Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.     

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.     

Zebrafish mutants identify an essential role for laminins in notochord formation

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.     

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.     

Immunohistochemical distribution of laminin-5 gamma2 chain and its developmental change in human embryonic and foetal tissues

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.     

Interaction between growth factors and retinoic acid in the induction of kidney tubulogenesis in tissue culture.

Kidney tubulogenesis is the initial step in renal organogenesis. The precise molecular determinants of this pattern formation are presently unknown, although soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. To define the molecular determinants of renal proximal tubule morphogenesis, primary cultures of rabbit renal proximal tubule cells in hormonally defined, serum-free media were treated with transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), and the retinoid, all trans-retinoic acid (RA), singly or in combination. Utilizing phase contrast and light and transmission electron microscopy, the simultaneous administration of TGF-beta 1 (10 ng/ml), EGF (1 nM), and RA (0.1 nM) transformed a confluent monolayer of renal proximal tubule cells within 5 to 6 days into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border. All three factors were necessary and sufficient to induce this phenotypic transformation. Further studies demonstrated that RA promoted the deposition of the A and B1 chains of laminin, a cell attachment protein of the basement membrane, in a small subset of proximal tubule cells in culture, as deduced by indirect immunofluorescent microscopy. Additional studies demonstrated that soluble purified laminin fully substituted for RA in this system to promote renal tubulogenesis when combined with TGF-beta 1 and EGF. These results demonstrate that the growth factors, TGF-beta 1 and EGF, and the retinoid, RA, promote tubulogenesis in adult renal proximal tubule cells in tissue culture in a manner reminiscent of inductive embryonic kidney morphogenesis. These observations define a coordinated interplay between growth factors and retinoids to induce pattern formation and morphogenesis. Furthermore, the demonstration of RA-induced laminin deposition as a critical event in this morphogenic process identifies laminin as a possible target protein for RA to act as a morphogen.     

Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.     

Engrailed-1 misexpression in chick embryos prevents apical ridge formation but preserves segregation of dorsal and ventral ectodermal compartments

Using lineage tracers, we recently showed dorsal and ventral ectodermal compartments along the sides of the body in chick embryos. The compartments are formed both in presumptive limb-forming regions where they position the apical ridge and also in presumptive interlimb (flank). Here we show, using a novel technique combining fate mapping and in situ hybridisation, that the ventral compartment coincides with the Engrailed-1 (En-1) domain of expression. This coincidence suggests that En-1 could maintain the ventral compartment and be necessary for apical ridge formation. To test this hypothesis, we ectopically expressed En-1 via retroviral transfer and then examined limb development and cell lineage restriction in the ectoderm. En-1 misexpression can completely prevent formation of both normal limbs and ectopic limbs induced in the flank by application of FGF-2. In both cases, there are no morphological signs of apical ectodermal ridge formation and expression of ridge-associated genes is undetectable. In striking contrast, the lineage restriction between dorsal and ventral ectoderm is not altered. Therefore, En-1 is involved in the regulation of ridge formation but not compartment maintenance.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1(-/-) embryos die within a day of implantation. We report the generation of mice lacking laminin alpha1 and laminin beta1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1(-/-) embryos are similar to Lamc1(-/-) embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1(-/-) embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (alpha5beta1gamma1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin alpha5 from a transgene improved the phenotype of Lama1(-/-) embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.     

Regional differences in the expression of laminin isoforms during mouse neural tube development

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.     

Beta1-integrin orients epithelial polarity via Rac1 and laminin

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.     

Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly

Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin-Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.     

The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.     

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.     

Immunohistochemical Distribution of Laminin-5 γ 2 Chain and its Developmental Change in Human Embryonic and Foetal Tissues

Immunohistochemical distribution of laminin γ 2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin γ 2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5–6 weeks of gestation. Between 6–7 and 12–13 weeks, laminin γ 2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin γ 2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin γ 2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin γ 2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its γ 2 chain in the morphogenesis of human tissues.     

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.     

An in vitro tubule assay identifies HGF as a morphogen for the formation of seminiferous tubules in the postnatal mouse testis.

We have been working with a recently immortalized Sertoli cell line, SF7, that appears to produce sleeves, or hollow tubules, when cultivated on a layer of growth factor-reduced Matrigel (GFR-Matrigel) in medium supplemented with serum. We tried to determine which components of GFR-Matrigel and serum provide the environment needed for tubule formation. While laminin and collagen IV were essential for the formation of flat cords, none of the basement membrane constituents, when taken alone or in combination, would support the formation of tubules in minimal culture medium. Moreover, none of the growth factors present in GFR-Matrigel could induce tubulogenesis. Recently, much attention has been focused upon the role of hepatocyte growth factor (HGF) and its receptor c-met in the induction of tubulogenesis by epithelial cells. Therefore, we investigated the expression of HGF/c-met in the mouse testis at different postnatal stages and in the adult and evaluated the contribution of HGF/c-met in the production of Sertoli cell tubules by SF7 and primary Sertoli cells in vitro. Our results confirm that laminin and collagen IV are essential for the formation of testicular cords and reveal that HGF/c-met are necessary for the further remodeling of these cords into tubules.