo implantation and placentation. We show here that pharmacological treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality. Implantation sites from plasminogen-deficient galardin-treated mice at 7.5 days post coitus (dpc) showed delay in both decidualization and invasion of maternal vessels into the decidua. At 8.5 dpc, half of the embryos were runted and still at the developmental stage of a 7.5 dpc embryo. Most embryos that escaped these initial defects eventually died, probably from defective vascularization and development of the labyrinth layer of the placenta, although a direct role on embryo development cannot be ruled out. These results demonstrate that the combination of MMPs and plasminogen is essential for the proper development of the placenta. Plasminogen deficiency alone and galardin treatment alone had much less effect and there was a pronounced synergism on both placental vascularization and embryonic lethality, indicating a functional overlap between plasminogen and MMPs.     

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.     

Lats2 is an essential mitotic regulator required for the coordination of cell division

Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.     

Generation of Axin1 conditional mutant mice

Axin1 is a critical negative regulator of the canonical Wnt-signaling pathway. It is a concentration-limiting factor in the β-catenin degradation complex. Axin1 null mutant mouse embryos died at embryonic day 9.5, precluding direct genetic analysis of the roles of Axin1 in many developmental and physiological processes using these mutant mice. In this study, we have generated mice carrying two directly repeated loxP sites flanking the exon 2 region of the Axin1 gene. We show that floxed-allele-carrying mice (Axin1( fx/fx) ) mice appear normal and fertile. Upon crossing the Axin1( fx/fx) mice to the CMV-Cre transgenic mice, the loxP-flanked exon 2 region that encodes the N-terminus and the conserved regulation of G-protein signaling domain was efficiently deleted by Cre-mediated excision in vivo. Moreover, we show that mouse embryos homozygous for the Cre/loxP-mediated deletion of exon 2 of the Axin1 gene display embryonic lethality and developmental defects similar to those reported for Axin1(-/-) mice. Thus, this Axin1(fx/fx) mouse model will be valuable for systematic tissue-specific dissection of the roles of Axin1 in embryonic and postnatal development and diseases.     

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.     

Rescue of keratin 18/19 doubly deficient mice using aggregation with tetraploid embryos

We have previously shown that the targeted deletions of both type I keratins (K) 18 and 19 cause lethality by embryonic day (e) 9.5 due to fragility and cytolysis of trophoblast giant cells. The development of the embryo proper appeared to be unaffected and its death was caused by nutrient deficiency. In order to address the function of keratins within the embryo proper, lethality due to extraembryonic tissue failure must be overcome. One approach to rescue doubly deficient embryos is by aggregating knockout embryos with tetraploid wild-type embryos. As a general tool, tetraploid aggregation can be used to rescue embryonic lethality caused by defects in extraembryonic tissues like the placenta, trophoblast or yolk sac. We rescued K18-/- K19-/- embryos until e11.5, using this approach, proving that the loss of the keratin cytoskeleton causes defects in the trophoblast giant cell layer, but has no effect on early development of the embryo proper.     

Inducible expression of V600EBraf using tyrosinase-driven Cre recombinase results in embryonic lethality

We recently demonstrated that expression of ^V600EBraf in mature mouse melanocytes induces melanoma. Here, we show that expression of ^V600EBraf using the tyrosinase promoter leads to an unexpected embryonic lethality, with the animals dying before, at, or shortly after birth. The mice suffer from a range of developmental defects in the skin, the brain, the eyes and the heart, tissues that are normally colonized by melanocytes. We show that the ^V600EBraf expressing cells are potential melanocytic precursors that are fully transformed, suggesting that ^V600EBraf stimulates proliferation and blocks differentiation of these cells. Our data suggests that the presence of these cells in the organs that are normally occupied by melanocytes leads to severe developmental disruption, resulting in catastrophic defects and leading to death of the individual.