Microwave-accelerated fixation and lacZ activity staining of testicular cells in transgenic mice.

Microwave energy has been used in conjunction with glutaraldehyde to rapidly fix testicular samples of transgenic mice (whole tubules, individual cells, and cryosections) as a preparation for histochemical bacterial beta-galactosidase activity staining. The results demonstrate that the microwave-enhanced aldehyde fixation step is a convenient and simple adaptation for routine analyses, with almost no artifactual consequences or gross distortions in morphology at the microscopic level. The entire procedure (from sacrificing the animal to microscopic observation of the blue spermatogenic cells) can be completed in 1 h.     

VavCre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages

Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific somatic tissues. We have generated vavCre transgenic mice, which can be used to inactivate genes specifically in adult hematopoietic and endothelial cells. In these animals, a Cre transgene is expressed under control of murine vav gene regulatory elements. To assess their usefulness, vavCre transgenic mice were bred with R26R mice, which express a lacZ reporter gene only in cells where Cre-mediated recombination has occurred. VavCre/R26R double-heterozygous offspring were analyzed by beta-galactosidase histochemistry and flow cytometry. VavCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs, including the hematopoietic progenitor-rich bone marrow. Recombination also occurred in most endothelial and germ cells, but only rarely in other cell types. The recombination in both hematopoietic and endothelial lineages may partly reflect their putative shared ontogeny and provides a unique tool for simultaneous pan-hematopoietic and endothelial mutagenesis.     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.     

Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.     

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta- galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.     

Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants.

The lacZ gene of Escherichia coli, coding for beta-galactosidase, is a widely used reporter gene for gene expression studies in microbial and animal systems. To demonstrate that it is also a powerful reporter gene in plants, lacZ was fused to 5' regulatory elements of several genes known to be functional in plant cells. By measuring LacZ activities in transgenic plants containing these gene constructs, we showed that the reporter is correctly monitoring the regulatory properties of the well-characterized promoters fused to lacZ. beta-Galactosidase was assayed directly in plant extracts when they contained high levels of LacZ or, when LacZ was expressed at low level, by separating the endogenous and LacZ activities electrophoretically and detecting the enzymes with a fluorogenic substrate. The most outstanding property of the marker is its amenability to histochemical detection. Due to its stability, LacZ can be fixed in the tissue with glutaraldehyde without loss of activity and detected with high resolution by using XGal. We could reveal expression patterns unnoticed earlier for many of the regulatory elements studied. The chlorophyll a/b binding protein gene, expressed at very high levels in green tissues, is also expressed at a low level in the vascular cylinder of the root. The Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and the TR2' gene was shown to be root specific in the intact plant and stimulated by wounding in the leaf tissue. The TR1' gene, fused to nptII, shows similar characteristics suggesting co-regulation of this tightly linked dual promoter.     

Developmental characterization and chromosomal mapping of a LacZ transgene expressed in the mouse apical ectodermal ridge

The apical ectodermal ridge (AER) has an essential role in limb morphogenesis involving the specification of the proximal-distal axis of the limb. During the analysis of transgenic mice that harbor a LacZ transgene, we detected strong expression of beta-galactosidase within the AER of developing embryos. In this mouse line, called Z16, the bacterial LacZ gene is linked to a Herpes simplex virus immediate early promoter that is normally silent in mice. Embryos from other independent mouse lines harboring the same DNA construct exhibited no AER specific staining. Thus, it appears that the LacZ transgene in the Z16 line is expressed in the AER in response to regulatory influences from genomic DNA flanking the integration site. By fluorescent in situ hybridization, the transgene insertion site was mapped to chromosome 12. Hemizygous and homozygous transgenic mice appear normal and are fertile. AER specific beta-galactosidase staining was detected by 9.5 days post coitum in the forelimb and hindlimb bud. beta-galactosidase staining could be seen throughout the development of the limbs up to 14.5 days post coitum when expression was restricted to the distal-most regions of the digits of the hindlimbs. The loss of beta-galactosidase staining between digits correlated with the onset of programmed cell death, or apoptosis, in the digit interzones. LacZ expression in this transgenic line represents a useful marker for studying AER function in limb specification during mouse embryogenesis.     

A novel <Emphasis Type="Italic">Flk1</Emphasis>-TVA transgenic mouse model for gene delivery to angiogenic vasculature

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.     

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.     

Morphofunctional studies of the glomerular wall in mice lacking entactin-1.

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the alpha(3)- and the alpha(v)-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of alpha(v) was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.     

Changes in expression of the neuropeptide Y Y1 receptor gene in the medial amygdala of transgenic mice during pregnancy and after delivery

Long-term administration of progesterone or allopregnanolone was previously shown to increase Y1 receptor gene expression in the medial amygdala of Y1R/LacZ transgenic mice, which harbor a construct comprising the murine Y1 receptor gene promoter and a lacZ reporter. We have now investigated the effects of physiological fluctuations in the cerebrocortical concentrations of neuroactive steroids during pregnancy on Y1R/LacZ transgene expression by quantitative histochemical analysis of beta-galactosidase activity. Cerebrocortical concentrations of progesterone and its metabolites allopregnanolone and allotetrahydrodeoxycorticosterone were increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Transgene expression in the medial amygdala was also increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Similar results were obtained after analysis of Y1R mRNA levels in the medial amygdala of pregnant mice by in situ hybridization. Administration of the 5alpha-reductase inhibitor finasteride to pregnant mice prevented both the increase in the cerebrocortical concentrations of neuroactive steroids as well as the increase in transgene expression. These data suggest that fluctuations in the brain concentrations of endogenous neuroactive steroids during pregnancy are associated with changes in Y1 receptor gene expression in the medial amygdala, further supporting a functional interaction between the GABAergic and NPY-Y1 receptor systems.     

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.     

Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes

Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.     

Preferential activity of Tie2 promoter in arteriolar endothelium

The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most reliable markers of the endothelial phenotype, across organisms, organs, and developmental stages. However, endothelium is intrinsically heterogeneous in origin, composition and function, presenting an arteriolar/venular asymmetry. In this regard, the expression of Tie2 along the vascular tree, although thought to be homogenous, has not been systematically investigated. Therefore we questioned whether the activity of Tie2 promoter is uniform in the microvascular endothelium. To this end, we analyzed in situ the expression of the markers beta-galactosidase [LacZ(Tie2)] and green fluorescent protein (GFP) [GFP(Tie2)], placed under the Tie2 promoter in transgenic mice, in whole mount tissue samples, which allow the simultaneous evaluation of its relative distribution in various microvascular compartments. In the mesenteries of LacZ(Tie2) and GFP(Tie2) mice, we found that the activity of Tie2 promoter is asymmetrically distributed, being much stronger in arteries and arterioles than on the venular side of the vascular tree. This observation was replicated in the diaphragm of LacZ(Tie2) mice. The capillaries presented a mosaic pattern of Tie2 promoter activity. Stimulation of angiogenesis either by wounding, or by intraperitoneal injection of Vascular Endothelial Growth Factor (VEGF), revealed that the arteriolar/venular asymmetry is established at endothelial cellular level early during new capillary formation, even before the starting of the microvascular blood flow. In conclusion, a strong Tie2 promoter activity qualifies as a novel marker of the arteriolar phenotype in microvascular endothelium.     

Cre-mediated somatic site-specific recombination in mice.

Conditional mutant mice equipped with heterologous recombination systems (Cre/lox or Flp/frt) are promising for studying tissue-specific gene function and for designing better models of human diseases. The utility of these mice depends on the cell target specificity, on the efficiency and on the control over timing of gene (in)activation. We have explored the utility of adenoviral vectors and transgenic mice expressing Cre under the control of tissue-specific promoters to achieve Cre/lox-mediated somatic recombination of the LacZ reporter gene, using a newly generated flox LacZ mouse strain. When adeno Cre viruses were administered via different routes, recombination and expression of LacZ was detected in a wide range of tissues. Whereas in liverbeta-galactosidase activity was quickly lost by turnover of expressing cells, even though the recombined allele was retained,beta-galactosidase in other tissues persisted for many months. Our data indicate that the flox LacZ transgenic line can be utilized effectively to monitor the level and functionality of Cre protein produced upon infection with adeno Cre virus or upon crossbreeding with different Cre transgenic lines.     

Flt1 and Flk1 mediate regulation of intraocular pressure and their double heterozygosity causes the buphthalmia in mice

Flt1 and Flk1 are receptor tyrosine kinases for vascular endothelial growth factor-A which play a crucial role in physiological and pathological angiogenesis. To study genetic interaction between the Flt1 and Flk1 genes, we crossed between Flt1 and Flk1 heterozygous (Flt1(+/-) and Flk1(+/-)) mice. We found that Flt1; Flk1 double heterozygous (Flt1(+/-); Flk1(+/-)) mice showed enlarged eyes similar to the buphthalmia detected in human congenital glaucoma with elevation of intraocular pressure (IOP). Actually, IOP was elevated in Flt1(+/-); Flk1(+/-) mice and also in Flt1 or Flk1 single heterozygous mice. However, none of these mutants showed hallmarks of glaucoma such as ganglion cell death and excavation of optic disc. To clarify the pathological causes for enlarged eyes and elevated IOP, we investigate the mice from matings between Flt1(+/-) and Flk1(+/-) mice. Flt1(+/-) mice showed enlarged Schlemm's canal and disordered collagen fibers in the sclera, whereas Flk1(+/-) mice showed atrophied choriocapillaris in the choroid. These tissues are a part of the main outflow and alternative uveoscleral outflow pathway of the aqueous humor, suggesting that these pathological changes found in Flt1(+/-) and Flk1(+/-) mice are associated with the buphthalmia in Flt1(+/-); Flk1(+/-) mice.