Technical Reference #1632

1632.

Phospholipase D1 is required for Angiogenesis of Intersegmental Blood Vessels in Zebrafish Xin-Xin Zeng; Xiangjin Zheng; Yun Xiang; Hyekyung Cho; Jason Jessen; Tao Zhong; Lilianna Solnica-Krezel; Alex Brown, Vanderbilt University, Developmental Biology, 328(1632), (2009)

Abstract:
Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies incultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functionsincluding intracellular vesicle traff

Keywords:
Phospholipids; phospholipases; notochord; somites; angiogenesis

Materials & Methods:
Cloning of zebrafish pld1 and RT-PCR Four zebrafish-expressed sequence tag (EST) clones (GenBank accession numbers: CK237875 CK029229 CD590334 and CF997495) encoding protein fragments with sequence similarity to human PLD1 were identified using NCBI tblastn. The 5′- and 3′-UTR regions of zebrafish pld1 were determined by 5′- and 3′-RACE PCR (Clontech) respectively. The total RNA from 2 dpf embryos was isolated using Trizol® reagent (Invitrogen). SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNAs. The full length ORF of zebrafish pld1 was amplified by PCR using a forward primer (5′-CACCATGAGTGATTCGGTGGAGAACCTGGACACC-3′) and a reverse primer (5′-TCAGGTCCAGATCTCGGTGGGCACCA-3′). The resulting PCR product was directly cloned into pENTR™/SD/DTOPO ® entry vector by Gateway® BP recombination reaction (Invitrogen). Subsequently pld1 ORF was transferred into pCS2 destination vector (a gift fromLawson lab) for in vitro transcription. Sequencing of the resulting plasmids confirmed the zebrafish pld1 ORF and the deduced amino acid sequences of the pld1 ORF match those in the GenBank database with the exception of M116L and I503M. To measure the zebrafish Pld1 activity in mammalian cells the pld1 ORF was amplified by PCR using a forward primer (5′-AACTGCAGTCACCATGAGTGATTCGGTG- 3′) and a reverse primer (5′-GGGGTACCTCAGGTCCAGATCTCGGTG- 3′) and the PCR product was subcloned into the Pst1 and Kpn1 sites of pEGFP-C1 vector (Clontech). The catalytic inactive mutant K846R was generated by a point mutation in the 2nd HKD motif using Quick Change II Site-directed mutagenesis kit (Stratagene). The resulting mutation was confirmed by sequencing. The total RNA from control or antisense morpholino oligonucleotides (MOs) injected embryos was isolated using Trizol® reagent (Invitrogen). The first strand cDNAs were synthesized using Thermoscript RT-PCR System (Invitrogen) according to the manufacturer's instructions. pld1 cDNAwas then amplified using the first strand cDNA. The following primers were used in the PCR reactions: forward primer 1: 5′-GCAGACATGAGTGATTCGGT-3′; forward primer 2: 5′-CTGAGCCCTGAGATCTTTCTGA- 3′; reverse primer 1: 5′-TCTTGACAAAGGCTGGATAGT- 3′; reverse primer 2: CTCCCACTCCTGTCTGAAGACT-3′. Measurement of Pld activity in vivo and in vitro TREx HEK293 cells were plated at 5×105 cells/well on poly-Llysine coated 6-well plates the day before transfection. Cells were transfected with vectors encoding hPLD1 wt hPLD1 K898R zPld1 wt zPld1 K846R or pEGFP-C1 vector alone (Clontech) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. All of the PLD1 expression plasmids were cloned in pEGFP-C1 vector. To measure PLD activity in vivo 30 h after transfection cells were radiolabeled with 3H-18:1 fatty acid in the serum free medium for 18 h. The majority of the label is incorporated as phosphatidylcholine which is the major substrate of PLD. After removing the labeling medium cellswere treated with either vehicle (basal) or 100 nMPMA (phorbol ester) for 30 min in the presence of 0.3% 1-butanol at 37 °C. The assays were conducted as described (Brown et al. 2007; Henage et al. 2006). The in vitro assay is a well-established biochemical assay (Brown et al.1993) used to measure PLD activity in reconstituted lipid vesicles. The details for making the lipid vesicles purification of recombinant proteins and measurements of kinetic parameters are described in detail in Brown et al. (2007). The analysis of lipids by mass spectrometry was performed essentially as previously described. Lipids were extracted from zebrafish embryos prepared for mass spectrometry analysis and statistically analyzed as detailed in Ivanova et al. (2007). The references and examples of lipid fragmentation used to identify molecular species can be found (along with downloads of relevant methods chapters) at http://www.lipidmaps.org/. Zebrafish strains and maintenance and chemical treatment The AB⁎ and TLWT zebrafish strains were maintained as described (Solnica-Krezel et al. 1994). Embryos were obtained from natural spawnings and staged according to morphology as described (Kimmel et al. 1995). A transgenic line expressing green fluorescent protein (GFP) under the control of the flk-1 promoter TG(flk1:GFP) was described by Cross et al. (2003). A transgenic line expressing green fluorescent protein (GFP) under the control of the fli-1 promoter TG (fli1:GFP) was described by Lawson and Weinstein (2002). To inhibit PLD signaling embryos were incubated with embryo media containing 0.3% 1-butanol or t-butanol at the indicated stages. In vitro transcription and translation In vitro transcription and translation of zebrafish Pld1 was performed by using the TNT Quick Coupled Reticulocyte Lysate system (Promega) according to the manufacturer's protocol. 0.5 μg flag-Pld1-pCS2 and various amount of MO1-pld1 (0 10 nM 0.5 μM and 5 μM) were added for each 25 μl reaction. The reactions were incubated at 30 °C for 90 min. 6 μl of the reaction samples was denatured loaded and separated on 8% SDS-PAGE gel and transferred to Immuno-P membrane. The synthesized zebrafish Pld1 protein was detected by anti-flag antibody. Whole mount in situ hybridization sectioning immunostaining and microscopy Whole mount in situ hybridization was performed as described previously (Thisse et al. 1993). pld1 antisense digoxigen-labeled in situ probe was synthesized using T7 RNA polymerase (Ambion) and precipitated with LiCl and EtOH and re-suspended in RNAase free water. Probe quantity was measured by spectro-photometry and quality was assayed using gel electrophoresis. Photographs of embryos were captured with a Zeiss Discovery V.12 microscope equipped with an Axiocam digital camera. For histological sections the embryos processed by in situ hybridizationwere embedded in JB-4 embedding solution and sectioned using Leica RM2265 Rotary Microtome. Whole mount immunohistochemistry was performed as described previously (Topczewska et al. 2001). Monoclonal Anti-Titin (Mouse) and Anti-Laminin (Rabbit) primary antibodies were purchased from Sigma®. Znp1 was obtained from the Developmental Studies Hybridoma Bank. TG(flk1:GFP)/TG(fli1:GFP) embryos were mounted laterally in 2% methylcellulose in glass bottom culture dishes (MatTek). The images were acquired using a Zeiss LSM 510 laser scanning inverted confocal microscope. Morpholino oligonucleotides and microinjections Three antisense morpholino oligonucleotides (MOs) (Gene Tools) MO1-pld1 (5′-CCAGGTTCTCCACCGAATCACTCAT-3′) MO2-pld1 (5′- TGTCTCATCACCTCTTAAGAAAGAG-3′) and MO3-pld1 (5′-GGTCCATCATACAAACCTGCTCTAT- 3′) were designed to specifically target the ATG start codon and inhibit translation the intron–exon junction and the exon–intron junction of the exon coding the first HKD motif respectively. A 5-mismatch MO (5mismatchMO2-pld1: 5′-TGTCTGATGACCTCTTAACAAACAG- 3′) corresponding to HKD1a was also designed as a control. Indicated dosages of MOs were injected into the yolk of one to two-cell stage embryos using a pneumatic picopump (WPI) or Eppendorf FemtoJet. Microinjection was performed as described previously (Zeng et al. 2007). Transplantation Genetic mosaic analyses were performed essentially as described (Yamashita et al. 2002). WT donor embryos were injected with 1% Rhodamine-dextran (Molecular Probes) at the one-cell stage. pld1 morphant host embryos were generated by injecting 6 ng MO2-pld1 into TG(fli1:GFP) transgenic embryos at the one-cell stage. For notochord transplantations between 5.7 and 6 hpf (hours post fertilization) when the embryonic shield became morphologically distinct 30–50 cells from the shield region were aspirated from one WT donor embryo using a transplantation needle. The group of the donor cells was immediately transplanted into the shield of a host embryo. To transplant somitic cells at the similar stage 30–50 deep cells were aspirated from the blastoderm margin next to the shield of one donor embryo and immediately transplanted into the equivalent region of a host embryo to ensure that the initial positions of the donor and host embryos were identical. The host embryos were analyzed at 2 dpf. Statistical analysis Calculations were made in Microsoft Excel. We report mean and standard error of mean (SEM) and the probability associated with Student's T-test (with 2-tailed distribution) and two samples of unequal variance. Results Cloning of zebrafish pld1 To clone a zebrafish Pld homolog the human PLD1 amino acid sequence was used to search the zebrafish EST and genomic sequence databases. Four ESTs sharing similarity with human PLD1 gene were found. The full-length pld1 cDNA was amplified by using RT-PCR and 5′-RACE (Rapid Amplification of cDNA Ends Clontech) and maintained in Gateway® Cloning system (Invitrogen). Multiple sequence alignments revealed a 64–68% shared identity at the protein level between zebrafish Pld1 and mammalian PLD1s (hPLD1 rPLD1 mPLD1) and a 50% shared identity between zebrafish Pld1 and mammalian PLD2s (hPLD2 rPLD2 mPLD2) (Supplementary Figs. 1A C). The phylogenetic tree of Pld1 and PLDs from other species is shown in Supplemental data (Supplementary Fig. 1B). Similar to mammalian PLDs Pld1 contains two conserved HxKxxxxD motifs (Supplementary Fig. 1A blue dotted rectangles) and also putative interaction sites for protein kinase C (PKC) and RhoA (Cai and Exton 2001; Kook and Exton 2005) (Supplementary Fig.1A the PKC binding site of zebrafish Pld1 is on 1–314 amino acids region; RhoA binding site is on 859–1010 amino acids region). The C-terminal portion of Pld1 is highly similar to mammalian PLD1 with more variation occurring in the N-terminal portion and the loop region of the protein. A 20-amino acid insertion at the extreme N-terminal region of mammalian PLD1 is absent in Pld1 (Supplementary Fig. 1A). We subsequently performed biochemical assays to compare activity of zebrafish Pld1 with the human enzyme. Similar to hPLD1 zebrafish Pld1 exhibited a low basal activity when expressed in the HEK293 TREx cells and it was robustly stimulated by phorbol ester (PMA) which is widely used as an activator of PKC and PLD catalytic activities (Fig.1A). A mutation K898R in human PLD1 HKD domain has been shown to produce a catalytically inactive protein (Sung et al. 1997). Likewise an HKD-mutant construct harboring a corresponding mutation K846R in the zebrafish Pld1 lacked activity in this assay (Fig. 1A). To further characterize the biochemical regulation of pld1 gene product we next performed in vitro PLD activity assays testing whether its activation is regulated by PKC and small GTPases as reported for the mammalian enzymes (Brown et al.1993; Colley et al. 1997; Singer et al. 1996). These assays demonstrated that zebrafish Pld1 similar to mammalian PLD1 enzymes can be activated by both PKCα and Arf1 (Fig.1B). The combination of PKCα and Arf1 resulted in an additive activation of wild-type Pld1. In contrast both human K898R and zebrafish K846R mutants did not reveal additional activity beyond that exhibited by the endogenous PLD in the control HEK293 cells transformed with the empty vector (Fig. 1B). The western blot in Fig.1C indicates the relative amounts of wild-type GFP-Pld1/PLD1 and mutated GFP-Pld1/PLD1 that were used in the activity assays. Taken together these cell culture and in vitro data demonstrate that the zebrafish pld1 gene encodes a bona fide Pld1.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
HEK-293