Corrinne G. Lobe

Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.

Summary: The Cre/loxP system has become an important tool in designing postintegrational switch mechanisms for transgenes in mice. The power and spectrum of application of this system depends on transgenic mouse lines that provide Cre recombinase activity with a defined cell type‐, tissue‐, or developmental stage‐specificity. We have developed a novel mouse line that acts as a Cre reporter. The mice, designated Z/EG (lacZ/EGFP), express lacZ throughout embryonic development and adult stages. Cre excision, however, removes the lacZ gene, which activates expression of the second reporter, enhanced green fluorescent protein. We have found that the double‐reporter Z/EG line is able to indicate the occurrence of Cre excision from early embryonic to adult lineages. The advantage of the Z/EG line is that Cre‐mediated excision can be monitored in live samples and that live cells with Cre‐mediated excision can be isolated using a single‐step FACS. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site‐specific recombinase system. genesis 28:147–155, 2000.

The post-natal heart contains a myocardial stem cell population.

The recent identification of stem cell pools in a variety of unexpected tissue sources has raised the possibility that a pluripotent stem cell population may reside in the myocardium and contribute to the post‐natal growth of this tissue. Here, we demonstrate that the post‐natal myocardium contains a resident verapamil‐sensitive side population (SP), with stem cell‐like activity. When growth of the post‐natal heart was attenuated through over‐expression of a dominant negative cardiac transcription factor (MEF2C), the resident SP cell population was subject to activation, followed by a consequent depletion. In addition, cardiac SP cells are capable of fusion with other cell types, but do not adopt the corresponding gene expression profile. These observations suggest that a responsive stem cell pool resides in the adult myocardium, and may influence adaptation of the post‐natal heart.

Early restriction of peripheral and proximal cell lineages during formation of the lung.

To establish the timing of lineage restriction among endodermal derivatives, we developed a method to label permanently subsets of lung precursor cells at defined times during development by using Cre recombinase to activate floxed alkaline phosphatase or green fluorescent protein genes under control of doxycycline-dependent surfactant protein C promoter. Extensive or complete labeling of peripheral lung, thyroid, and thymic epithelia, but not trachea, bronchi, or gastrointestinal tract occurred when mice were exposed to doxycycline from embryonic day (E) 4.5 to E6.5. Nonoverlapping cell lineages of conducting airways (trachea and bronchi), as distinct from those of peripheral airways (bronchioles, acini, and alveoli), were established well before formation of the definitive lung buds at E9–9.5. At E11.5, the labeled precursors of peripheral lung were restricted to relatively few cells along the bronchial tubes and clusters in bronchial tips and lateral buds. Thereafter, these cells underwent marked expansion to form the entire gas-exchange region in the lung. This study demonstrates early restriction of endodermal progenitor cells forming peripheral as compared with proximal airways, identifies distinct cell lineages in conducting airways, and distinguishes neuroepithelial and tracheal–bronchial gland cell lineages from those lining peripheral regions of the lung. This system for conditional gene addition or deletion is useful for the study of lung morphogenesis and gene function in vivo, and identifies progenitor cells that may serve as useful targets for cell or gene replacement for pulmonary disorders.

Glypican-3 Promotes the Growth of Hepatocellular Carcinoma by Stimulating Canonical Wnt Signaling

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. GPC3 is expressed by most hepatocellular carcinomas but not by normal hepatocytes and benign liver lesions. We report here that GPC3 stimulates the in vitro and in vivo growth of hepatocellular carcinoma cells by increasing autocrine/paracrine canonical Wnt signaling. Co-immunoprecipitation experiments showed that GPC3 is able to form complexes with Wnts, and cell-binding assays indicated that GPC3-expressing cells have an increased capacity to bind Wnt. Collectively, these results suggest that GPC3 stimulates Wnt activity by facilitating the interaction of this polypeptide with its signaling receptors. Surprisingly, in contrast to the current model that proposes that Wnt-glypican binding is mediated by the heparan sulfate chains, we found that the nonglycanated GPC3 core protein can form complexes with Wnts. Furthermore, we showed that the glycosaminoglycan chains are not required for the stimulatory effect on Wnt signaling and hepatocellular carcinoma growth.

Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition

Synaptic activity drives synaptic rearrangement in the vertebrate nervous system; indeed, this appears to be a main way in which experience shapes neural connectivity. One rearrangement that occurs in many parts of the nervous system during early postnatal life is a competitive process called ‘synapse elimination’. At the neuromuscular junction, where synapse elimination has been analysed in detail, muscle fibres are initially innervated by multiple axons, then all but one are withdrawn and the ‘winner’ enlarges. In support of the idea that synapse elimination is activity dependent, it is slowed or speeded when total neuromuscular activity is decreased or increased, respectively. However, most hypotheses about synaptic rearrangement postulate that change depends less on total activity than on the relative activity of the competitors. Intuitively, it seems that the input best able to excite its postsynaptic target would be most likely to win the competition, but some theories and results make other predictions. Here we use a genetic method to selectively inhibit neurotransmission from one of two inputs to a single target cell. We show that more powerful inputs are strongly favoured competitors during synapse elimination.

Ectopic Notch Activation in Developing Podocytes Causes Glomerulosclerosis

Genetic evidence supports an early role for Notch signaling in the fate of podocytes during glomerular development. Decreased expression of Notch transcriptional targets in developing podocytes after the determination of cell fate suggests that constitutive Notch signaling may oppose podocyte differentiation. This study determined the effects of constitutive Notch signaling on podocyte differentiation by ectopically expressing Notchs intracellular domain (NOTCH-IC), the biologically active, intracellular product of proteolytic cleavage of the Notch receptor, in developing podocytes of transgenic mice. Histologic and molecular analyses revealed normal glomerular morphology and expression of podocyte markers in newborn NOTCH-IC-expressing mice; however, mice developed severe proteinuria and showed evidence of progressive glomerulosclerosis at 2 wk after birth. Features of mature podocytes were lost: Foot processes were effaced; expression of Wt1, Nphs1, and Nphs2 was downregulated; cell-cycle re-entry was induced; and the expression of Pax2 was increased. In contrast, mice with podocyte-specific inactivation of Rbpsuh, which encodes a protein essential for canonical Notch signaling, seemed normal. In addition, the damaging effects of NOTCH-IC expression were prevented in transgenic mice after simultaneous conditional inactivation of Rbpsuh in murine podocytes. These results suggest that Notch signaling is dispensable during terminal differentiation of podocytes but that constitutive (or inappropriate) Notch signaling is deleterious, leading to glomerulosclerosis.

Conditional genome alteration in mice

The recent ability to inactivate specific genes in mice has significantly accelerated our understanding of molecular, cellular, and even behavioral aspects of normal and disease processes. However, this ability has also demonstrated the extreme complexity of genetic determination in mammals, in particular, that genes in the same family or pathway can be functionally redundant and that a given gene often has multiple roles. Thus, inactivation of a gene often does not indicate its complete spectrum of functions. To circumvent this problem, many new tools and novel applications of classic techniques have been developed to place spatial and temporal restrictions on the genomic alterations. These approaches include chimera and mosaic studies, organ transplantation, complementation assays, dominant negative mutants, conditional gene knockouts, and lineage‐specific gene rescue. Not only has this opened up more sophisticated ways to make genomic alterations, but it has provided the opportunity to create animal models for sporadic human genetic diseases. BioEssays 20:200–208, 1998.

Contrasting effects of VEGF gene disruption in embryonic stem cell‐derived versus oncogene‐induced tumors

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF‐deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF‐null tumors (528ras, 528neu) originated from VEGF−/− embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene‐driven tumor angiogenesis. Both ras and neu oncogenes down‐regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP‐1)]. This is functionally important as administration of an antiangiogenic TSP‐1 peptide (ABT‐526) markedly inhibited growth of VEGF−/− tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell‐derived VEGF in certain cases of ‘adult’ tumor angiogenesis, and thus highlight the importance of considering VEGF‐independent as well as VEGF‐dependent pathways when attempting to block this process pharmacologically.

Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida

Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan−/− males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan−/− males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.

Grg1 Acts as a Lung-Specific Oncogene in a Transgenic Mouse Model

Groucho proteins are transcriptional corepressors that are recruited to gene regulatory regions by numerous transcription factors. Long isoforms, such as Grg1, have all the domains of the prototype Drosophila Groucho. Short Groucho proteins, such as Grg5, have only the amino-terminal Q and G/P domains. We generated Grg1 and Grg5 transgenic mice and found that Grg1 overexpression induces lung adenocarcinoma, whereas Grg5 overexpression does not. Coexpression of Grg5 with Grg1 reduces tumor burden. Grg1 and Grg5 both diminish p53 protein levels; however, only Grg1 overexpression induces elevated levels of ErbB1 and ErbB2 receptor tyrosine kinases. The molecular and biological changes that accompany tumor progression in Grg1 transgenic mice closely reiterate events seen in human lung cancer. We also found that within a human lung tumor tissue array, a significant number of carcinomas overexpress Grg1/TLE1. Our data suggest that Grg1 overexpression contributes to malignancy in human lung cancers.