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(Circulation Research. 2008;102:1057.)
© 2008 American Heart Association, Inc.

Molecular Medicine

An Acyltransferase Controls the Generation of Hematopoietic and Endothelial Lineages in Zebrafish

Jing-Wei Xiong^*, Qingming Yu^*, Jiaojiao Zhang, John D. Mably

From the Nephrology Division (J.-W.X., Q.Y., J.Z.) and Cardiovascular Research Center (J.-W.X., J.D.M.), Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass. Present address for J.D.M.: Cardiovascular Research, Children’s Hospital Boston, Mass.

Correspondence to Jing-Wei Xiong, The Nephrology Division, Massachusetts General Hospital-East, Harvard Medical School, 149 13th St, Room 8216, Charlestown, MA 02129. E-mail Xiong{at}cvrc.mgh.harvard.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hematopoietic and endothelial cells develop from a common progenitor, the hemangioblast, or directly from mesodermal cells. The molecular pathway that regulates the specification of both cell lineages remains elusive. Here, we show that a lysocardiolipin acyltransferase, lycat, is critical for the establishment of both hematopoietic and endothelial lineages. We isolated lycat from the deletion interval of cloche, a zebrafish mutant that has dramatically reduced hematopoietic and endothelial cell lineages. Reduction of lycat mRNA levels in wild-type zebrafish embryos decreases both endothelial and hematopoietic lineages. lycat mRNA rescues blood lineages in zebrafish cloche mutant embryos. E165R and G166L mutations in the highly conserved catalytic domain in Lycat abolish its function in zebrafish hematopoiesis. Epistasis analysis supports that lycat acts upstream of scl and etsrp in zebrafish hemangioblast development. These data indicate that lycat is the earliest known player in the generation of both endothelial and hematopoietic lineages.

Key Words: acyltransferase • hemangioblast • hematopoiesis • vasculogenesis • zebrafish • cloche

	Introduction

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It was proposed nearly a century ago that a common progenitor generates both the hematopoietic and endothelial lineages.¹ This hypothesis was based on the observation that blood and endothelial cells develop within close proximity of each other in the extraembryonic yolk sac. Using in vitro mouse and human embryonic stem cell (ESC) differentiation assay, a blast colony–forming cell was characterized that clonally generates both endothelial and hematopoietic cells in the presence of vascular endothelial growth factor and bone morphogenetic protein 4.^2–4 The blast colony–forming cell was later isolated in vivo from the posterior primitive streak of midgastrulation mouse embryos.⁵ A single-cell fate mapping in the zebrafish gastrula by uncaging fluorescein dextran suggests that hemangioblasts are interspersed with hematopoietic and endothelial progenitors in the ventral–lateral mesoderm.⁶ However, several studies in mice have shown that endothelial and hematopoietic lineages are independently derived from mesodermal cells.^7,8

The zebrafish cloche (clo) mutant has significantly reduced endothelial and hematopoietic lineages, as well as no endocardium.⁹ cloche is thought to act at the level of the hemangioblast and represents the first single gene mutation that almost eliminates both endothelial and hematopoietic lineages. Epistasis analyses have shown that cloche acts upstream of zbp-89 (a Krüppel-like zinc finger–containing transcription factor), scl (a basic helix–loop–helix transcription factor), lmo2 (a LIM-containing protein), gata1 (GATA binding protein 1), fli1 (an ETS domain–containing protein), flk1 (fetal liver kinase 1), and etsrp (a ETS1-related protein) in hematopoietic and endothelial cell development in zebrafish.^9–17 In addition, genome-wide microarray analyses have revealed that cloche specifically regulates a panel of hematopoietic and endothelial genes.^18–20 All of these early studies have proved that cloche is the earliest gene acting in hemangioblast development. However, it remains unknown what is the molecular nature of the cloche gene.

Here, we have cloned lycat as a candidate gene for the cloche locus in zebrafish. The mouse homolog of Lycat is strongly expressed in the heart and is enriched in the Flk1⁺/Scl^– and Flk1⁺/Scl⁺ hemangioblast populations in embryoid bodies.^21,22 We have previously reported that mouse Lycat gene plays essential roles in hemangioblast, hematopoietic, and endothelial lineage development using loss-of-function (siRNA knockdown) and gain-of-function (over- and ectopic expression) analyses in mouse ESC differentiation system.²¹ Both mouse and zebrafish lycat genes encode a transmembrane acyltransferase with a C-terminal endoplasmic reticulum localization signal. The mouse Lycat protein has acyltransferase enzymatic activities using lysocardiolipin as a substrate.²² Protein acyltransferase has emerged as an important player in regulating protein trafficking, sorting, and development.^23,24 The porcupine (Porc) gene in fly was isolated and characterized as an endoplasmic reticulum–localized acyltransferase that is required for addition of palmitate to Wnt3a and Wnt1 for producing fully functional Wnt proteins.^25,26 Skinny hedgehog (Ski) was found to be another acyltransferase that is required for palmitoylation and biological activity of the Hedgehog.²⁷ Therefore, some protein acyltransferases can be important signaling components regulating embryonic patterning and organogenesis. We therefore analyzed the zebrafish lycat gene function in hemangioblast, endothelial, and hematopoietic lineage development. Morpholino-mediated knockdown of zebrafish lycat results in the reduction of both endothelial and hematopoietic lineages and elimination of the endocardium, which is very similar to that in cloche. Microinjection of zebrafish lycat mRNA into cloche mutants partially rescues the cloche phenotype. Lycat acts upstream of several known hemangioblastic genes. Although lycat is deleted in the spontaneous cloche^m39 allele, we have not found causative mutations in the open reading frame of lycat from 2 ethylnitrosourea-induced cloche alleles. This study suggests that lycat is the earliest known player in hematopoietic and endothelial (including endocardial) cell development, probably acting on the level of hemangioblasts.

	Materials and Methods

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Isolating lycat From the cloche Genetic Interval and Zebrafish Strains
The cloche alleles clo^m39 and clo^m378 have been previously described.^9,28 A new ethylnitrosourea-induced allele, clo^fv087b, was also used, which was isolated in the laboratory of Mark C. Fishman in 2000. Transheterozygous zebrafish crosses from any 2 of the 3 cloche alleles produce 25% identical cloche mutant embryos, supporting that the 3 are allelic. Genetic mapping of cloche was carried out using hybrid F₁ from crosses clo^m39xTL, clo^m378xTL, and clo^fv087bxWIK. cloche was mapped on the telomere of chromosome 13, close to the microsatellite markers Z17223, Z22194, and Z10362, using bulked segregation analysis, and the lycat gene was cloned from the clo^fv087b genetic interval (Figure I in the online data supplement, available at http://circres.ahajournals.org). Transgenic lines Tg(flk1:GFP) and Tg(gata1:GFP)^29,30 (a gift from Dr Shuo Lin, University of California, Los Angeles) were crossed with heterozygous clo^fv087b/+ fish, and heterozygous Tg(flk1:GFP)/+; clo^fv087b/+ and Tg(gata1:GFP)/+; clo^fv087b/+ individuals were identified. Zebrafish were raised and handled in accordance with the Massachusetts General Hospital institutional guidelines.

Morpholinos and Capped mRNA Injections
Three antisense morpholinos were synthesized to target the zebrafish lycat ATG site (lycat-MO1): AACACACACCACGAGGAGACACCAT; the second splicing donor site (lycat-MO2): TAAGCTCTGCGTACCACAGGTAAG; the third splicing donor site (lycat-MO3): CTGAACACACACACTGACCGAAGC; and a control morpholino (Ctr-MO): GCAGCGGGCACTGCTGGTGGAAGT (Gene Tools, LLC). etsrp-MO (an untranslated region morpholino) (TTGGTACATTTCCATATCTTAAAGT) and scl-MO (an untranslated region morpholino) (GCTCGGATTTCAGTTTTTCCATCAT) were reported.¹⁶ Zebrafish lycat, mouse Lycat, and zebrafish scl and etsrp were cloned and linearized, and capped mRNA was synthesized using Ambion mMESSAGE mMACHINE mRNA transcription synthesis kits. Morpholinos or mRNA were injected into 1- or 2-cell-stage wild-type, clo^fv087b; Tg(flk1:GFP), or clo^fv087b; Tg(gata1:GFP) embryos. Injected embryos were incubated at 28.5°C for examination of phenotypes or were fixed for in situ hybridization. The pictures were taken using a Leica MZ16 fluorescence microscope.

RNA In Situ Hybridization and Histology
In situ hybridization and histology were done using antisense lycat, flk1, fli1, etsrp, scl, lmo2, and gata1 RNA probes.^11–14,16

Reverse Transcription–PCR
Zebrafish embryos were pooled from Ctr-MO; clo mutant embryos from clo^fv087, clo^m378, or clo^m39 heterozygous crosses; or lycat-MO3 morphant embryos at 18 or 30 hours postfertilization (hpf). Wild-type TL embryos were collected at different time points. Total RNA was isolated from embryos using TRIzol reagents (Invitrogen). First-strand cDNA was synthesized using the Superscript II RT system (Invitrogen). Semiquantitative PCR for zebrafish lycat, flk1, and β-actin were done with Qiagen Taq polymerases and quantitative PCR were done with TaqMan or SYBR Green probes using the 7000 Sequence Detection System (ABI Prism).

Site-Directed Mutagenesis and Rescue of gata1 Expression in Homozygous clo^m39 Mutant Embryos
The wild-type lycat cDNA was used for site-directed mutagenesis. Mutations were created using the GeneEditor in vitro Site-Directed Mutagenesis System (Promega) following the instructions of the manufacturer. The E165R-G166L mutation was generated by a substitution of glutamic acid (E) to arginine (R) at amino acid 165 and glycine (G) to leucine (L) at amino acid 166. The following primers were used for making E165R-G166L: 5'-TGCAGCTGCTGCTGTTCCCTcgGctCACCGACCTCA-3' (forward) and 5'-AGGGAACAGCAGCAGCTGCACCGGCTCTCT-3' (reverse). Capped mRNAs were synthesized from wild-type lycat cDNA and E165R-G166L cDNA templates using mMESSAGE mMACHINE SP6 and T7 Kits (Ambion). mRNA was injected into 1- or 2-cell-stage clo^m39 or clo^fv087b; Tg(gata1:GFP) embryos from heterozygous clo crosses. Injected embryos were incubated at 28.5°C and later fixed with 4% paraformaldehyde. gata1 expression in the injected embryos was examined by in situ hybridization. Embryos were subsequently genotyped by PCR with the marker Z1412 that is deleted in the clo^m39 allele or with marker D, a CA repeat marker for clo^fv087b allele (supplemental Figure I). Marker D is as follows: forward primer, 5'-TCTGATTGGCCACTGAGG-3'; reverse primer, 5'-GCGTGTGTGTAACCCGTCTA-3'. Marker Z1826 is outside of the deletion interval in the clo^m39 allele, which was used as DNA quality control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antisense lycat Morpholinos Reduce Endothelial and Hematopoietic Lineages
Embryos injected with any 1 of the 3 antisense morpholinos targeting the zebrafish lycat exon 2 splice donor site (lycat-MO2 or MO2), the exon 3 splice donor site (lycat-MO3 or MO3), and the translational start site (lycat-MO1 or MO1), respectively, significantly reduced blood cells and blood vessels and eliminated the endocardium (Figure 1, supplemental Figure II, and supplemental Table I). Embryos injected with the lycat-MO3 generated a mutant transcript retaining the fourth intron sequence, leading to out-of-frame translation after the amino acid G122 (Figure 1b, arrowhead MT; sequencing data not shown). Injection of lycat-MO3 morpholinos into the Tg(flk1:GFP) or the Tg(gata1:GFP) transgenic embryos dramatically reduced flk1- or gata1-expressing cells in the morphants, respectively (Figure 1h and 1k). Eighty-eight percent (132/150) of Tg(flk1:GFP) embryos microinjected with lycat-MO3 showed hematopoietic, endothelial, and endocardial defects (supplemental Table Ib). Coinjection of the zebrafish lycat-MO3 and mouse Lycat mRNA rescued the morpholino-induced reduction of flk1- and gata1-expressing cells in the injected embryos (Figure 1i and 1l; supplemental Table I), confirming that the morphant phenotype was caused by the knockdown of the zebrafish lycat gene. These data indicate that mouse Lycat is a functional ortholog of zebrafish lycat.

View larger version (92K): [in this window] [in a new window]   Figure 1. Antisense lycat morpholinos mimic the cloche mutant phenotype and lycat mRNA rescues morpholino-mediated knockdown. a, The lycat gene exons and introns organization including black boxes of exons 1 to 7 and solid lines of introns. 43F indicates a forward primer; 599R, a reverse primer; these were used to amplify a 557 bp of the wild-type lycat transcript in b. MO1 indicates a ATG morpholino; MO2, a splice morpholino targeting the exon 2 donor site; MO3, a splice morpholino targeting the exon 3 donor site. b, RT-PCR analysis of wild-type and mutant transcripts by 43F and 599R primers in Ctr-MO–injected (lane 4) and MO3-injected (lane 5) embryos at 20 hpf and lycat plasmid DNA as a positive (lane 2) and water as a negative (lane 3) PCR control. A 731-bp mutant transcript (arrowhead MT) (lane 5) is found, but the 557 bp wild-type transcript is not detected in MO3 morphant embryos. The 557-bp wild-type transcript is detected in Ctr-MO–injected embryos (lane 4) and lycat plasmid DNA control (lane 2) (arrowhead WT). Lane 1 indicates the 100-bp DNA ladder (New England Biolabs). c through f, Lycat MO3-injected (0.3 mmol/L) morphants at 36 hpf (e) and at 48 hpf (f) and 0.5 mmol/L Ctr-MO–injected embryo at 36 hpf (c) and at 48 hpf (d). g through i, Strong flk1:GFP signals in vessels of Ctr-MO–injected control embryo (g), very little flk1:GFP signals in 0.3 mmol/L MO3–injected embryo (h), and rescued flk1:GFP signals in the embryo coinjected with 0.3 mmol/L MO3 and 300 ng/µL lycat RNA (i) at 24 hpf. j through l, Strong gata1:GFP signal in the blood islands of Ctr-MO–injected control embryo (j), very little gata1:GFP signal in 0.3 mmol/L MO3–injected embryo (k), and rescued gata1:GFP signal in the embryo coinjected with 0.3 mmol/L MO3 and 300 ng/µL lycat RNA (l). m through p, The scl expression in the intermediate cell mass in control-injected embryo (m), decreased scl in embryos injected with 0.3 mmol/L MO3 (n), 0.4 mmol/L MO3 (o), and 0.5 mmol/L MO3 (p) at 20 hpf by RNA in situ analysis. q through t, The fli1 expression in vessels in uninjected control embryo (q), significantly decreased fli1 in the homozygous clom39 embryo (r), 0.5 mmol/L MO1–injected embryo (s), and 0.5 mmol/L MO2–injected embryo (t) at 20 hpf by RNA in situ analysis.

The zebrafish lycat-MO3 reduced the expression of scl in the intermediate cell mass in a dose-dependent manner (Figure 1m through 1p). Morphants injected with lycat-MO1 or MO1 (Figure 1s), lycat-MO2 or MO2 (Figure 1t), or lycat-MO3 or MO3 (not shown) showed significantly reduced fli1 expression in vessels. The fli1 expression in lycat-MO2 (Figure 1t) or lycat-MO3 morphants (not shown) is similar to that in homozygous clo embryo (Figure 1r), whereas very-low-level fli1 expression remained in trunk vessels in lycat-MO1 morphants (Figure 1s). Lycat-MO2 morphant embryos have similar fli1 expression as that in cloche mutant embryos; therefore, the expression level of fli1 in Figure 1t represents experimental variation. The lycat-MO3 is the most potent one among the three antisense lycat morpholinos tested; therefore, lycat-MO3 is used for subsequent experiments. In summary, morpholino-mediated lycat knockdown results in a similar phenotype as that in cloche, suggesting that lycat is essential for hemangioblast, endothelial, and hematopoietic lineage development and that flk1, scl, gata1, and fli1 function downstream of lycat.

Zebrafish and Mouse Lycat but Not an Enzyme-Defective Lycat Partially Rescues Hematopoiesis in cloche Mutant Embryos
We found that both zebrafish and mouse Lycat mRNA partially rescued cloche embryos (Figure 2). In cloche mutant embryos from clo^fv087b/+; Tg(gata1:GFP)^tg/tg sibling crosses, the gata1:GFP expression was remarkably increased after injection with lycat mRNA (Figure 2c and 2d). Although the rescued cloche embryos lacked an intact circulation, they had significant number of blood cells, whereas uninjected cloche mutants had very few blood cells (data not shown). Using Tg(gata1:GFP) as a marker, the lycat mRNA injection was able to rescue 38.5% (35/91) of cloche mutant embryos (supplemental Table I), genotyped with marker D (supplemental Figure Ia and data not shown). We failed to rescue the flk1⁺ and etsrp⁺ endothelial cells in cloche embryos injected with lycat mRNA (not shown). These data are not entirely supportive of lycat as the cloche gene, although ectopic mRNA expression of a developmentally regulated gene does not always rescue its genetic mutant phenotype in zebrafish.

View larger version (39K): [in this window] [in a new window]   Figure 2. Zebrafish lycat and mouse Lycat RNA could partially rescue hematopoietic cells in cloche. Strong gata1:GFP expression in the blood island (arrow) of WT (a), very little gata1:GFP in clo mutant injected with the zebrafish antisense lycat RNA (arrow) (b), and rescued gata1:GFP in clo mutant with zebrafish sense lycat RNA (c) and with mouse sense Lycat RNA (d) at 20 hpf. There is neuronal gata1-gfp expression (arrowhead) that is unrelated to clo function. e through j, One-cell embryos from heterozygous clom39 crosses are injected with wild-type lycat (lycat) or catalytic mutant (E165R-G166L) RNA. Staged embryos are fixed and subjected to RNA in situ analysis with antise- nse gata1 probes. Control wild-type (uninjected) embryos have strong gata1 expression in the intermediate cell mass (e and h), and cloche mutant embryos (f, g, i, and j) are partially rescued by wild-type lycat RNA (g and j) but not by mutant E165R-G166L lycat RNA (f and i).

Recent studies have shown that acyltransferase from bacteria to mammals contains highly conserved catalytic motifs including H(X)₄D and EGTR.²² The zebrafish Lycat protein contains both H(RTRL)D (amino acids 85 to 90) and EGTD (amino acids 165 to 168) motifs (supplemental Figure Ib, shadowed). We examined whether the EGTR motif in Lycat is essential for hematopoiesis in zebrafish. We generated a mutant Lycat with substitutions of both E165R and G166L (named E165R-G166L) by site-directed mutagenesis. We found that the E165R-G166L mutant lycat mRNA rescued hematopoietic gata1 expression only in 2% (2/89) of homozygous clo^m39 embryos, whereas the wild-type lycat mRNA rescued 47% (31/66) of cloche embryos (Figure 2g and 2j). Gata1-expressing homozygous cloche embryos were confirmed by genotyping with marker Z1412 deleted in the clo^m39 allele and a control marker Z1826 outside of the clo^m39 deletion interval (not shown). These data establish that the catalytic activity of the Lycat protein is required for its function in hematopoietic development in zebrafish.

Zebrafish lycat Acts Upstream of scl and etsrp to Specify the Hemangioblast
To determine the role of lycat gene in zebrafish hemangioblast development, we examined the interactions of lycat with scl and etsrp in the lateral plate mesoderm (LPM) in early-somitogenesis-stage embryos. The flk1, etsrp, scl, lmo2, and gata1 genes were not expressed or significantly reduced in both anterior and posterior LPM in lycat-MO3 morphants (87% [47/54] have reduced flk1; 73% [38/52] have reduced etsrp; 96% [45/47] have reduced scl; 80% [32/40] have significantly reduced lmo2; and 95% [37/39] have reduced gata1; Figures 3b, 3l, 4b, 4g, and 4k). fli1 was not expressed in the anterior LPM in lycat-MO3 morphant embryo (49% [18/37]) (Figure 4h). The expression profile of these genes in lycat-MO3 morphants is very similar to that in homozygous clo embryos (data not shown). scl mRNA rescued flk1 (66%, 40/61), fli1 (88%, 44/50), etsrp (100%, 62/62), lmo2 (59%, 26/44), and gata1 (95%, 40/42) expression in lycat-MO3 morphants (Figures 3c, 3i, 3m, 4 h, and 4l). In addition, overexpression of scl mRNA in lycat-MO3 morphants led to ectopic fli1 and lmo2 expression (Figures 3i and 4h) and increased the more lateral stripe of flk1- and etsrp-expression domains (Figure 3c and 3m, arrowheads) and expanded gata1-expression domain (Figure 4l). Similarly, overexpression of etsrp mRNA in MO3 morphants also rescued flk1 (69%, 24/35), fli1 (80%, 28/35), scl (78%, 25/32), lmo2 (53%, 16/30), and gata1 (82%, 23/28) expression, as well as led to ectopic flk1, fli1, scl, lmo2, gata1 expression (Figures 3d, 3j, 4c, 4i, and 4m). Therefore, lycat acts upstream of both scl and etsrp and is essential for fli1, flk1, etsrp, scl, lmo2, and gata1 expression during development of hemangioblasts, endothelial, and hematopoietic cells.

View larger version (145K): [in this window] [in a new window]   Figure 3. Zebrafish lycat acts upstream to scl and etsrp in hemangioblast and angioblast development. The flk1, fli1, and etsrp were detected by RNA in situ hybridization with embryos at 8-somite stage. All embryos have flat-mounted dorsal view, with the anterior on the top. a through f, flk1 is expressed in the anterior LPM (alpm) and posterior LPM (plpm) of Ctr-MO embryo (a). The flk1 expression in the alpm and plpm is reduced in 0.3 mmol/L MO3–injected embryo (b). The lack of flk1 expression in MO3 morphant embryo (b) could be rescued by coinjection of 0.3 mmol/L MO3 and scl RNA (0.1 mg/mL) (c), as well as rescued by coinjection of 0.3 mmol/L MO3 and etsrp RNA (0.1 mg/mL) (d). In addition, coinjection of MO3 and scl RNA increases flk1 expression in the more lateral flk1-expressing stripe in which flk1 is normally very weak (c, arrowhead). The flk1 expression in the anterior and posterior LPM is eliminated or significantly reduced in 1.0 mmol/L etsrp-MO–injected embryo (e) and could not be rescued by coinjection of 1.0 mmol/L etsrp-MO and lycat RNA (0.3 mg/mL) (f). g through j, fli1 is expressed in the anterior LPM and the posterior LPM in Ctr-MO embryo (g). The anterior LPM is dramatically reduced in 0.3 mmol/L MO3–injected embryo (h). The MO3 morphant phenotype could be rescued by coinjection of 0.3 mmol/L MO3 and scl RNA (0.1 mg/mL) (i) or of 0.3 mmol/L MO3 and etsrp RNA (0.1 mg/mL) (j). In addition, overexpression of either scl or etsrp could generate ectopic fli1 expression (arrowheads) in MO3 morphant embryos (i and j). However, the lack of fli1 in etsrp-MO morphants could not be rescued by coinjection of 1.0 mmol/L etsrp-MO and lycat RNA (0.3 mg/mL) (not shown). k through m, etsrp expression in the anterior and posterior LPM in Ctr-MO embryo (k) is dramatically reduced, with a few etsrp-expressing cells in the alpm in 0.3 mmol/L MO3–injected embryo (l). The MO3 morphant phenotype could be rescued by coinjection of 0.3 mmol/L MO3 and scl RNA (0.1 mg/mL) (m). In addition, coinjection of MO3 and scl RNA increases etsrp expression in the more lateral stripe (m, arrowhead).

View larger version (63K): [in this window] [in a new window]   Figure 4. Zebrafish lycat acts upstream to scl and etsrp in hemangioblast and hematopoietic development. The scl, lmo2, and gata1 expression was detected by RNA in situ hybridization with embryos at 8-somite stage. a through i, Flat-mounted dorsal view, with the anterior on the top. j through o, The posterior view, with the anterior on the top. a through e, scl is expressed in the anterior LPM and posterior LPM of Ctr-MO embryo (a) and has no or very little scl in 0.3 mmol/L MO3–injected embryo (b). The lack of scl expression in MO3 morphant embryo (b) could be rescued by coinjection of 0.3 mmol/L MO3 and etsrp RNA (0.1 mg/mL) (c). The scl expression in the anterior part (arrowheads) of the posterior LPM is dramatically reduced in 1.0 mmol/L etsrp-MO–injected embryo (d, arrowheads) and could not be rescued by coinjection of 1.0 mmol/L etsrp-MO and lycat RNA (0.3 mg/mL) (e). f through i, lmo2 expression in the anterior and posterior LPM in Ctr-MO embryo (f) is reduced Figure 4 (Continued). in 0.3 mmol/L MO3–injected embryo (g). The MO3 morphant phenotype could be rescued by coinje- ction of 0.3 mmol/L MO3 and scl RNA (0.1 mg/mL) (h) or of 0.3 mmol/L MO3 and etsrp RNA (0.1 mg/mL) (i). In addition, overexpression of either scl or etsrp could generate ectopic lmo2 expression in MO3 morphant embryos (h and i, arrowheads). j through o, gata1-expressing LPM in Ctr-MO embryo (j) is dramatically reduced in 0.3 mmol/L MO3–injected embryo (k). The MO3 morphant phenotype could be fully rescued by coinjection of 0.3 mmol/L MO3 and scl RNA (0.1 mg/mL) (l), as well as partially rescued with ectopic gata1 expression by coinjection of 0.3 mmol/L MO3 and etsrp RNA (0.1 mg/mL) (m). The gata1 expression is reduced in 1.0 mmol/L scl-MO–injected embryo (n) and could not be fully rescued by coinjection of 1.0 mmol/L scl-MO and lycat RNA (0.3 mg/mL) (o).

To further examine the relationship between lycat and etsrp, etsrp morpholinos were used to knockdown etsrp function in early somitogenesis embryos.^16,17 In etsrp morphant (etsrp-mo) embryos, the flk1 expression was significantly reduced (93%, 28/30) in both anterior and posterior LPM (Figure 3e), fli1 was reduced in the anterior LPM (not shown), scl was reduced (79%, 45/57) in the anterior part of the posterior LPM (Figure 4d, arrowheads), and lmo2 and gata1 expressions were not affected (not shown). The etsrp morphant phenotypes could not be rescued by overexpression of lycat mRNA (Figures 3f and 4e) (100% [28/28] have reduced flk1 expression; 77% [20/26] have reduced scl expression). Together with the rescue of lycat-MO3 morphant phenotypes by etsrp mRNA (Figures 3d, 3j, 4c, 4i, and 4m), our data support that lycat acts upstream of etsrp in hemangioblast development and etsrp is critical for flk1-expressing angioblastic lineage and plays a role in generation of gata1- and lmo2-expressing hematopoietic lineages.

Antisense morpholino knockdown of scl was also applied to examine the scl and lycat relationship in hemangioblast development. scl morphant showed reduced gata1 expression (44%, 32/72) (Figure 4n) compared with that of control embryo (Figure 4j). This scl morphant phenotype could not be rescued by coexpression of lycat RNA (60%, 41/68) (Figure 4o). We have also carried out knockdown of both scl isoforms (scl and sclβ) as described³¹ and have found that lycat RNA could not rescue gata1 expression in scl and sclβ morphant embryos (data not shown). The lmo2 expression was neither affected in embryos injected with scl morpholinos nor embryos injected with both scl morpholino and lycat RNA (not shown). Therefore, scl acts downstream of lycat and is essential for the derivation of gata1-expressing hematopoietic lineages.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have isolated and characterized lycat as an important hemangioblastic gene and a cloche candidate gene in zebrafish. Lycat is among the acyltransferase family members that have 3 putative transmembrane domains and a highly conserved acyltransferase domain (supplemental Figure Ib). Our loss-of-function (morpholino knockdown) and gain-of-function (ectopic RNA expression) analyses have firmly demonstrated the roles of lycat in hemangioblast, endothelial (including the endocardium), and hematopoietic lineage development. This observation is further supported by our previous report on mouse Lycat function in these lineages during ESC differentiation.²¹ Mouse Lycat, or Alcat1, is also characterized as a lysocardiolipin acyltransferase that plays an important role in the cardiolipin remodeling pathway.²² However, our functional analyses of Lycat in zebrafish and ESC systems strongly suggest that Lycat may also act as a protein acyltransferase that regulates important hemangioblastic proteins. Identifying lycat is a highly significant finding and will lead to novel molecular insights into the origin and formation of hemangioblasts. Second, lycat is within the cloche genetic interval, and the lycat morphant phenotype is remarkably similar to that of the cloche mutant. Therefore, lycat remains as a strong candidate for the cloche locus. Although this report has not provided direct evidence for this, identification of lycat and many close genetic markers related to the cloche locus certainly moves the field a step closer to ultimately revealing the molecular nature of cloche.

Genetic studies in Drosophila and Caenorhabditis elegans have identified acyltransferases that are required for the generation of active morphogens, Wingless, and Hedgehog.^26,27 Hedgehogs and Wnts are involved in hematopoiesis and vasculogenesis,^26,32,33 as well as many other important functions. Therefore, endoplasmic reticulum–associated, transmembrane acyltransferases are important for regulating embryonic patterning and organogenesis in different species. Zebrafish Lycat is predicted as an endoplasmic reticulum–localized transmembrane acyltransferase. Mouse Lycat has acyltransferase activity for Cardiolipin and may modify additional proteins.²² We have shown that mouse Lycat is enriched in the Flk1⁺/Scl^– hemangioblasts and plays an important role in hematopoietic and endothelial cell development during ESC differentiation in vitro.²¹ It is possible that Lycat could act as a protein acyltransferase modifying 1 or several signaling components that control hematopoietic and endothelial specification in the early embryo. The identification of Lycat targets will reveal the mechanism of Lycat in this process. There is no consensus sequence for an acyltransferase target domain making it challenge to identify potential Lycat targets.³⁴ Future studies are required to determine whether zebrafish Lycat directly modifies important known molecules, such as the Hedgehogs, Wnts, Etsrp, Scl, Runx1, and vascular endothelial growth factor pathway components.^16,17,26,33 Another area of interest is to discover novel Lycat targets that influence hemangioblast development by using an unbiased genome-wide proteomics approach.³⁵

The zebrafish clo mutant has significantly reduced hematopoietic and endothelial lineages.⁹ Gain-of-function analysis in zebrafish suggests that scl, lmo2, and etsrp are involved in specifying the hemangioblast from early lateral posterior mesoderm.^{11,14,16,17,36,37} However, morpholino knockdown or genetic mutant analyses of these genes have revealed that none of them is absolutely required for the formation of both endothelial and hematopoietic lineages in zebrafish embryos. Our data support that lycat is required for flk1-, etsrp-, fli1-, gata1-, scl-, and lmo2-expressing cell formation (Figures 1, 3, and 4). To our knowledge, lycat is the first hemangioblastic gene required for both endothelial and hematopoietic lineages in zebrafish embryos. Both scl and etsrp mRNA rescue lycat morphant phenotype in endothelial and hematopoietic development (Figures 3 and 4), whereas lycat mRNA fails to rescue etsrp or scl morphant phenotypes (Figures 3f, 4e, and 4o), supporting the notion that lycat is upstream of both scl and etsrp. Our studies and others also support that etsrp, downstream to lycat, is essential for flk1-expressing endothelial and gata1-expressing hematopoietic lineages, whereas scl is essential for generation of the gata1-expressing hematopoietic lineages and plays minor roles in flk1-expressing endothelial lineages (Figures 3 and 4 and data not shown).¹⁷

The zebrafish lycat zygotic expression pattern was unclear by RNA in situ hybridization probably because of very low levels of expression (data not shown). Zebrafish lycat mRNA was detected in embryos from 1-cell-stage to 72 hpf by RT-PCR (data not shown). Using RNA in situ analysis with antisense lycat probes, we did find transgenic lycat mRNA expression in Tg(flk1:lycat) embryos, in which lycat is driven by the zebrafish flk1 promoter (data not shown), supporting that the antisense lycat probes that we used were good and that the uncertainness in detecting endogenous lycat mRNA may be attributable to its low level of expression (data not shown). Future studies need to fill this gap as well. We fully anticipate that deciphering Lycat targeting proteins and its expression pattern presumably in hemangioblast domains will lead to both molecular insights in hemangioblast development and therapeutic utility in regenerative medicine.

	Acknowledgments

 
J.-W.X. acknowledges advice, encouragement, and enthusiastic support from Dr Mark C. Fishman. The zebrafish lycat cDNA was isolated by J.-W.X. in the laboratory of Dr Fishman at the Cardiovascular Research Center, Massachusetts General Hospital. We acknowledge Drs Shuo Lin, Randy Peterson, and M. Amin Arnaout, as well as members of the Massachusetts General Hospital zebrafish laboratories, for their comments on the manuscript and valuable suggestions during this study.

Sources of Funding

This project was supported by NIH grant K01-AG19676, the Harvard Stem Cell Institute, March of Dimes Birth Defects Foundation, and Massachusetts General Hospital Nephrology Division funding (to J.-W.X.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

Original received September 10, 2007; revision received March 14, 2008; accepted March 21, 2008.

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