Abstract
Autotaxin, a lysophospholipase D encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid in the extracellular space. Lysophosphatidic acid acts on specific G protein-coupled receptors, thereby regulating cell growth, migration, and survival. Previous studies have revealed that Enpp2(-/-) mouse embryos die at about embryonic day (E) 9.5 because of angiogenic defects in the yolk sac. However, what cellular defects occur in Enpp2(-/-) embryos and what intracellular signaling pathways are involved in the phenotype manifestation remain unknown. Here, we show that Enpp2 is required to form distinctive large lysosomes in the yolk sac visceral endoderm cells. From E7.5 to E9.5, Enpp2 mRNA is abundantly expressed in the visceral endoderm cells. In Enpp2(-/-) mouse embryos, lysosomes in the visceral endoderm cells are fragmented. By using a whole embryo culture system combined with specific pharmacological inhibitors for intracellular signaling molecules, we show that lysophosphatidic acid receptors and the Rho-Rho-associated coiled-coil containing protein kinase (ROCK)-LIM kinase pathway are required to form large lysosomes. In addition, electroporation of dominant negative forms of Rho, ROCK, or LIM kinase also leads to the size reduction of lysosomes in wild-type visceral endoderm cells. In Enpp2(-/-) visceral endoderm cells, the steady-state levels of cofilin phosphorylation and actin polymerization are reduced. In addition, perturbations of actin turnover dynamics by actin inhibitors cytochalasin B and jasplakinolide result in the defect in lysosome formation. These results suggest that constitutive activation of the Rho-ROCK-LIM kinase pathway by extracellular production of lysophosphatidic acid by the action of autotaxin is required to maintain the large size of lysosomes in visceral endoderm cells.
Highlights
That produces lysophosphatidic acid (LPA)2 [1,2,3,4]
Perturbations of actin turnover dynamics by cytochalasin B or jasplakinolide result in the defect in lysosome formation in visceral endoderm (VE) cells. These findings suggest that the control of actin turnover dynamics through the Rho-Rho-associated coiled-coil containing protein kinase (ROCK)-LIM kinase (LIMK) pathway by way of autotaxin-LPA signaling is required for the regulatory processes of lysosome biogenesis
We demonstrated that autotaxin-LPA signaling is required to form distinctive large lysosomes in yolk sac VE cells
Summary
Animal Experiments—All the experiments using animals were approved by the Animal Care and Use Committee of the University of Tsukuba and performed under its guidelines. The linearized vector was electroporated into mouse embryonic stem cells (E14 clone derived from 129/Ola), and neomycin-resistant colonies were selected by PCR. Whole embryos were mounted on a glass-bottom dish (Matsunami, Osaka, Japan), allowing observation of the yolk sac using a laser scanning confocal microscope (LSM510; Zeiss) at room temperature. To compare fluorescence signal intensity, confocal images of yolk sacs in cryostat sections were collected with all laser settings constant. Electroporation of Whole Embryos—Whole embryos were dissected at E7.5 and transferred to Hanks’ balanced salt solution (Invitrogen) kept at 37 °C. One embryo was inserted into a small hole of an agarose mold (2% agarose/phosphate-buffered saline, 7-mm square) placed in Hanks’ balanced salt solution. The embryo was rinsed with Hanks’ balanced salt solution and cultured as described above. DN-cofilin (cofilin (S3E)) and CA-cofilin (cofilin (S3A)) [22, 23] were fused to yellow fluorescent protein at their N termini in the expression vector derived from pEGFP-C1 (Clontech)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
