Bertolesi GE et al. (2012), Identification and expression analysis of GPAT ...

XB-ART-45254

Gene Expr Patterns 2012 Jan 01;127-8:219-27. doi: 10.1016/j.gep.2012.04.002.

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Identification and expression analysis of GPAT family genes during early development of Xenopus laevis.

Bertolesi GE , Iannattone S , Johnston J , Zaremberg V , McFarlane S .

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Production of lysophosphatidic acid (LPA) is the first step in the de novo pathway for glycerolipid biosynthesis, which is mainly catalyzed by the glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15). DHAPAT (EC2.3.1.42) also contributes in a minor way, using dihydroxyacetone phosphate as substrate. Final products and intermediates of the glycerolipid synthesis pathway are the main structural components of cellular membranes, and provide signalling molecules that regulate diverse biological processes, including cell proliferation, differentiation and growth. Here we identified the four orthologs of the mammalian GPATs (1-4) and DHAPAT in Xenopus, including a novel, short variant of GPAT2, and analyzed their expression pattern during embryonic development. Xenopus GPAT1/2 localized to mitochondria, while GPAT3/4 associated with the endoplasmic reticulum. All are similarly expressed in the early embryonic nervous system. A more tissue specific pattern emerges during organogenesis, including liver expression for GPAT1/4, and testis expression for GPAT2. All acyltransferases were expressed in kidney, though GPAT3 was excluded from the pronephric ducts. Our results suggest important roles of GPATs and DHAPAT during early organogenesis.

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Species referenced: Xenopus laevis
Genes referenced: agpat1 agpat2 agpat3 agpat4 ambp canx eef1a1 gnpat gpam gpat2 gpat3 gpat4 myc odc1 tbx2 tbx3 vax1 vax2

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	Fig. 2. Subcellular localization of GPATs and DHAPAT. (A) RT-PCR products for GPATs and DHAPAT generated from total cDNA obtained from XTC and A6 cells. (B and C) Confocal microscopy of Cos-7 cells overexpressing the indicated myc-tagged GPATs visualized by using anti-myc antibodies (green) and either the mitochondrial marker, Mitotracker Red CM-H2HRos (B), or the ER marker calnexin (C) (red).
	Fig. 3. Expression of GPATs and DHAPAT at different stages of X. laevis embryonic development. (A) RT-PCR analysis showing the expression of GPAT1 to 4 and DHAPAT at the indicated stage of development, from eggs (E) to stage 42. The stages corresponding to cleavage (seg), gastrulation, neurulation and organogenesis are indicated. ODC, whose levels remain constant during development, was used as a loading control, while EF1α was used as a positive control. (B) Densitometry analysis of 3 independent PCR reactions is shown. Results were normalized to levels at stage 42, and are presented as the ratio between each GPAT and DHAPAT with ODC at the corresponding embryonic stage. Smooth curves integrating the average of the nine nearest neighbors are shown with dashes.
	Fig. 4. mRNA expression of GPATs and DHAPAT during neurulation and early organogenesis detected by whole-mount in situ hybridization. Expression is first detected at late gastrulation (stage 12/13) and early neurulation. All the GPATs and DHAPAT are expressed in a region around the blastopore (b) and along the anterior-posterior embryonic axis, towards the dorsal midline (dm). At stage 17, GPATs and DHAPAT are expressed in the nervous system, including the neural tube (nt), hindbrain (hb), the forebrain (fb) and the eye primordia (ep). This pattern becomes more evident at stage 19. Nervous system expression is observed in dorsal and anterior views of embryos at stage 20 and older. Vax2 and ET are two genes encoding transcription factors involved in early eye development, and were used for reference. Stage 20/22 left panel, dorsal view; Stage 17, 19, 20/22 right panel and 23, anterior view.
	Fig. 5. mRNA expression of GPATs and DHAPAT in embryos and in kidney between stages 32 and 37/38 as detected by whole-mount in situ hybridization. mRNA for all GPATs and DHAPAT are present in the kidney (K), as well as in the nervous system, including the eye (E), otic vesicle (OV), spinal cord (SC) and branchial arches (BA). GPAT1 and GPAT4 are also expressed in the liver (L). Lateral views at the indicated stages (anterior to left). GPAT1, GPAT2, GPAT4, and DHAPAT are expressed in the pronephric tubules (arrows) and ducts (arrowheads), but GPAT3 is not expressed in the pronephric ducts (white arrowhead).
	Fig. 6. mRNA expression of GPAT1 and GPAT2 in different organs detected by whole- mount in situ hybridization. (A) GPAT1 is expressed during kidney and liver organogenesis. Whole-mount in situ hybridization with the GPAT1 antisense riboprobe of embryos at the indicated stages is shown in the left column. Higher magnifications of the kidney and liver regions are shown in a lateral view in the right two panels (anterior to left). Eye (E), kidney (K) (early expression detected at stage 26) and liver (L) (early expression detected at stage 33/34) are shown. (B) GPAT1 and GPAT2 show a different expression pattern at stage 40. GPAT1 and GPAT2 are both expressed in the nervous system (E, eye; B, brain; and SC, spinal cord). Further, GPAT1 is strongly expressed in liver (L) and kidney (K), whereas GPAT2 is expressed in presumptive gonad (testis-T) and weakly in kidney.
	Supplementary Figure 3. mRNA expression of GPAT1 in the liver. GPAT1 is expressed in kidney (K), eye (E), otic vesicle (OV), spinal cord (SC) and liver (L), as confirmed with an antisense riboprobe against the liver specific marker AMBP.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 35 and 36, as assayed by in situ hybridization, lateral view: anterior left, dorsal up.
	AMBP ( alpha-1-microglobulin/bikunin precursor ) gene expression in Xenopus laevis embryos, NF stage 35 and 36, assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 26, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 37 and 38, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	gpam ( glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 40, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gpat2 (glycerol-3-phosphate acyltransferase, mitochondrial ) gene expression in Xenopus laevis embryos, NF stage 40, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.