Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells

Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2-cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine X HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion.     

Primordial germ cells in the mouse embryo during gastrulation

With the aid of a whole-mount technique, we have detected a small cluster of alkaline phosphatase (ALP)-positive cells in whole mounts of mid-primitive-streak-stage embryos, 7-7 1/4 days post coitum (dpc). Within the cluster, about 8 cells contain a small cytoplasmic spot, intensely stained for ALP activity and possibly associated with an active Golgi complex. The cluster lies just posterior to the definitive primitive streak in the extraembryonic mesoderm, separated from the embryo by the amniotic fold. Towards the end of gastrulation, the number of cells containing the ALP-positive spot rises to between 50 and 80. Thereafter the number of cells in the extraembryonic cluster declines, and similar cells start to be seen in the mesoderm of the primitive streak and then in the endoderm. At 8 dpc, about 125 ALP-stained cells are found, mainly in the hindgut endoderm and also at the base of the allantois, their appearance and location at this stage agreeing closely with previous reports on primordial germ cells (PGCs). Embryos from which the cluster area has been removed at the 7-day stage are devoid of PGCs after culture for 48 h, whereas the excised tissue is rich in PGCs. We argue that the cells in the cluster are indeed primordial germ cells, at a stage significantly earlier than any reported previously. This would indicate that the PGC lineage in the mouse is set aside at least as early as 7 dpc, possibly as one of the first 'mesodermal' cell types to emerge, and that its differentiation, as expressed by ALP activity, is gradual.     

Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis

Abstract. Hyaluronan was localized in postimplantation mouse embryos using CD44, the principal hyaluronan receptor. The specificity of CD44 receptor-globulin labelling was confirmed using Streptomyces hyaluronidase, anti-chondroitin sulfate antibody, and other receptor globulins. Our major findings are summarized as follows:
1. Implantation of the blastocyst into the uterine wall triggers a rapid loss of hyaluronan from the extracellular matrix of decidual cells on the anti-mesometrial side of the uterus.
2. Hyaluronan appears early in development in the yolk cavity, and the basement membranes of primitive ectoderm and primitive endoderm.
3. During gastrulation, mesodermal cells enter a hyaluronan-rich environment, but lack a pericellular hyaluronan coat themselves.
4. In limb bud embryos, hyaluronan is present throughout the cranial mesenchyme, but is generally not present in the branchial bars, somites, or limb buds.
5. At mid-gestation, hyaluronan is present in the axial skeleton, craniofacial mesenchyme, endocardial cushions of the heart, smooth muscle of the gastrointestinal tract, and connective tissue throughout the body.
The pattern of hyaluronan expression in the day 13 fetus is nearly identical to the published distribution of transforming growth factor β (TGF β), suggesting a close functional relationship between these molecules. Together, the results suggest that hyaluronan is involved in the formation of early mesoderm, differentiation of craniofacial mesenchyme, and morphogenesis of the axial skeleton.     

Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis

Abstract. Hyaluronan was localized in postimplantation mouse embryos using CD44, the principal hyaluronan receptor. The specificity of CD44 receptor-globulin labelling was confirmed using Streptomyces hyaluronidase, anti-chondroitin sulfate antibody, and other receptor globulins. Our major findings are summarized as follows:

• 1. 

  Implantation of the blastocyst into the uterine wall triggers a rapid loss of hyaluronan from the extracellular matrix of decidual cells on the anti-mesometrial side of the uterus.
• 2. 

  Hyaluronan appears early in development in the yolk cavity, and the basement membranes of primitive ectoderm and primitive endoderm.
• 3. 

  During gastrulation, mesodermal cells enter a hyaluronan-rich environment, but lack a pericellular hyaluronan coat themselves.
• 4. 

  In limb bud embryos, hyaluronan is present throughout the cranial mesenchyme, but is generally not present in the branchial bars, somites, or limb buds.
• 5. 

  At mid-gestation, hyaluronan is present in the axial skeleton, craniofacial mesenchyme, endocardial cushions of the heart, smooth muscle of the gastrointestinal tract, and connective tissue throughout the body.

The pattern of hyaluronan expression in the day 13 fetus is nearly identical to the published distribution of transforming growth factor β (TGF β ), suggesting a close functional relationship between these molecules. Together, the results suggest that hyaluronan is involved in the formation of early mesoderm, differentiation of craniofacial mesenchyme, and morphogenesis of the axial skeleton.     

Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo

Early sequential expression of mouse Hox genes is essential for their later function. Analysis of the relationship between early Hox gene expression and the laying down of anterior to posterior structures during and after gastrulation is therefore crucial for understanding the ontogenesis of Hox-mediated axial patterning. Using explants from gastrulation stage embryos, we show that the ability to express 3' and 5' Hox genes develops sequentially in the primitive streak region, from posterior to anterior as the streak extends, about 12 hours earlier than overt Hox expression. The ability to express autonomously the earliest Hox gene, Hoxb1, is present in the posterior streak region at the onset of gastrulation, but not in the anterior region at this stage. However, the posterior region can induce Hoxb1 expression in these anterior region cells. We conclude that tissues are primed to express Hox genes early in gastrulation, concomitant with primitive streak formation and extension, and that Hox gene inducibility is transferred by cell to cell signalling. Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis showed that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.     

The genome-wide molecular regulation of mouse gastrulation embryo

The diverse morphologies among vertebrate species stems from the evolution of a basic body plan that is constituted by a spatially organized ensemble of tissue lineage progenitors. At gastrulation, this body plan is established through a coordinated morphogenetic process and the delineation of tissue lineages that are driven by the activity of the genome. To explore the molecular mechanisms, in a comprehensive context, it is imperative to glean an understanding of the region-and population-specific genetic activity underpinning this fundamental developmental process. In this review, we outline the recent progress and the future directions in studies of genome activity for the regulation of mouse embryogenesis at gastrulation.     

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.     

Monoclonal antibodies to laminin reveal the heterogeneity of basement membranes in the developing and adult mouse tissues

Two monoclonal antibodies raised against laminin isolated from a mouse parietal yolk sac cell line were used for immunohistochemical studies of basement membranes of the mouse embryo and various fetal and adult tissues. No immunoreactivity with either of the two monoclonal antibodies could be detected in the preimplantation-stage embryos, although it has been shown that these embryos contain extracellular laminin reactive with the conventional polyclonal antilaminin antibodies. Reichert's membrane in early postimplantation stages of development reacted with the monoclonal antibody LAM-I but not with the antibody LAM-II. However, from day 8 of pregnancy onward the Reichert's membrane reacted with both antibodies. Basement membranes of the embryo proper were unreactive with both monoclonal antibodies until day 12 of pregnancy. By day 14 some basement membranes of the fetal tissues became reactive with one or both monoclonal antibodies, whereas others remained still unreactive. In the 17-d fetus and the newborn mouse most of the basement membranes reacted with both monoclonal antibodies, whereas others still reacted with only one. Similar heterogeneity in the immunoreactivity of basement membranes of various tissues was noted in the adult mouse as well. These results indicate that the immunoreactivity of laminin in the extracellular matrix changes during development and that the basement membranes in various anatomic locations display heterogeneity even in the adult mouse.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

Summary The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques.After preembedding immunostaining for laminin using JgG-PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A-PO, staining of the l. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the l. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the l. fibroreticularis and the l. rara but not in the l. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques. After preembedding immunostaining for laminin using IgG--PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A--PO, staining of the 1. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the 1. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the 1. fibroreticularis and the 1. rara but not in the 1. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

PTEN抑制胚胎原肠胚形成期EMT的过程

PTEN(Phosphatase and tensin homolog)是一种重要的抑癌基因,具有非常广泛的生物学活性,例如在细胞的生长发育、迁移、凋亡和信号传导等均发挥重要作用。PTEN基因表达始于在胚胎早期的上胚层,而后主要出现在神经外胚层和胚胎中胚层结构,表明PTEN可能参与胚胎早期发育过程的细胞迁移、增殖和分化。文章主要应用在体改变早期胚胎PTEN的表达水平来观察其对上胚层至中胚层细胞转换—EMT(Epithe-lial-mesenchymal transition)的作用。首先,原位杂交结果提示,内源性PTEN表达在原条以及之后的中胚层细胞结构如体节等。在体PTEN转染实验,体外培养至HH3期的鸡胚胎,转染Wt PTEN-GFP或移植Wt PTEN-GFP原条组织至未转染的同时期的宿主胚胎相同部位后,观察到PTEN转染细胞大都由上胚层迁移至原条并滞留于原条,不再参与中胚层细胞形成。移植实验也得到相似结果,发现在Wt PTEN-GFP阳性原条组织移植后很少细胞迁移出原条。另外在原肠胚期PTEN siRNA降调胚胎一侧PTEN基因后,降调侧中胚层细胞数明显少于正常侧。上述研究结果均提示PTEN基因在胚胎原肠胚期三胚层形成过程中可能具有抑制EMT的作用。     

Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.

Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.

Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.     

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.     

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.     

Appearance and distribution of collagens and laminin in the early mouse embryo

Appearance and distribution of the different collagen types and the noncollagenous glycoprotein laminin was studied during early mouse development from unfertilized ova to 8-day embryos using indirect immunofluorescence techniques. Laminin was first detected intracellularly in the 16-cell compacted morula and appeared also intercellularly along cell contours. Type IV collagen was first seen in the blastocyst mainly in the inner cell mass. After implantation intense fluorescence for both of these proteins was found in all the embryonic and extraembryonic basement membranes. The interstitial collagens type I and III were first detected in the 8-day embryo closely codistributed in tissues of mesodermal origin including the head and heart mesenchymes and in basement membranes bounded by mesodermal structures. The results establish a developmental sequence for the appearance of basement membrane and extracellular matrix glycoproteins in early mouse development. The distribution of laminin suggests the presence of extracellular matrix material already in compacted morulae. The appearance of type IV collagen coincides with differentiation of the primitive endoderm and assembly of the first embryonal basement membrane. The appearance of the interstitial collagens during mesoderm differentiation indicates a stage when mesoderm acquires connective tissue characteristics.     

Sensitive radioimmunoassays for 7 S collagen and laminin: Application to serum and tissue studies of basement membranes

Radioimmunoassays were developed for the basement membrane components, 7 S collagen and fragments of the noncollagenous protein laminin, which allowed quantitative analysis of as little as 0.1–0.4 ng of these proteins. Similar materials could be detected by these assays in serum, in kidney digests, and in the medium of cell cultures obtained from mice and rats. Distinct changes in the amounts of antigen in serum and kidney were observed during aging and in mice inoculated with a basement membrane tumor.     

Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes.

The presence of laminin in authentic basement membranes was examined at the level of a large pepsin-resistant fragment P1. This strongly antigenic fragment has been recently isolated from a mouse tumour basement membrane. By using antibodies to mouse laminin P1 for identification it was possible to isolate a homologous fragment P1 (Mr about 250 000) and a related component Pa (Mr about 70 000--90 000) from pepsin digests of human placenta and kidney. The fragments were in half-cystine (90--130 residues/1000) and carbohydrate and showed strong binding to concanavalin A. Reduction of disulphide bonds produced several smaller peptide chains, indicating a complex pepsin cleavage. Immunological assays demonstrated partial antigenic identity between laminin fragments obtained from mouse and human tissue, and suggested that fragment Pa may originate from a protein not completely identical with laminin. The results showed that laminin is an abundant component of tissue rich in basement membranes, which has been previously suggested by immunohistological studies.