In vivo notch reactivation in differentiating cochlear hair cells induces sox2 and prox1 expression but does not disrupt hair cell maturation

COVER PHOTOGRAPH: Lineage tracing of Prox1 expressing inner ear cochlear cells in Prox1CreEGFP/+; Rosa26‐EYFPloxp/+ mice at P23. All cochlear hair cells are labeled with calbindin in red. EYFP in green historically records the cochlear cells experiencing Prox1 expression during the development, which include hair cells and supporting cells (both inside and outside the organ of Corti). From Liu et al., Developmental Dynamics 241:684–696, 2012.     

Murine Notch1 is required for lymphatic vascular morphogenesis during development

BACKGROUND: The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)‐specific conditional Notch1 knockout mice crossed with an inducible Prox1CreER^T2 driver. RESULTS: LEC‐specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1‐mutant LECs, whereas proliferation was increased. RNA‐seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1‐mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated. CONCLUSIONS: The lymphatic phenotype of LEC‐specific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis. Developmental Dynamics 243:957–964, 2014. © 2014 Wiley Periodicals, Inc.     

Preclinical transgenic and patient‐derived xenograft models recapitulate the radiological features of human adamantinomatous craniopharyngioma

To assess the clinical relevance of transgenic and patient‐derived xenograft models of adamantinomatous craniopharyngioma (ACP) using serial magnetic resonance imaging (MRI) and high resolution post‐mortem microcomputed tomography (μ‐CT), with correlation with histology and human ACP imaging. The growth patterns and radiological features of tumors arising in Hesx1^Cre/+;Ctnnb1^lox(ex3)/+ transgenic mice, and of patient‐derived ACP xenografts implanted in the cerebral cortex, were monitored longitudinally in vivo with anatomical and functional MRI, and by ex vivo μ‐CT at study end. Pathological correlates with hematoxylin and eosin stained sections were investigated. Early enlargement and heterogeneity of Hesx1^Cre/+;Ctnnb1^lox(ex3)/+ mouse pituitaries was evident at initial imaging at 8 weeks, which was followed by enlargement of a solid tumor, and development of cysts and hemorrhage. Tumors demonstrated MRI features that recapitulated those of human ACP, specifically, T₁‐weighted signal enhancement in the solid tumor component following Gd‐DTPA administration, and in some animals, hyperintense cysts on FLAIR and T₁‐weighted images. Ex vivo μ‐CT correlated with MRI findings and identified smaller cysts, which were confirmed by histology. Characteristic histological features, including wet keratin and calcification, were visible on μ‐CT and verified by histological sections of patient‐derived ACP xenografts. The Hesx1^Cre/+;Ctnnb1^lox(ex3)/+ transgenic mouse model and cerebral patient‐derived ACP xenografts recapitulate a number of the key radiological features of the human disease and provide promising foundations for in vivo trials of novel therapeutics for the treatment of these tumors.     

Role of bone morphogenetic proteins on cochlear hair cell formation: Analyses of Noggin and Bmp2 mutant mice

The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1^cre/+ and Gfi1^cre/+. Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1^cre/+;Bmp2^lox/? mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4. Developmental Dynamics 239:505–513, 2010. Published 2010 Wiley‐Liss, Inc.     

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

Coordinated lymphangiogenesis is critical in lymph node development and maturation

Background: Lymph node (LN) formation requires multiple but coordinated signaling from intrinsic and extrinsic cellular components during embryogenesis. However, the contribution and role of lymphatic vessels (LVs) in LN formation and maturation are poorly defined. Here, using lymphatic‐specific reporters, Prox1‐GFP mice and Vegfc^+/LacZ mice, we analyzed migration, assembly, and ingrowth of lymphatic endothelial cells (LECs) in LNs during pre‐ and postnatal development. Results: Prox1⁺ LECs form string‐like connections rather than lymph sac–like structures until E14.5, but the LEC coverage around LN anlagen completes before birth. Compared to wild‐type littermates, Vegfc^+/LacZ mice had markedly smaller LNs in neonates and adults, presumably due to the decrease in LTi cell clusters and migrating Prox1⁺ LECs during embryogenesis. In addition, Vegfc‐haploinsufficiency or inhibition of VEGFR3 signaling led to an impairment of LN LV ingrowth, resulting in a significant decrease in LN volume. These data indicate that VEGF‐C/VEGFR3 signaling plays a substantial role in normal LN formation through proper migration and organization of LECs. Conclusions: Taken together, our results provide compelling evidence that the contribution of LVs through VEGF‐C/VEGFR3 signaling is critical in LN development and maturation. Developmental Dynamics 245:1189–1197, 2016. © 2016 Wiley Periodicals, Inc.     

Disappearance of centroacinar cells in the Notch ligand‐deficient pancreas

Notch signaling has been shown to contribute to murine pancreatic development at various stages. Delta‐like 1 (Dll1) or Jagged1 (Jag1) are the Notch ligands that solely function to trigger this signaling during the pancreatic bud stage (~e9.5) or after birth, respectively. However, it has not been elucidated whether these Notch ligands are required at the later stage (e10.5–18.5) when the particular pancreas structures form. Here, we detected the dual expression of Dll1 and Jag1 in the epithelium after e10.5, which was restricted to the ductal cell lineage, including centroacinar cells expressing Sox9, CD133 and Hes1 but not the ductal cell markers Hnf1β and DBA, at e18.5. To evaluate the significance of the Notch ligands during this period, we established double‐floxed mice of Dll1 and Jag1 genes with Ptf1a‐Cre knock‐in allele and examined the effects on development. The abrogation of both ligands but not a single one led to the loss of centroacinar cells, which was due to the decrease in cell proliferation and the increase in cell death, as well as to the reduction of Sox9. These results suggested that Dll1 and Jag1 function redundantly and are necessary to maintain the centroacinar cells as an environmental niche in the developing pancreas.     

Man1, an inner nuclear membrane protein,regulates left–right axis formation by controlling nodal signaling in a node‐independent manner

Man1, an inner nuclear membrane protein, regulates transforming growth factor β signaling by interacting with receptor‐associated Smads. In Man1‐deficient (Man1^Δ/Δ) embryos, vascular remodeling is perturbed by misregulation of Smad activity. Here, we show that Man1^Δ/Δ embryos exhibit abnormal heart morphogenesis including the looping abnormality. We searched for the molecular basis underlying the heart abnormalities and found that the left side‐specific genes responsible for left–right (LR) asymmetry, Nodal, Lefty2, and Pitx2, were expressed bilaterally in the lateral plate mesoderm and that their expression was enhanced significantly in mutants. Notably, Lefty1, a marker for the midline barrier, was maintained in Man1^Δ/Δ mutants. Crossing Man1^Δ/+ with Nodal hypomorphs (Nodal^neo/+), in which Nodal signaling in the node is disrupted, to generate double homozygous embryos (Man1^Δ/Δ; Nodal^neo/neo) revealed that the bilateral Nodal was retained in Man1^Δ/Δ; Nodal^neo/neo embryos. These results suggest that Man1 regulates LR asymmetry by controlling Nodal signaling in a node‐independent manner. Developmental Dynamics 237:3565–3576, 2008. © 2008 Wiley‐Liss, Inc.     

Histological study in the brain of the <Emphasis Type="Italic">reelin</Emphasis>/<Emphasis Type="Italic">Dab1</Emphasis>-compound mutant mouse

The Reelin (Reln)-deficient mouse (reeler) and the Dab1-deficient mouse (yotari) are autosomal recessive mutant mice characterized by cerebellar ataxia. Previously, we reported that Reelin and Dab1 proteins have slightly different functions during the development of the cerebral cortex. To analyze the functional roles of Reelin and Dab1 proteins in detail, we attempted to generate a reelin/Dab1 compound-mutant mouse by breeding heterozygote reeler and yotari mice. We examined the cytoarchitecture of the cerebral and cerebellar cortices and the hippocampus of wild-type (Reln ^+/+ ; Dab1 ^+/+ ), double-heterozygote (Reln ^rl/+ ; Dab1 ^yot/+ ), reeler (Reln ^rl/rl ; Dab1 ^+/+ , Reln ^rl/rl ; Dab1 ^yot/+ ), yotari (Reln ^+/+ ; Dab1 ^yot/yot , Reln ^rl/+ ; Dab1 ^yot/yot ), and double-compound-deficient (Reln ^rl/rl ; Dab1 ^yot/yot ) mice. Nissl staining demonstrated that no abnormality was recognized in the mice of reelin/Dab1 double-heterozygote (Reln ^rl/+ ; Dab1 ^yot/+ ). The reelin/Dab1-compound mutant mouse (Reln ^rl/rl ; Dab1 ^yot/yot ) showed histological abnormalities in the cerebral and cerebellar cortices and the hippocampus, in addition to those of reeler and yotari mice. We injected HRP into the lumbar cord of these animals with various gene compositions to examine the distribution pattern of corticospinal tract (CST) neurons. CST neurons of the reelin/Dab1-compound mutant mice were not confined to layer V, but scattered throughout the motor cortex. This quantitative and statistical analysis shows that the distribution pattern of CST neurons of the reelin/Dab1-compound mutant mouse differs from those of either of the reeler or yotari counterparts. Taken together, although Reelin/Dab1 signal transduction is a primary cascade in neurons during developmental periods, other signaling cascades (e.g., the Cdk-5/Dab1 pathway) may lie in a parallel fashion to Reelin/Dab1 signal transduction.