The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1vil−/−) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1vil−/− enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1vil−/− epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylomicrons and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.
Loss of kindlin-3 impairs activation of β1, β2, and β3 integrin classes, resulting in osteopetrotic defects in osteoclast adhesion and spreading.
The blood cell–specific kindlin-3 protein is required to activate leukocyte and platelet integrins. In line with this function, mutations in the KINDLIN-3 gene in man cause immunodeficiency and severe bleeding. Some patients also suffer from osteopetrosis, but the underlying mechanism leading to abnormal bone turnover is unknown. Here we show that kindlin-3–deficient mice develop severe osteopetrosis because of profound adhesion and spreading defects in bone-resorbing osteoclasts. Mechanistically, loss of kindlin-3 impairs the activation of β1, β2, and β3 integrin classes expressed on osteoclasts, which in turn abrogates the formation of podosomes and sealing zones required for bone resorption. In agreement with these findings, genetic ablation of all integrin classes abolishes the development of podosomes, mimicking kindlin-3 deficiency. Although loss of single integrin classes gives rise to podosomes, their resorptive activity is impaired. These findings show that osteoclasts require their entire integrin repertoire to be regulated by kindlin-3 to orchestrate bone homeostasis.
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.
Ap-2 transcription factors comprise a family of 5 closely related sequence-specific DNA binding proteins that play pivotal and non-redundant roles in embryonic organogenesis. To investigate the function of Ap-2δ, wδe analyzed its expression during embryogenesis and generated Ap-2δ-deficient mice. In line with the specific expression pattern of Ap-2δ in the mesencephalic tectum and the dorsal midbrain, Ap-2δ-deficient mice failed to maintain the colliculus inferior, a derivative of the dorsal midbrain, as a consequence of increased apoptotic cell death. To identify specific Ap-2δ target genes in cells of the developing dorsal midbrain, we performed whole genome analysis of cDNA expression levels. This approach identified a set of 12 putative target genes being expressed in the developing midbrain, including the transcription factors Pitx2, Mef2c, Bhlhb4 and Pou4f3. Using chromatin immunoprecipitation (CHIP) we showed that some of these genes are direct targets of Ap-2δ. Consistently, we demonstrate that Ap-2δ occupies and activates the Pou4f3 and Bhlhb4 promoters. In addition, known Pou4f3 target genes were downregulated in the posterior midbrain of Ap-2δ-deficient mice. Despite the absence of a central part of the auditory pathway, the presence of neuronal responses to sounds in the neocortex of Ap-2δ-deficient mice indicates that auditory information from the brainstem still reaches the neocortex. In summary, our data define Ap-2δ as an important transcription factor, specifying gene expression patterns required for the development of the posterior midbrain.
Patent ductus arteriosus (PDA) is one of the most common forms of congenital heart disease. Mutations in transcription factor TFAP2B cause Char syndrome, a human disorder characterized by PDA, facial dysmorphysm and hand anomalies. Animal research data are needed to understand the mechanisms. The aim of our study was to elucidate the pathogenesis of Char syndrome at the molecular level.
Gene expression of Tfap2b during mouse development was studied, and newborns of Tfap2b-deficient mice were examined to identify phenotypes. Gel shift assays had been carried out to search for Tfap2 downstream genes. Promoters of candidate genes were cloned into a reporter construct and used to demonstrate their regulation by Tfap2b in cell transfection. In situ hybridizations showed that the murine transcription factor Tfap2b was expressed during the entire development of mouse ductus arteriosus. Histological examination of ductus arteriosus from Tfap2b knockout mice 6 hours after birth revealed that they were not closed. Consequently, the lungs of Tfap2b−/− mice demonstrated progressive congestion of the pulmonary capillaries, which was postulated to result secondarily from PDA. In addition, Tfap2b was expressed in the limb buds, particularly in the posterior limb field during development. Lack of Tfap2b resulted in bilateral postaxial accessory digits. Further study indicated that expressions of bone morphogenetic protein (Bmp) genes, which are reported to be involved in the limb patterning and ductal development, were altered in limb buds of Tfap2b-deficient embryos, due to direct control of Bmp2 and Bmp4 promoter activity by Tfap2b.
Tfap2b plays important roles in the development of mouse ductus arteriosus and limb patterning. Loss of Tfap2b results in altered Bmp expression that may cause the heart-limb defects observed in Tfap2b mouse mutants and Char syndrome patients. The Tfap2b knockout mouse may add to the very limited available animal models of PDA.
In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggesting that there are more factors working in the trophoblast lineage. Here, we report that the transcription factor Tcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that the molecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating that they are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish the extraembryonic trophoblast maintenance program in murine embryos.
ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1ad−/−) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1ad−/− mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1ad−/− adipocytes, suggesting that lipid droplet growth is defective in Arfrp1ad−/− mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1ad−/− mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.
The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models.
Here, we investigate vinculin-ΔEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-ΔEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-ΔIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-ΔIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-ΔEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-ΔEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-ΔEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites.
Vinculin-ΔEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.
Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B subunits. In heterologous expression systems, B subunits alone cannot form functional CNG channels, but they confer a number of channel properties when coexpressed with A subunits. To investigate the importance of the CNGB subunits in vivo, we deleted the CNGB1 gene in mice. In the absence of CNGB1, only trace amounts of the CNGA1 subunit were found on the rod outer segment. As a consequence, the vast majority of isolated rod photoreceptors in mice lacking CNGB1 (CNGB1−/−) failed to respond to light. In electroretinograms (ERGs), CNGB1−/− mice showed no rod-mediated responses. The rods also showed a slow-progressing degeneration caused by apoptotic death and concurred by retinal gliosis. Cones were primarily unaffected and showed normal ERG responses up to 6 months, but they started to degenerate in later stages. At the age of ~1 year, CNGB1−/− animals were devoid of both rods and cones. Our results show that CNGB1 is a crucial determinant of native CNG channel targeting. As a result of the lack of rod CNG channels, CNGB1−/− mice develop a retinal degeneration that resembles human retinitis pigmentosa.
cyclic nucleotide-gated channel; CNGB1; channel trafficking; rod photoreceptor; retinitis pigmentosa; apoptosis
Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.
Kindler Syndrome (KS), characterized by transient skin blistering followed by abnormal pigmentation, skin atrophy, and skin cancer, is caused by mutations in the FERMT1 gene. Although a few KS patients have been reported to also develop ulcerative colitis (UC), a causal link to the FERMT1 gene mutation is unknown. The FERMT1 gene product belongs to a family of focal adhesion proteins (Kindlin-1, -2, -3) that bind several β integrin cytoplasmic domains. Here, we show that deleting Kindlin-1 in mice gives rise to skin atrophy and an intestinal epithelial dysfunction with similarities to human UC. This intestinal dysfunction results in perinatal lethality and is triggered by defective intestinal epithelial cell integrin activation, leading to detachment of this barrier followed by a destructive inflammatory response.
Mutations in FERMT1, coding for the Kindlin-1 protein, cause Kindler Syndrome in humans, characterized by skin blistering, atrophy, and cancer. Recent reports showed that some Kindler Syndrome patients additionally suffer from ulcerative colitis. However, it is unknown whether this is caused by loss of Kindlin-1 or by unrelated abnormalities such as infections or additional mutations. We ablated the Fermt1 gene in mice to directly analyze the pathological consequences and the molecular mode of action of Kindlin-1. Kindlin-1–deficient mice develop a severe epidermal atrophy, but lack blisters. All mutant mice die shortly after birth from a dramatic, shear force-induced detachment of intestinal epithelial cells followed by a profound inflammation and organ destruction. The intestinal phenotype is very similar to, although more severe than, the one observed in Kindler Syndrome patients. In vitro studies revealed that impaired integrin activation, and thus impaired adhesion, to the extracellular matrix of the intestinal wall causes intestinal epithelial cell detachment. Therefore, we demonstrate that intestinal epithelial cells require adhesive function of integrins to resist the shear force applied by the stool. Furthermore, we provide evidence that the colitis associated with Kindler Syndrome is caused by a dysfunction of Kindlin-1 rather than by a Kindlin-1–independent event.
GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8+/+, Slc2a8+/−, and Slc2a8−/− mice, respectively) resulting in a deviation (p = 0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.
GLUT8; glucose transport; sperm motility; mitochondrial membrane potential
The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype.
A ~17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue.
We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.
Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin β subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.
The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells. However, its function within the nervous system has remained unknown. Here, we show that both the fly and mouse SWS proteins can rescue the defects that arise in sws mutant flies, whereas a point mutation in the proposed active site cannot restore SWS function. Overexpression of catalytically active SWS caused formation of abnormal intracellular membraneous structures and cell death. Cell-specific expression revealed that not only neurons but also glia depend autonomously on SWS. In wild-type flies, endogenous SWS was detected by immmunohistochemistry in the endoplasmic reticulum (the primary site of PtdCho processing) of neurons and in some glia. sws mutant flies lacked NTE-like esterase activity and had increased levels of PtdCho. Conversely, overexpression of SWS resulted in increased esterase activity and reduced PtdCho. We conclude that SWS is essential for membrane lipid homeostasis and cell survival in both neurons and glia of the adult Drosophila brain and that NTE may play an analogous role in vertebrates.
Drosophila; NTE; neurodegeneration; organophosphate-induced delayed neuropathy; phospholipids; ER
Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.
Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrPSc, the infectious and protease-resistant form of the cellular prion protein (PrPC). We generated lentivectors expressing PrPC-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrPSc accumulation. After intracranial injection, lentiviral shRNA reduced PrPC expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrPC. Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.
Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE+/− mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.
MIA/CD-RAP is a small, soluble protein secreted from malignant melanoma cells and from chondrocytes. Recent evidence has identified MIA/CD-RAP as the prototype of a small family of extracellular proteins adopting an SH3 domain-like fold. It is thought that interaction between MIA/CD-RAP and specific epitopes in extracellular matrix proteins regulates the attachment of tumor cells and chondrocytes. In order to study the consequences of MIA/CD-RAP deficiency in vivo, we generated mice with a targeted gene disruption. The complete absence of MIA/CD-RAP mRNA and protein expression was demonstrated by reverse transcriptase, Western blot analysis, and enzyme-linked immunosorbent assay measurements of whole-embryo extracts. MIA−/− mice were viable and developed normally, and histological examination of the organs by means of light microscopy revealed no major abnormalities. In contrast, electron microscopic studies of cartilage composition revealed subtle defects in collagen fiber density, diameter, and arrangement, as well as changes in the number and morphology of chondrocytic microvilli. Taken together, our data indicate that MIA/CD-RAP is essentially required for formation of the highly ordered ultrastructural fiber architecture in cartilage and may have a role in regulating chondrocyte matrix interactions.
Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.
Somatostatin receptor type II expression in the mammalian brain displays a spatially and temporally very restricted pattern. In an investigation of the molecular mechanisms controlling these patterns, we have recently shown that binding of the transcription factor SEF-2 to a novel initiator element in the SSTR-2 promoter is essential for SSTR-2 gene expression. Further characterization of the promoter identified a species-conserved TC-rich enhancer element. By screening a mouse brain cDNA expression library, we cloned a cDNA encoding the transcription factor MIBP1. MIBP1 interacts specifically with both the TC box in the SSTR-2 promoter and with the SEF-2 initiator-binding protein to enhance transcription from the basal SSTR-2 promoter. We then investigated SSTR-2, SEF-2, and MIBP1 mRNA expression patterns in the developing and adult murine brain by Northern blotting and in situ hybridization. While SEF-2 is widely expressed in many neuronal and nonneuronal tissues, MIBP1 expression overlapped precisely with expression of SSTR-2 in the frontal cortex and hippocampus. In summary, our data for the first time define a regulatory role for the transcription factor MIBP1 in mediating spatially and temporally regulated SSTR-2 expression in the brain.
AP-2 transcription factors have been suggested to exert key regulatory functions in vertebrate embryonic development, in tumorigenicity of various cancer cell types, and in controlling cell cycle and apoptotic effector genes. In this study, we investigated transcriptional regulation of the AP-2α gene promoter mediated by an autoregulatory element (referred to as A32) with a core consensus AP-2 binding site at position −336 relative to the mRNA initiation site. AP-2 and multiple different nuclear proteins in HeLa and Neuro2A cell extracts form specific bandshifts with the A32 element. By screening a mouse brain cDNA expression library, we isolated two different cDNAs encoding the transcription factor BTEB-1 and a novel zinc finger protein, AP-2rep. AP-2rep reveals a modular structure with homology to transcription factors of the wt-1/egr-1-family. AP-2rep, BTEB-1, and AP-2 interact in a mutually exclusive manner with overlapping binding sites in the A32 element. Transfection studies revealed that BTEB-1 is a strong activator of AP-2α promoter activity, whereas cotransfected AP-2α resulted in moderate autoactivation of promoter activity. In contrast, AP-2rep confers strong transcriptional repression to the AP-2α gene, and we observed an excellent correlation between induction of AP-2rep mRNA expression and downregulation of AP-2α mRNA during development of the kidney. In summary, we have identified multiple transcription factors and cloned from an expression library a novel zinc finger silencing factor, AP-2rep, mediating positive and negative regulation of AP-2α expression through a set of overlapping cis-regulatory promoter elements.