BMP receptor-activated Smads confer diverse functions during the development of the dorsal spinal cord.

Bone Morphogenetic Proteins (BMPs) have multiple activities in the developing spinal cord: they specify the identity of the dorsal-most neuronal populations and then direct the trajectories of dorsal interneuron (dI) 1 commissural axons. How are these activities decoded by dorsal neurons to result in different cellular outcomes? Our previous studies have shown that the diverse functions of the BMPs are mediated by the canonical family of BMP receptors and then regulated by specific inhibitory (I) Smads, which block the activity of a complex of Smad second messengers. However, the extent to which this complex translates the different activities of the BMPs in the spinal cord has remained unresolved. Here, we demonstrate that the receptor-activated (R) Smads, Smad1 and Smad5 play distinct roles mediating the abilities of the BMPs to direct cell fate specification and axon outgrowth. Smad1 and Smad5 occupy spatially distinct compartments within the spinal cord, with Smad5 primarily associated with neural progenitors and Smad1 with differentiated neurons. Consistent with this expression profile, loss of function experiments in mouse embryos reveal that Smad5 is required for the acquisition of dorsal spinal neuron identities whereas Smad1 is critical for the regulation of dI1 axon outgrowth. Thus the R-Smads, like the I-Smads, have discrete roles mediating BMP-dependent cellular processes during spinal interneuron development.

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

Lipoprotein receptor-related protein in brain and in cultured neurons of mice deficient in receptor-associated protein and transgenic for apolipoprotein E4 or amyloid precursor protein.

The role of the receptor-associated protein in controlling the expression of the low-density lipoprotein receptor-related protein was analysed in brain and in cultured neurons of receptor-associated protein - / - mice. In addition, the effect of two important ligands of lipoprotein receptor-related protein in brain, i.e. apolipoprotein E and amyloid precursor protein, was examined by crossing the receptor-associated protein - / - mice with transgenic mice overexpressing these proteins specifically in neurons. The immunohistochemical localization of lipoprotein receptor-related protein and receptor-associated protein in wild-type mouse brain was demonstrated to be congruent over all structures, including the cortex and hippocampus. In primary hippocampal neurons, lipoprotein receptor-related protein was distributed somatodendritically and receptor-associated protein was concentrated perinuclearly. In hippocampal neurons from receptor-associated protein - / - mice, lipoprotein receptor-related protein was redistributed over the cell body at the expense of the dendrites. In the absence of receptor-associated protein, maturation of lipoprotein receptor-related protein is slow, resulting in accumulation of the uncleaved 600,000 mol. wt precursor. Neither the added expression of apolipoprotein E4 nor that of amyloid precursor protein in cultured neurons influenced the maturation of lipoprotein receptor-related protein, in either the presence or absence of receptor-associated protein. This result shows that receptor-associated protein is not needed to allow co-expression of lipoprotein receptor-related protein with these ligands in neurons. Furthermore, the typical ramified neuronal morphology of cultured primary neurons and the histology and architecture of the brain were normal in receptor-associated protein - / - mice and in all of the double transgenic mice. Finally, we demonstrated that the survival of receptor-associated protein - /- hippocampal neurons was normal and unaffected by the genotype of the glial feeder cells, whether they were derived from wild-type mice or from mice deficient in receptor-associated protein or apolipoprotein E. These results show that, despite the dramatic effect on maturation and cellular localization of lipoprotein receptor-related protein, the absence of receptor-associated protein did not result in any notable physiological, functional or morphological effects.

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency.

Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1(-/-) mice, PS2(-/-) mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1(+/-) PS2( -/-) mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.

Analysis of expression of genes involved in apolipoprotein E-based lipoprotein metabolism in pregnant mice deficient in the receptor-associated protein, the low density lipoprotein receptor, or apolipoprotein E.

Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP(-/-) mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP(-/-) pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.

alpha2-macroglobulin- and murinoglobulin-1- deficient mice. A mouse model for acute pancreatitis.

Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.

Humanization of mouse 5-hydroxytryptamine1B receptor gene by homologous recombination: in vitro and in vivo characterization.

We replaced the coding region of the murine 5-hydroxytryptamine (5-HT)1B receptor by the human 5-HT1B receptor using homologous recombination in embryonic stem cells and generated and characterized homozygous transgenic mice that express only the human (h) 5-HT1B receptor. The distribution patterns of h5-HT1B and murine (m) 5-HT1B receptor mRNA and binding sites in brain sections of transgenic and wild-type mice were identical as measured by in situ hybridization histochemistry and radioligand receptor autoradiography. When measured in parallel under identical conditions, the h5-HT1B receptor expressed in mouse brain had the same pharmacological characteristics as that in human brain. Stimulation by 5-HT1B agonists of [35S]guanosine-5'-O-(3-thio)triphosphate binding in brain sections demonstrated the functional coupling of the h5-HT1B receptor to G proteins in mouse brain. In tissue slices from various brain regions, electrically stimulated [3H]5-HT release was not modified by 5-HT1B agonists in tissue from either transgenic and wild-type mice; a 5-HT1B antagonist enhanced electrically stimulated [3H]5-HT release in wild-type mouse brain, but was ineffective in the transgenics. The centrally active 5-HT1A/5-HT1B agonist RU24969 induced hypothermia but did not increase locomotor activity in the transgenic mice. The ineffectiveness of RU24969 in the transgenic mice could be due to the lower affinity of the compound for the h5-HT1B receptor compared with the m5-HT1B receptor. The present study demonstrates a complete replacement of the mouse receptor by its human receptor homolog and a functional coupling to G proteins. However, modulation of [3H]5-HT release could not be shown. Furthermore, behavioral effects were not clearly observed, which may be due to a lack of appropriate tools.

Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Murine alpha-macroglobulins demonstrate divergent activities as neutralizers of transforming growth factor-beta and as inducers of nitric oxide synthesis. A possible mechanism for the endotoxin insensitivity of the alpha2-macroglobulin gene knock-out mouse.

alpha2-Macroglobulin null mice demonstrate increased resistance to endotoxin challenge (Umans, L., Serneels, L., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1995) J. Biol. Chem. 270, 19778-19785). We hypothesized that this phenotype might reflect the function of murine alpha2M (malpha2M) as a neutralizer of transforming growth factor-beta (TGF-beta) and inducer of nitric oxide synthesis in vivo. When incubated with wild-type mouse plasma, TGF-beta1 and TGF-beta2 bound only to malpha2M. Alternative TGF-beta-binding proteins were not detected in plasma from alpha2M(-/-) mice. Wild-type mouse plasma, but not plasma from alpha2M(-/-) mice, inhibited TGF-beta1 binding to TGF-beta receptors on fibroblasts. Purified malpha2M bound TGF-beta1 and TGF-beta2 with similar affinity; the KD values were 28 +/- 4 and 33 +/- 4 nM, respectively. Murinoglobulin, the second murine alpha-macroglobulin, bound both TGF-beta isoforms with 30-fold lower affinity. Malpha2M counteracted the activities of TGF-beta1 and TGF-beta2 in an endothelial cell growth assay. Malpha2M also induced NO synthesis when incubated with RAW 264.7 cells, an activity which probably results from the neutralization of autocrine TGF-beta activity. Human alpha2M induced NO synthesis comparably to malpha2M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-beta is a property of malpha2M, which is not redundant in the murine alpha-macroglobulin family or in murine plasma. Malpha2M is the only murine alpha-macroglobulin that promotes NO synthesis. The absence of malpha2M, in alpha2M(-/-) mice, may allow TGF-beta to more efficiently suppress excessive iNOS expression following endotoxin challenge.

Targeted inactivation of the mouse alpha 2-macroglobulin gene.

The mouse alpha 2-macroglobulin gene was inactivated in embryonic stem cells by homologous recombination. Liver alpha 2-macroglobulin mRNA and plasma protein was absent in homozygotes and reduced to 50% in heterozygotes. alpha 2-Macroglobulin-deficient mice were viable and produced normally sized litters with normal sex ratio over 3 generations. Characterization of adult homozygotes included diets with different fat content, treatments with endotoxin, bleomycin, carbon tetrachloride, and ethionine to test for immune system, lung, liver, and pancreas toxicity, respectively. Knock-out mice were more resistant to endotoxin but more sensitive to a choline-free diet supplemented with ethionine. Regulation of murinoglobulin mRNA expression during pregnancy was analyzed as a possible back-up mechanism for the deficiency in alpha 2-macroglobulin. In addition, expression of mRNA was studied, coding for alpha 2-macroglobulin receptor/lipoprotein receptor-related protein, low density lipoprotein receptor, and very low density lipoprotein receptor and for some common ligands, i.e. apolipoprotein E, lipoprotein lipase, and the 44-kDa heparin binding protein. Their differential regulation in the knock-out mice relative to C57B1 mice was evident and is discussed. The impressive 15-fold increase in maternal liver murinoglobulin mRNA at partum in the knock-out mice indicated increased consumption, compared to only 4-fold in normal mice. Thus, murinoglobulin appears as the major proteinase inhibitor around partum, obviously solicited to a much greater extend in alpha 2-macroglobulin-deficient mice.

Cloning, characterization, and chromosomal localization to 4p16 of the human gene (LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein and structural comparison with the murine gene coding for the 44-kDa heparin-binding protein.

We report the molecular cloning of the human gene (symbol LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein (A2MRAP), as well as the gene coding for the 44-kDa heparin-binding protein (HBP-44), its murine counterpart. For both, genomic cosmid clones were isolated, and for the human gene a bacteriophage P1 clone containing the entire A2MRAP gene was also retrieved. The genes were characterized after subcloning: in both species, the known coding part of the cDNA is encoded by eight exons, and the position of the boundaries of the exons was conserved. The human LRPAP1 locus was assigned to chromosome 4 by PCR of human-hamster hybrid cell lines and by fluorescence in situ hybridization to band 4p16.3. This maps closely to the variable constitutional deletions of the short arm of chromosome 4, observed cytogenetically in patients with the Wolf-Hirschhorn syndrome. Metaphase spreads of two such patients were analyzed by fluorescence in situ hybridization with an LRPAP1 genomic probe. The first patient, with karyotype 46,XY,del4(p14-p16.1), had retained both copies of the LRPAP1 gene. In contrast, the other patient, with karyotype 46,XY,del4(p15.3-pter), displayed no signal for LRPAP1 on the deleted chromosome.

Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein.

The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR.

Molecular cloning of the mouse gene coding for alpha 2-macroglobulin and targeting of the gene in embryonic stem cells.

We have cloned the mouse gene coding for alpha 2-macroglobulin in overlapping lambda clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5' flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phase A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

Study of the synthesis and secretion of normal and artificial mutants of murine amyloid precursor protein (APP): cleavage of APP occurs in a late compartment of the default secretion pathway.

Amyloid precursor protein (APP) secretase plays a pivotal role in the processing of APP since its activity precludes the formation of amyloid peptide in Alzheimer's Disease. The identity and the subcellular localization of this enzyme are at this moment unknown. It is also unclear how APP escapes the activity of this enzyme when amyloid is formed. We have previously shown that APP-secretase activity is not inhibited by exogenously added proteinase inhibitors of different specificity (De Strooper, B., F. Van Leuven, and H. Van Den Berghe. 1992. FEBS (Fed. Eur. Biochem. Soc.) Lett. 308:50-53). We show here that the primary amine methylamine inhibits the secretion of APP into the medium. Furthermore, we show that a truncated form of APP, devoid of the cytoplasmic domain, is more efficiently cleaved and secreted than wild-type APP, which together with the methylamine block, shows that APP-secretase is located in a late compartment of the default constitutional secretion pathway. The sorting signals in the cytoplasmic domain of APP are therefore important in the deviation of APP from the secretase pathway. Finally we show that mutation of Arg609 to Asp in combination with Lys612 to Glu makes APP a less efficiently cleaved substrate for APP-secretase. The results are discussed in the context of recent findings on the targeting of APP and a parallel is drawn with some lysosomal glycoproteins that follow similar pathways.

Molecular cloning and sequencing of the murine alpha-2-macroglobulin receptor cDNA.

We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.