Laser surgery for mouse geneticists.
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Animals↗
Biomedical subjects
Thomas Gridley
Publications and source records attributed to Thomas Gridley.
Critical role of endothelial Notch1 signaling in postnatal angiogenesis.
Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1(+/-) mice, but not Notch4(-/-) mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.
Amyloid Precursor Protein Secretases↗
Mutation of a ubiquitously expressed mouse transmembrane protein (Tapt1) causes specific skeletal homeotic transformations.
L5Jcs1 is a perinatal lethal mutation uncovered in a screen for ENU-induced mutations on mouse chromosome 5. L5Jcs1 homozygotes exhibit posterior-to-anterior transformations of the vertebral column midsection, similar to mice deficient for Hoxc8 and Hoxc9. Positional cloning efforts identified a mutation in a novel, evolutionarily conserved, and ubiquitously expressed gene dubbed Tapt1 (Transmembrane anterior posterior transformation 1). TAPT1 is predicted to contain several transmembrane domains, and part of the gene is orthologous to an unusual alternatively spliced human transcript encoding the cytomegalovirus gH receptor. We speculate that TAPT1 is a downstream effector of HOXC8 that may act by transducing or transmitting extracellular information required for axial skeletal patterning during development.
Amino Acid Sequence↗
Snail1 gene function during early embryo patterning in mice.
Originally identified as one of two zygotically expressed genes required for gastrulation in Drosophila, the Snail gene and other family members play critical roles in vertebrate development. Functionally, these genes are thought to drive epithelial-mesenchymal transitions at several points during development, and also during the metastatic progression of cancer. Although the Snai2-null mouse is viable and fertile, the early embryonic lethality of Snai1-null mice has precluded the detailed analysis of Snai1 function after gastrulation. We have recently generated a conditional allele of the Snai1 gene and examined its function during the formation of the neural crest and establishment of the left-right axis. We uncovered new details regarding Snai1 function during gastrulation and left-right asymmetry determination, while surprisingly showing that neither the Snai1 nor Snai2 genes are essential for neural crest cell delamination. These results shed new light on the role of Snail family genes in early mouse development, and raise interesting questions concerning the diversity of gene function among vertebrate species.
Alleles↗
Snail family genes are required for left-right asymmetry determination, but not neural crest formation, in mice.
Snail family genes encode zinc finger transcriptional repressors that are key regulators of epithelial-mesenchymal transitions in vertebrates, including the transitions that generate the mesoderm and neural crest. Here, we show that, contrary to observations in frog and avian embryos, the Snail family genes Snail (Snai1) and Slug (Snai2) are not required for formation and delamination of the neural crest in mice. However, embryos with conditional inactivation of Snai1 function exhibit defects in left-right asymmetry determination. This work demonstrates that although some aspects of Snail family gene function, such as a role in left-right asymmetry determination, appear to be evolutionarily conserved, their role in neural crest cell formation and delamination is not.
Alleles↗
The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear.
In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.
Alleles↗
Jag2-Notch1 signaling regulates oral epithelial differentiation and palate development.
During mammalian palatogenesis, palatal shelves initially grow vertically from the medial sides of the paired maxillary processes flanking the developing tongue and subsequently elevate and fuse with each other above the tongue to form the intact secondary palate. Pathological palate-mandible or palate-tongue fusions have been reported in humans and other mammals, but the molecular and cellular mechanisms that prevent such aberrant adhesions during normal palate development are unknown. We previously reported that mice deficient in Jag2, which encodes a cell surface ligand for the Notch family receptors, have cleft palate associated with palate-tongue fusions. In this report, we show that Jag2 is expressed throughout the oral epithelium and is required for Notch1 activation during oral epithelial differentiation. We show that Notch1 is normally highly activated in the differentiating oral periderm cells covering the developing tongue and the lateral oral surfaces of the mandibular and maxillary processes during palate development. Oral periderm activation of Notch1 is significantly attenuated during palate development in the Jag2 mutants. Further molecular and ultrastructural analyses indicate that oral epithelial organization and periderm differentiation are disrupted in the Jag2 mutants. Moreover, we show that the Jag2 mutant tongue fused to wild-type palatal shelves in recombinant explant cultures. These data indicate that Jag2-Notch1 signaling is spatiotemporally regulated in the oral epithelia during palate development to prevent premature palatal shelf adhesion to other oral tissues and to facilitate normal adhesion between the elevated palatal shelves.
Animals↗
The long and short of it: somite formation in mice.
A fundamental characteristic of the vertebrate body plan is its segmentation along the anterior-posterior axis. This segmental pattern is established during embryogenesis by the formation of somites, the transient epithelial blocks of cells that derive from the unsegmented presomitic mesoderm. Somite formation involves a molecular oscillator, termed the segmentation clock, in combination with gradients of signaling molecules such as fibroblast growth factor 8, WNT3A, and retinoic acid. Disruption of somitogenesis in humans can result in disorders such as spondylocostal dysostosis, which is characterized by vertebral malformations. This review summarizes recent findings concerning the role of Notch signaling in the segmentation clock, the complex regulatory network that governs somitogenesis, the genes that cause inherited spondylocostal dysostosis, and the mechanisms that regulate bilaterally symmetric somite formation.
Animals↗
Generation of a Snail1 (Snai1) conditional null allele.
Members of the Snail gene superfamily, which encode zinc finger transcriptional repressors, play critical roles in the establishment of the vertebrate body plan. The Snail1 (Snai1) gene promotes epithelial-mesenchymal transitions during development and disease progression, and Snai1 null mouse embryos exhibit defects in gastrulation. However, the early embryonic lethality of Snai1 null embryos precludes the study of Snai1 function in other developmental contexts or diseases. To overcome this restriction, we generated a Snai1 conditional null allele by flanking the promoter and first two exons of the Snai1 gene with loxP sites. Cre-mediated deletion of the Snai1(flox) allele generates the Snai1(del2) allele, which behaves genetically as a Snai1 null allele. This conditional null allele will enable investigation of Snai1 function in a variety of developmental and pathological contexts.
Alleles↗
Generation of new Notch2 mutant alleles.
The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice.
Alleles↗
Loss of notch activity in the developing central nervous system leads to increased cell death.
Many cells in the mammalian brain undergo apoptosis as a normal and critical part of development but the signals that regulate the survival and death of neural progenitor cells and the neurons they produce are not well understood. The Notch signaling pathway is involved in multiple decision points during development and has been proposed to regulate the survival and apoptosis of neural progenitor cells in the developing brain; however, previous experiments have not resolved whether Notch activity is pro- or anti-apoptotic. To elucidate the function of Notch signaling in the survival and death of cells in the nervous system, we have produced single and compound Notch conditional mutants in which Notch1 and Notch3 are removed at different times during brain development and in different populations of cells. We show here that a large number of neural progenitor cells, as well as differentiating neurons, undergo apoptosis in the absence of Notch1 and Notch3, suggesting that Notch activity promotes the survival of both progenitors and newly differentiating cells in the developing nervous system. Finally, we show that postmitotic neurons do not require Notch activity indefinitely to regulate their survival since elevated levels of cell death are observed only during embryogenesis in the Notch mutants and are not detected in neonates.
Animals↗
Not all lunatic fringe null female mice are infertile.
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Animal Husbandry↗
The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear.
The mammalian auditory sensory epithelium, the organ of Corti, contains sensory hair cells and nonsensory supporting cells arranged in a highly patterned mosaic. Notch-mediated lateral inhibition is the proposed mechanism for creating this sensory mosaic. Previous work has shown that mice lacking the Notch ligand JAG2 differentiate supernumerary hair cells in the cochlea, consistent with the lateral inhibitory model. However, it was not clear why only relatively modest increases in hair cell production were observed in Jag2 mutant mice. Here, we show that another Notch ligand, DLL1, functions synergistically with JAG2 in regulating hair cell differentiation in the cochlea. We also show by conditional inactivation that these ligands probably signal through the NOTCH1 receptor. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate, rather than through excess proliferation. Although these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the organ of Corti. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.
Animals↗
Notch signaling coordinates the patterning of striatal compartments.
Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.
Animals↗
Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells.
Radial glia function during CNS development both as neural progenitors and as a scaffolding supporting neuronal migration. To elucidate pathways involved in these functions, we mapped in vivo the promoter for Blbp, a radial glial gene. We show here that a binding site for the Notch effector CBF1 is essential for all Blbp transcription in radial glia, and that BLBP expression is significantly reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These results identify Blbp as the first predominantly CNS-specific Notch target gene and suggest that it mediates some aspects of Notch signaling in radial glia.
Animals↗
Mutations in snail family genes enhance craniosynostosis of Twist1 haplo-insufficient mice: implications for Saethre-Chotzen Syndrome.
In Drosophila, mutations in the Twist gene interact with mutations in the Snail gene. We show that the mouse Twist1 mutation interacts with Snai1 and Snai2 mutations to enhance aberrant cranial suture fusion, demonstrating that genetic interactions between genes of the Twist and Snail families have been conserved during evolution.
Acrocephalosyndactylia↗
Developmental transcription factor slug is required for effective re-epithelialization by adult keratinocytes.
During re-epithelialization of cutaneous wounds, keratinocytes recapitulate several aspects of the embryonic process of epithelial-mesenchymal transition (EMT), including migratory activity and reduced intercellular adhesion. The transcription factor Slug modulates EMT in the embryo and controls desmosome number in adult epithelial cells, therefore, we investigated Slug expression and function during cutaneous wound re-epithelialization. Slug expression was elevated in keratinocytes bordering cutaneous wounds in mice in vivo, in keratinocytes migrating from mouse skin explants ex vivo, and in human keratinocytes at wound margins in vitro. Expression of the related transcription factor Snail was not significantly modulated in keratinocytes during re-epithelialization in vitro. Epithelial cell outgrowth from skin explants of Slug knockout mice was severely compromised, indicating a critical role for Slug in epithelial keratinocyte migration. Overexpression of Slug in cultured human keratinocytes caused increased cell spreading and desmosomal disruption, both of which were most pronounced at wound margins. Furthermore, in vitro wound healing was markedly accelerated in keratinocytes that ectopically expressed Slug. Taken together, these findings suggest that Slug plays an important role during wound re-epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development.
Animals↗
Snail regulates p21(WAF/CIP1) expression in cooperation with E2A and Twist.
Snail, a zinc-finger transcriptional repressor, is essential for mesoderm and neural crest cell formation and epithelial-mesenchymal transition. The basic helix-loop-helix transcription factors E2A and Twist have been linked with Snail during embryonic development. In this study, we examined the role of Snail in cellular differentiation through regulation of p21(WAF/CIP1) expression. A reporter assay with the p21 promoter demonstrated that Snail inhibited expression of p21 induced by E2A. Co-expression of Snail with Twist showed additive inhibitory effects. Deletion mutants of the p21 promoter revealed that sequences between -270 and -264, which formed a complex with unidentified nuclear factor(s), were critical for E2A and Snail function. The E2A-dependent expression of the endogenous p21 gene was also inhibited by Snail.
Basic Helix-Loop-Helix Proteins↗