Fgfr2 is integral for bladder mesenchyme patterning and function

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme ( Fgfr2BM−/−). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic ( E) day 16.5 ( E16.5) Fgfr2BM−/− bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal ( P) day 1 ( P1) to P30, Fgfr2BM−/− bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2BM−/− bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2BM−/− bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2BM−/− bladders compared with controls. Moreover, E16.5 Fgfr2BM−/− bladders exhibit higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling.

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Fgfr2 is integral for bladder mesenchyme patterning and function

AJP Renal Physiology ◽

10.1152/ajprenal.00624.2014 ◽

2015 ◽

Vol 308 (8) ◽

pp. F888-F898 ◽

Cited By ~ 2

Author(s):

K. A. Walker ◽

Y. Ikeda ◽

I. Zabbarova ◽

C. M. Schaefer ◽

D. Bushnell ◽

...

Keyword(s):

Cell Fate ◽

Ex Vivo ◽

Smooth Muscle Actin ◽

Three Dimensional ◽

Growth Factor Receptor ◽

Immunoblot Analysis ◽

Type Ia ◽

And Function ◽

Passive Stretch

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor (Fgfr)2 is necessary for kidney and ureter mesenchymal development. The objective of the present study was to determine the role of Fgfr2 in the bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in the bladder mesenchyme ( Fgfr2 BM−/−). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblot analysis, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with control bladders, embryonic day 16.5 (E16.5) Fgfr2 BM−/− bladders had thin muscle layers with less α-smooth muscle actin and thickened lamina propria with increased collagen type Ia and IIIa that intruded into the muscle. The reciprocal changes in mutant layer thicknesses appeared partly due to a cell fate switch. From postnatal days 1 to 30, Fgfr2 BM−/− bladders demonstrated progressive muscle loss and increased collagen expression. Postnatal Fgfr2 BM−/− bladder sheets exhibited decreased agonist-mediated contractility and increased passive stretch tension versus control bladder sheets. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2 BM−/− versus control bladders. Mechanistically, whereas Shh expression appeared normal, mRNA and protein readouts of hedgehog activity were increased in E16.5 Fgfr2 BM−/− versus control bladders. Moreover, E16.5 Fgfr2 BM−/− bladders exhibited higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, compared with control bladders. In conclusion, loss of Fgfr2 in the bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility.

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Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

Journal of Experimental Medicine ◽

10.1084/jem.20071800 ◽

2007 ◽

Vol 204 (13) ◽

pp. 3103-3111 ◽

Cited By ~ 188

Author(s):

Brian G. Petrich ◽

Patrizia Marchese ◽

Zaverio M. Ruggeri ◽

Saskia Spiess ◽

Rachel A.M. Weichert ◽

...

Keyword(s):

Platelet Adhesion ◽

Ex Vivo ◽

Focal Adhesions ◽

Β1 Integrin ◽

Normal Morphology ◽

And Function ◽

Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

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Mesenchymal Igf2 is a major paracrine regulator of pancreatic growth and function

10.1101/714121 ◽

2019 ◽

Author(s):

Constanze M. Hammerle ◽

Ionel Sandovici ◽

Gemma V. Brierley ◽

Nicola M. Smith ◽

Warren E. Zimmer ◽

...

Keyword(s):

Ex Vivo ◽

Whole Body ◽

Imprinted Genes ◽

Paracrine Signalling ◽

Genetic Mechanisms ◽

Pancreatic Epithelium ◽

And Function ◽

Maternal Glucose

AbstractThe genetic mechanisms that determine the size of the adult pancreas are poorly understood. Here we demonstrate that many imprinted genes are highly expressed in the pancreatic mesenchyme, and explore the role of Igf2 in-vivo. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy. Surprisingly, mesenchymal mass is unaffected, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Furthermore, increased IGF2 activity specifically in the mesenchyme, through Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Ex-vivo exposure of primary acinar cells to exogenous IGF2 increases cell proliferation and amylase production through AKT signalling. We propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

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Mechanical mapping of mammalian follicle development using Brillouin microscopy

10.1101/2021.02.21.432113 ◽

2021 ◽

Author(s):

Chii Jou Chan ◽

Carlo Bevilacqua ◽

Robert Prevedel

Keyword(s):

Material Properties ◽

Ex Vivo ◽

Three Dimensional ◽

Follicle Development ◽

Mammalian Development ◽

Oocyte Growth ◽

Intact Mouse ◽

Matrix Deposition

AbstractIn early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides a novel approach to study the role of mechanics in follicle morphogenesis and pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

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Mechanical mapping of mammalian follicle development using Brillouin microscopy

Communications Biology ◽

10.1038/s42003-021-02662-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Chii Jou Chan ◽

Carlo Bevilacqua ◽

Robert Prevedel

Keyword(s):

Material Properties ◽

Ex Vivo ◽

Three Dimensional ◽

Follicle Development ◽

Mammalian Development ◽

Oocyte Growth ◽

Intact Mouse ◽

Matrix Deposition

AbstractIn early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides an alternative approach to study the role of mechanics in follicle morphogenesis and might pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

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The Spinebot—A Robotic Device to Intraoperatively Quantify Spinal Stiffness

Journal of Medical Devices ◽

10.1115/1.4049915 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Philippe Büchler ◽

Jonas Räber ◽

Benjamin Voumard ◽

Steve Berger ◽

Brett Bell ◽

...

Keyword(s):

Ex Vivo ◽

Three Dimensional ◽

Axial Rotation ◽

Flexion Extension ◽

Torque Load ◽

The Moment ◽

Reference Measurements ◽

Spine Function ◽

And Function

Abstract Degenerative spine problems and spinal deformities have high socio-economic impacts. Current surgical treatment is based on bony fusion that can reduce mobility and function. Precise descriptions of the biomechanics of normal, deformed, and degenerated spinal segments under in vivo conditions are needed to develop new approaches that preserve spine function. This study developed a system that intraoperatively measures the three-dimensional segmental stiffness of patient's spine. SpineBot, a parallel kinematic robot, was developed to transmit loads to adjacent vertebrae. A force/torque load cell mounted on the SpineBot measured the moment applied to the spinal segment and calculated segmental stiffnesses. The accuracy of SpineBot was characterized ex vivo by comparing its stiffness measurement of five ovine specimens to measurements obtained with a reference spinal testing system. The SpineBot can apply torques up to 10 N·m along all anatomical axes with a total range of motion of about 11.5 deg ± 0.5 deg in lateral bending, 4.5 deg ± 0.3 deg in flexion/extension, and 2.6 deg ± 0.5 deg in axial rotation. SpineBot's measurements are noisier than the reference system, but the correlation between SpineBot and reference measurements was high (R2 > 0.8). In conclusion, SpineBot's accuracy is comparable to that of current reference systems but can take intraoperative measurements. SpineBot can improve our understanding of spinal biomechanics in patients who have the pathology of interest, and take these measurements in the natural physiological environment, giving us information essential to developing new “nonfusion” products.

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EML1 is essential for retinal photoreceptor localization and light detection

10.21203/rs.3.rs-821540/v1 ◽

2021 ◽

Author(s):

Deepak Poria ◽

Chi Sun ◽

Andrea Santeford ◽

Michel Kielar ◽

Rajendra S. Apte ◽

...

Keyword(s):

Calcium Binding ◽

Light Adaptation ◽

Ex Vivo ◽

Outer Nuclear Layer ◽

Retina Development ◽

Rods And Cones ◽

Cone Sensitivity ◽

And Function

Abstract Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.

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The Role of the 3Rs for Understanding and Modeling the Human Placenta

Journal of Clinical Medicine ◽

10.3390/jcm10153444 ◽

2021 ◽

Vol 10 (15) ◽

pp. 3444

Author(s):

Joana Costa ◽

Ruth Mackay ◽

Sophie-Christine de Aguiar Greca ◽

Alessandro Corti ◽

Elisabete Silva ◽

...

Keyword(s):

Human Placenta ◽

Ex Vivo ◽

Structure And Function ◽

Collective Impact ◽

And Function ◽

Animal Welfare Legislation ◽

Made In

Modeling the physiology of the human placenta is still a challenge, despite the great number of scientific advancements made in the field. Animal models cannot fully replicate the structure and function of the human placenta and pose ethical and financial hurdles. In addition, increasingly stricter animal welfare legislation worldwide is incentivizing the use of 3R (reduction, refinement, replacement) practices. What efforts have been made to develop alternative models for the placenta so far? How effective are they? How can we improve them to make them more predictive of human pathophysiology? To address these questions, this review aims at presenting and discussing the current models used to study phenomena at the placenta level: in vivo, ex vivo, in vitro and in silico. We describe the main achievements and opportunities for improvement of each type of model and critically assess their individual and collective impact on the pursuit of predictive studies of the placenta in line with the 3Rs and European legislation.

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The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000116 ◽

2012 ◽

Vol 82 (3) ◽

pp. 228-232 ◽

Cited By ~ 7

Author(s):

Mauro Serafini ◽

Giuseppa Morabito

Keyword(s):

Antioxidant Capacity ◽

Dietary Intervention ◽

Ex Vivo ◽

The Body ◽

Dietary Polyphenols ◽

Enzymatic Antioxidant ◽

Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

Molecular Brain ◽

10.1186/s13041-021-00738-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

M. Asada-Utsugi ◽

K. Uemura ◽

M. Kubota ◽

Y. Noda ◽

Y. Tashiro ◽

...

Keyword(s):

Memory Function ◽

Three Dimensional ◽

Excitatory Synapses ◽

Missense Mutations ◽

Glutamatergic Synapses ◽

Learning Tasks ◽

Transmission Electron ◽

Nmdar Activation ◽

And Function

AbstractN-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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