Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice.

The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

Sequence, recombinant expression and tissue localization of two novel extracellular matrix proteins, fibulin-3 and fibulin-4.

Fibulin-1 and fibulin-2 have previously been identified as basement membrane and microfibrillar proteins with a broad binding repertoire for other extracellular ligands. Here we report on the cloning and sequence analysis of human fibulin-3 (487 residues), also known as protein S1-5, and fibulin-4 (443 residues). These novel members of this protein family are most closely related to fibulin-1C. They consist of a C-terminal globular domain III, also shared by the fibrillins, a central rod-like element composed of five calcium-binding epidermal growth factor-like (EG) modules (domain II) and an N-terminal interrupted EG module (domain I) which replaces the anaphylatoxin-like modules of the other fibulins. This predicted domain structure was supported by electron microscopy of fibulin-4, which demonstrated short rods. Northern blots showed that both novel fibulins are expressed in several human tissues to a variable extent and that they are up-regulated in quiescent fibroblasts. Specific antibodies which were raised against each of the novel fibulins did not cross-react with fibulin-1. Immunohistology of adult mouse tissues showed that fibulin-3, fibulin-4 and fibulin-1 have overlapping but distinct extracellular tissue localizations. A particularly prominent feature was the staining of variable sets of large and small blood vessels.

Sequence and expression of a novel member (LTBP-4) of the family of latent transforming growth factor-beta binding proteins.

Overlapping cDNA clones from human heart and melanoma libraries were used to establish the 1587-residue sequence of a novel protein (LTBP-4) belonging to the family of extracellular microfibrillar proteins which also bind transforming growth factor-beta. LTBP-4 consists of 20 EG modules, 17 of them with a consensus sequence for calcium binding, 4 TB modules with 8 cysteines and several proline-rich regions. Northern blots demonstrated a single 5 kb mRNA which is highly expressed in heart but also present in skeletal muscle, pancreas, placenta and lung. The modular structure predicts that LTBP-4 should be a microfibrillar protein which probably also binds TGF-beta.

Functional expression of human intestinal Na+-dependent and Na+-independent nucleoside transporters in Xenopus laevis oocytes.

We have shown previously that the human jejunal brush border membrane expresses both the N1 (cif) and the N2 (cit) Na+-dependent (concentrative) nucleoside transporters but not the Na+-independent (facilitative) nitrobenzylmercaptopurineriboside (NBMPR)-sensitive (es) transporter (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). In the present study, we have demonstrated that when Xenopus laevis oocytes are microinjected with human jejunal mRNA, four nucleoside transporters are expressed simultaneously, namely the N1 and N2 Na+-dependent nucleoside transporters and the es and the NBMPR-insensitive (ei) Na+-independent transporters. The expressed Na+-dependent nucleoside transporters showed substrate specificity identical to that previously described by us using jejunal brush border membrane vesicles (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). The expressed es and ei Na+-independent transporters demonstrated broad substrate selectivity with both purines and pyrimidines capable of inhibiting the uptake of guanosine and thymidine mediated by this transporter. The expressed Na+-dependent nucleoside transporters mediated the transport of their respective nucleoside substrates with a high affinity and a low capacity, whereas the es and the ei transporters mediated the transport of nucleosides with a low affinity and a high capacity. Collectively, these observations suggest that the Na+-independent nucleoside transporters are expressed in the basolateral membrane of the human jejunal epithelium. Based on these data, we hypothesize that the concentrative transporters in the brush border membrane and equilibrative transporters in the basolateral membrane are arranged in series in the human jejunal epithelium to allow efficient vectorial transport of nucleosides from the lumen to the blood. The simultaneous expression of four nucleoside transporters in X. laevis oocytes establishes a basis for molecular cloning of these four human nucleoside transporters.

Evidence for a clustered genomic organization of FAX-zinc finger protein encoding transcription units in Xenopus laevis.

Finger associated box-zinc finger proteins (FAX-ZFPs) constitute a subfamily of the many C2H2 type ZFPs in Xenopus laevis. FAX is a highly conserved protein domain connected to variable zinc finger clusters. Three different FAX-ZFPs encoding genomic fragments were isolated and subjected to a detailed structural characterization. All three exhibit a common, highly conserved exon/intron organization, with the variant zinc finger clusters organized in a single exon for each gene analysed. Two of the three genomic fragments contain a second FAX-ZFP encoding (partial) transcription unit each. Further evidence for a clustered organization of FAX-ZFP transcription units is provided by Southern blot analysis of large genomic restriction fragments separated by transverse field gel electrophoresis, and by in situ hybridization on intact chromosomes. Comparative sequence analysis of the genes isolated reveals an exceptional degree of DNA sequence conservation in both exon and intron regions in one part of the FAX encoding region, suggesting that recent gene conversion has led to the combination of these sequence elements with DNA segments including regions encoding variant zinc finger clusters. Overexpression of the FAX domain by itself or of a full-length FAX-ZFP in X. laevis embryos by means of mRNA injection does not interfere with the normal developmental program, suggesting general and not cell specific/regulatory functions for X. laevis FAX-ZFPs.

The maternal store of zinc finger protein encoding mRNAs in fully grown Xenopus oocytes is not required for early embryogenesis.

A large family of C2H2 (Krüppel-like) zinc finger protein genes is maternally transcribed in Xenopus oocytes; many of the corresponding mRNAs are actively translated post-fertilization, before the onset of zygotic activation of transcription. With the aim of asking if any of these stored mRNAs have a function in Xenopus development, we made use of antisense oligonucleotide mediated, targeted RNA destruction. Injected oocytes lose the entire pool of C2H2 zinc finger protein encoding mRNAs. They are indistinguishable from control oocytes in their abilities to mature in vitro and to be fertilized in vitro. Embryos generated from such oocytes develop normally until tadpole stage. These findings do not rule out the possibility that C2H2 zinc finger protein genes are involved in developmental control in Xenopus. However, they do suggest that the biological function for at least some of the early expressed zinc finger proteins in Xenopus differs in important aspects from the way Krüppel or other DNA binding factors act as developmental regulators in Drosophila.