Developmental Biology 289 (2006) 482 – 493 www.elsevier.com/locate/ydbio Genomes & Developmental Control Fog1 is required for cardiac looping in zebrafish

R. Zaak Walton c, Ashley E.E. Bruce a, Harold E. Olivey c, Khalid Najib c, Vanitha Johnson c, Judy U. Earley c, Robert K. Ho b, Eric C. Svensson c,*

a Department of Zoology, University of Toronto, Chicago, IL 60637, USA b Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA c Department of Medicine, Section of Cardiology, University of Chicago, 5841 S. Maryland Ave, MC6088, Chicago, IL 60637, USA

Received for publication 23 September 2005, revised 14 October 2005, accepted 16 October 2005 Available online 28 November 2005

Abstract

To further our understanding of FOG function during cardiac development, we utilized zebrafish to examine FOG’s role in the early steps of heart morphogenesis. We identified fragments of three fog in the zebrafish genomic database and isolated full-length coding sequences for each of these genes by using a combination of RT-PCR and 5V-RACE. One gene was similar to murine FOG-1 (fog1), while the remaining two were similar to murine FOG-2 (fog2a and fog2b). All Fog were able to physically interact with GATA4 and function as transcriptional co- repressors. Whole-mount in situ hybridization revealed fog1 expression in the heart, the hematopoietic system, and the brain, while fog2a and fog2b expression was restricted to the brain. Injection of zebrafish embryos with a morpholino directed against fog1 resulted in embryos with a large pericardial effusion and an unlooped heart tube. This looping defect could be rescued by co-injection of mRNA encoding murine FOG-1, but not by mRNA encoding FOG-1 lacking the FOG repression motif. Taken together, these results demonstrate the importance of FOG proteins for zebrafish cardiac development and suggest a previously unappreciated role for FOG proteins in heart looping that is dependent on the FOG repression motif. D 2005 Elsevier Inc. All rights reserved.

Keywords: Transcription; Repression; GATA; FOG; Zfpm2; Zfpm1; Heart; Cardiogenesis; Development; Danio rerio

Introduction result in GATA factors with intact DNA binding but impaired binding to their co-factors, the friend of GATA (FOG) proteins The GATA family of transcriptional activators contains 6 (Mehaffey et al., 2001; Nesbit et al., 2004; Nichols et al., members, three of which are expressed predominantly in cells 2000). These observations highlight the importance of FOG- of the hematopoietic system (GATA1, 2, and 3) and three that GATA interactions for the development of specific organ are expressed predominantly in heart, gut, and lung (GATA4, 5, systems and suggest that FOG proteins themselves may be and 6) (Crispino, 2005; Heicklen-Klein et al., 2005; Molkentin, important in human disease. 2000; Pikkarainen et al., 2004; Sorrentino et al., 2005). There GATA factors are also known to be important for cardiac are several reports of mutations within these genes leading to development. Cardiac development is a tightly regulated human disease. Mutations in GATA1, for example, lead to process requiring the combinatorial interaction of multiple familial dyserythropoietic anemia and thrombocytopenia transcription factors in a temporally and spatially restricted (Mehaffey et al., 2001; Nichols et al., 2000), while mutations fashion (Brand, 2003; Bruneau, 2002; Cripps and Olson, 2002; in GATA3 lead to HDR (hypoparathyroidism, sensorineural Fishman and Chien, 1997). Work in flies, fish, and mice has deafness, renal anomaly) syndrome (Nesbit et al., 2004; Van demonstrated that GATA factors are required for proper heart Esch et al., 2000). Some of the reported mutations result in a formation and can physically interact with many other GATA that is unable to bind DNA, but other mutations transcriptional regulators of cardiac development including Nkx2.5, SRF, MEF2, NFATc, Tbx5, and FOG proteins (Belaguli et al., 2000; Durocher et al., 1997; Garg et al., * Corresponding author. Fax: +1 773 702 2681. 2003; Molkentin et al., 1998; Morin et al., 2000; Svensson et E-mail address: [email protected] (E.C. Svensson). al., 1999; Tevosian et al., 1999). This work has provided the

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.10.040 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493 483 foundation for understanding the molecular basis of human FOG-1 but not by a mutant version of FOG-1 lacking another congenital heart disease. As with GATA1 and GATA3, conserved domain, the FOG repression motif, demonstrating mutations in GATA4 have been described in humans (Garg the importance of this domain for Fog function in vivo. et al., 2003). These mutations disrupt GATA4’s ability to bind to DNA and interact with the Tbx5. Patients Materials and methods with such mutations have defects in cardiac morphogenesis characterized predominantly by atrial septal defects. Zebrafish strains and care Like the GATA family, members of the FOG family of transcriptional co-factors are expressed in the heart during The zebrafish strain used in this work was *AB. Zebrafish were cared for in accordance with the policies of the animal resources center at the University of development and are required for proper heart formation across Chicago. Zebrafish embryos were staged as described (Kimmel et al., 1995). species from fruit flies to mice (Cantor and Orkin, 2005). All FOG proteins are multi-type zinc finger proteins that are Isolation of fog cDNAs characterized by their ability to bind to GATA factors and co- activate or co-repress transcription of target genes (Holmes et Partial sequences encoding the C-terminus of three fog genes in zebrafish al., 1999; Lu et al., 1999; Svensson et al., 1999; Tevosian et al., were obtained by a BLAST search of the zebrafish genomic database using 1999; Tsang et al., 1997). In mice and humans, there are two the murine FOG-1 and FOG-2 protein sequences. To isolate the 5V end of each fog cDNA, we first isolated total RNA from 96 h post-fertilization (hpf) FOG genes, FOG-1 and FOG-2. Mice with a targeted zebrafish embryos using Trizol (Invitrogen, Carlsbad, CA). Next, rapid disruption of FOG-1 die at embryonic day (ED) 11.5 of severe amplification of cDNA ends (RACE) was performed using a commercially anemia secondary to a block in the differentiation of available kit (CLONTECH, Palo Alto, CA) to isolate cDNA from the 5V end erythrocytes, making it difficult to ascertain the role of FOG- of each fog gene. RACE products were subcloned into pGEM-T Easy 1 in cardiovascular development (Tsang et al., 1998). However, (Promega, Madison, WI) and ten RACE clones per fog gene were sequenced to identify the longest transcripts. The full-length coding sequence for each of an endothelial lineage-specific disruption of the FOG-1 gene these transcripts was deposited into Genbank with accession numbers results in mice that die at ED 14.5 of congenital heart defects DQ015975 for fog1, DQ015976 for fog2a, and DQ015977 for fog2b. that include double outlet right ventricle, a common AV valve, Expression vectors for each of the fog cDNAs were constructed by using the and ventricular and atrial septal defects, demonstrating the RT-PCR and primers (fog1,5V-CGGGATCCTCAGGGTTCTCGTG- importance of FOG-1 for cardiac development (Katz et al., TTTACTGTGG and 5V-CGGAATTCCTAAACTGTGGGAATAGGTCAGCG; fog2a,5V-CGGGATCCAGATACAGATACACACACGCGTGC and 5V- 2003). Mice with a targeted disruption in the FOG-2 gene also CCGCTCGAGAAGTGCTCCAGTAGCTAATGTCGG; fog2b,5V- CGGAATT- die in mid-gestation of congenital heart malformations that CAGTTGTGCTGCTGCAGCTCTACGG and 5V-ATTTGCGGCCGCTG- include defects similar to those seen in the FOG-1 endothelial- CAGTTGTCAAGGGTGGACAACG) to amplify the entire coding region of specific disruption (double outlet right ventricle, a common AV each fog gene. These fragments were then inserted into the BamHI/EcoRI, valve, ventricular, and atrial septal defects) as well as left BamHI/XhoI, or EcoRI/NotI sites of pcDNA3, respectively. All constructs were verified by sequencing. ventricular wall hypoplasia, and the failure to form coronary arteries (Svensson et al., 2000b; Tevosian et al., 2000). In vitro binding reactions Together, these results suggest the importance of both FOG-1 and FOG-2 for murine cardiac development. The BL21 strain of E. coli was transformed with an expression Although mutations in the FOG genes might be predicted to construct encoding glutathione-S-transferase (GST) fused to the zinc fingers cause congenital heart disease, there is only one report to date of murine GATA4 as described previously (Svensson et al., 1999). Fusion of congenital heart disease associated with sequence variations protein was purified using glutathione sepharose beads and incubated with in vitro translated, 35S-labeled Fog proteins in binding buffer (150 mM in human FOG genes (Pizzuti et al., 2003). Interpretation of NaCl, 50 mM Tris, pH7.5, 0.1% NP-40, 1 mM hME, 10 mM ZnSO4, this work is hampered in part by incomplete knowledge of the 0.25% BSA) as described previously (Svensson et al., 1999). Resultant important functional domains of FOG proteins. Such function- complexes were washed extensively, resolved by SDS-PAGE, and visualized ally important domains should be evolutionally conserved in by autoradiography. species as diverse as mice and zebrafish. To identify such domains, and to provide a better foundation for understanding Transfections the molecular basis of congenital heart disease, we sought to NIH 3T3 fibroblasts were transfected using Superfect reagent (Qiagen, identify and characterize fog genes in the Danio rerio genome. Valencia, CA) following the manufacturer’s protocol. Briefly, 1.5 Â 105 cells One of these genes, fog1, is similar to murine FOG-1 and were plated onto 12-well plates 18 h prior to transfection. On the day of expressed in the developing heart, intermediate cell mass transfection, 1.5 Ag of plasmid DNA was incubated with 3 Ag of Superfect (ICM), and nervous system of zebrafish embryos. The other reagent in 75 Al of OptiMEM for 10 min, and then 400 Al growth media was added, and the entire mixture was applied to the washed cells for 3 h at two genes, fog2a and fog2b, are similar to murine FOG-2, and 37-C, 5% CO2. Following this incubation, cells were washed with PBS, and both are expressed in the developing brain. All zebrafish Fog 1 ml of growth media was applied. Forty-eight hours after transfection, cells proteins can bind to GATA4 via highly conserved zinc finger and media were harvested. Cell lysate was assayed for protein concentration motifs and repress GATA-mediated transactivation of a cardiac- (BioRad, Hercules, CA) and for h-galactosidase activity (Promega) using h restricted promoter. Zebrafish depleted of Fog1 using an commercially available reagents. Corrected -galactosidase activity was calculated by dividing the measured h-galactosidase activity by the protein antisense morpholino develop congenital heart disease, with concentration of the lysate. Human growth hormone concentration in the cell the developing heart tube failing to undergo cardiac looping. media was determined using an ELISA (Roche, Indianapolis, IN). Relative This phenotype could be rescued by overexpression of murine promoter activity was calculated by dividing the growth hormone concen- 484 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493

tration by the corrected h-galactosidase activity. In each experiment, truncation of FOG-1 (pcDNA FOG-1231 – 995) using a commercially available transfections were performed in triplicate. Each experiment was repeated kit (Message Machine, Ambion, Austin, TX) (Svensson et al., 2000a). This 4 times. RNA was diluted to 80 ng/Al in a solution containing 0.5 mM fog1 morpholino, 0.2 M KCl, and 0.2 mg/ml Fast Green and used to inject one-cell embryos as In situ hybridization above.

Fragments of fog1 (925–2602 bp), fog2a (2976–3708 bp), fog2b (2443– Histology 3089 bp), or pitx2 (452–1471 bp) were amplified by the PCR and inserted into pGEM-T Easy (Promega). Cmlc2 in pGEM-T was obtained from Dr. Deborah Zebrafish embryos were dehydrated in a graded ethanol series and Yelon. In situ probes for tbx5 and tbx20 have been described previously (Ahn embedded in Durcupan resin (Sigma-Aldrich, St. Louis, MO). Five-micrometer et al., 2000, 2002). Antisense digoxigenin-labeled riboprobes were prepared sections were obtained using a Reichert-Jung ultramicrotome and stained with using these constructs and a commercially available kit (Promega) supple- 0.27% Basic Fuchsin. Sections were photographed using a Zeiss Axiophot mented with digoxigenin-UTP (Roche). Whole-mount in situ hybridizations microscope. were performed as follows. Zebrafish embryos were grown in the presence of 0.003% 1-phenyl-2-thiourea (PTU) for 24 to 72 hpf to reduce pigment Results formation, fixed in 4% paraformaldehyde at 4-C for 24 to 48 h, washed with phosphate-buffered saline (PBS) and PBST (PBS + 0.1% Tween-20) and then Isolation of fog cDNAs from zebrafish dehydrated in a graded methanol series. Embryos were then re-hydrated in PBST and digested with 10 Ag/ml of proteinase K at room temperature for 3 to 40 min, depending on the age of the embryo. Following digestion, embryos In both the mouse FOG-1 and FOG-2 genes, the final exon were re-fixed in 4% paraformaldehyde for 30 min at room temperature and then is large (>2 kb) and encodes a majority of the protein. This washed four times with PBST. Embryos were then pre-hybridized in Hyb mix same genomic structure is also conserved in the human and (5 Â SSC, 65% formamide, 50 Ag/ml heparin, 0.1% Tween-20, 0.5 mg/ml chicken FOG genes (E.C.S., unpublished observations). We tRNA) for 3 h at 70-C followed by hybridization with the digoxigenin-labeled riboprobe for 16 h at 70-C. Subsequently, embryos were washed with a graded reasoned that this might also be true in the zebrafish genome. series of Hyb mix/2 Â SSC buffers followed by two 30-min washes in 0.05 Â As an initial step in identifying FOG genes in zebrafish, we SSC, all at 70-C. Embryos were then washed with a graded series of 0.05 Â performed a translated BLAST search of the zebrafish genome SSC/PBST buffers at room temperature and then transferred to incubation (version 5, Sanger Institute) using the murine FOG-2 amino buffer (PBST + 2% sheep serum + 2 mg/ml BSA) for 1 h at room temperature. acid sequence. We identified 3 loci located on An alkaline phosphatase conjugated sheep anti-digoxigenin Fab fragment (Roche) was added (1:5000 dilution) and incubated for 16 h at 4-C. Following 16, 18, and 19 that had a large open reading frame that encoded this incubation, embryos were washed 10 times with PBST for 15 min each at a protein that was homologous to the C-terminus of FOG-2 and room temperature and then 3 times for 5 min each with AP buffer (100 mM Tris appeared to be the final exon of three distinct zebrafish fog pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20). Next, nitroblue genes. To isolate the 5V end of the corresponding cDNA for tetrazolium (NBT) was added to a final concentration of 0.44 mg/ml and 5- each of these genes, we used 5V rapid amplification of cDNA bromo, 4-chloro, 3-indolylphosphate (BCIP) was added to a final concentration of 0.165 mg/ml and embryos incubated at room temperature until staining was ends (5V-RACE). This allowed us to identify a complete open observed. Color development was stopped by serial washes with PBST and the reading frame and primary amino acid sequence by conceptual addition of 5 mM EDTA. Embryos were cleared with an overnight incubation translation for each of the fog genes. A comparison of the in glycerol and photographed using a Nikon Eclipse E600 microscope. amino acid sequence encoded by each of the zebrafish fog genes to the murine FOG-1 and FOG-2 sequences revealed that In vitro translations the gene located on 18 was most similar to murine FOG-1 and thus designated fog1. The predicted One microgram of pcDNAfog1, pcDNAfog2a, or pcDNAfog2b was in vitro transcribed and translated in the presence of [35S]methionine using a primary amino acid sequence revealed a polypeptide of 1191 commercially available kit (Promega) in the absence or presence of 20 ng/ amino acids that was 42% identical to murine FOG-1 and Al morpholino. Reactions were subsequently resolved by SDS-PAGE and contained 10 conserved zinc finger motifs (blue shading, Fig. subjected to autoradiography. 1). While murine FOG-1 has only 9 zinc fingers, a previously identified partial cDNA for a FOG protein from Xenopus (most Morpholino injections likely a FOG-1 orthologue) was also found to have 10 zinc fingers and shared 38% amino acid identity with zebrafish fog1 Morpholinos specific to the 5V untranslated region of fog1 (5V-GATCC- GTCCTCCGAGGCGACTAGCA), fog2a (5V-AATGAGAGGTTATTATGGAT- (Deconinck et al., 2000). In addition, the previously identified CATCC), fog2b (5V-TTCATCCAAACACAGATAAACGCTC) and a 5-base C-terminal binding protein (CtBP) interaction motif and mismatch control (5V-TTAATCGAAAGACAGATTAACGGTC) were synthe- nuclear localization signal are conserved between mouse, frog, sized by Gene Tools, LLC (Philomath, OR). Morpholinos were also designed to and zebrafish fog1 genes (orange and green shading, Fig. 1). target the exon 4/intron 4 splice donor site of fog1 (CCTTCATGTCCCCCT- Finally, the recently described N-terminal FOG repression TACCTCACTG) or fog2a (TTAAACGAGATGGACTGTACCTGTG) or the exon 5/intron 5 splice donor site of fog2b (AGACAAAGCAATCTCACCTT- motif was also conserved in zebrafish fog1 (red shading, Fig.

TACTG). Morpholinos were resuspended in dH2O at a concentration of 3 mM 1), suggesting that fog1 may function as a transcriptional co- and stored at À20-C. Immediately prior to use, the morpholinos were diluted to repressor (Lin et al., 2004). 0.1 mM to 0.5 mM in 0.2 M KCl and 0.2 mg/ml Fast Green as described The gene on was most similar to FOG-2 previously (Oates and Ho, 2002). One-cell embryos were injected with and named fog2a, while the third fog gene located on approximately 3.8 nl of this solution (0.4–1.9 pmol morpholino) into the streaming yolk, and successful injections were confirmed by observation of chromosome 19 was more similar to FOG-2 than FOG-1 and Fast Green in the yolk. For rescue experiments, mRNA was transcribed from thus named fog2b. Zebrafish Fog2a was 63% identical at the templates encoding murine FOG-1 (pcDNA FOG-1) or an N-terminal amino acid level to murine FOG-2, while zebrafish Fog2b was R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493 485

Fig. 1. Fog1 is the zebrafish orthologue of murine FOG-1. Shown is a ClustalW alignment of the primary amino acid sequence obtained through conceptual translation of the murine FOG-1 and zebrafish fog1 cDNAs. Gray boxes indicate amino acid identities. Blue shading indicates the positions of the zinc finger motifs. Red shading indicates the position of the FOG repression motif, green shading indicates the potential nuclear localization signal, orange shading indicates the CtBP interaction motif, and yellow shading indicates the position of other regions of high sequence conservation among FOG family members. 486 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493

Fig. 2. Fog2a and fog2b are zebrafish homologues of murine FOG-2. Shown is a ClustalW alignment of the primary amino acid sequence obtained through conceptual translation of the murine FOG-2 and zebrafish fog2a and fog2b cDNAs. Gray shading indicates amino acid identities. Blue shading indicates the positions of the zinc finger motifs. Red shading indicates the position of the FOG repression motif, green shading indicates the potential nuclear localization signal, orange shading indicates the CtBP interaction motif, and yellow shading indicates the position of other regions of high sequence conservation among FOG family members. R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493 487

43% identical to murine FOG-2. As with FOG-1, an alignment of the FOG-2 proteins from mice and zebrafish reveals the highest degree of conservation of the proteins lies in the zinc finger motifs and specifically fingers 1, 5, 6, 7 and 8 (Fig. 2). In addition, the FOG repression motif, CtBP binding site, and nuclear localization motif are also conserved. Finally, there are two regions that are conserved across all FOG proteins from all species examined to date: a domain N-terminal to the first zinc finger and a domain N-terminal to the fifth zinc finger (yellow shading, Figs. 1 and 2). The functional significance of these domains is currently unclear.

Fog proteins physically interact with and repress GATA factors

One of the properties common to all FOG proteins is the ability to interact with GATA factors. To test if the zebrafish Fog proteins can also bind to GATA factors, we used an in vitro binding assay as described previously (Svensson et al., 2000a). As can be seen in Fig. 3A, zebrafish Fog1, Fog2a, and Fog2b bind to a protein containing the N- and C-terminal zinc fingers of GATA4 fused to GST but not to GST alone (compare columns 2 and 3), demonstrating that all of the zebrafish Fog proteins are capable of binding to the zinc fingers of murine GATA4, as has been reported for other FOG proteins (Fox et al., 1999; Lu et al., 1999; Svensson et al., 1999; Tevosian et al., 1999). To test the functional significance of the interaction of the Fog proteins with GATA4, we transiently transfected NIH 3T3 fibroblasts with an expression plasmid for GATA4 and a reporter plasmid containing 638 bp of the ANF promoter Fig. 3. Zebrafish Fog proteins bind and repress GATA4. Zebrafish Fog proteins driving expression of human growth hormone. We have can physically interact with GATA4. In panel A, in vitro translated, 35S-labeled previously shown that GATA4 strongly transactivates this Fog1, Fog2a, or Fog2b proteins (column 1) were incubated with bacterially produced GST (column 2) or GST-GATA4 (column 3) fusion proteins and promoter, and the addition of either murine FOG-1 or FOG-2 purified using glutathione sepharose beads. The resultant complexes were represses this transactivation (Lin et al., 2004; Svensson et al., resolved by 7% SDS-PAGE and detected by autoradiography. A physical 2000a). Like murine FOG proteins, all of the zebrafish Fog interaction of GATA4 and Fog proteins is indicated by the presence of 35S- proteins can also repress GATA4 in this cell and promoter labeled protein in column 3. Zebrafish Fog proteins can repress GATA4 context (Fig. 3B). Fog1 blocked GATA4-mediated activation of mediated transcriptional activation. In panel B, NIH 3T3 fibroblasts were T transfected with a reporter plasmid containing 638 bp of the ANF promoter the ANF promoter by 87 5%, while Fog2a and Fog2b driving expression of human growth hormone (hGH). In addition, expression inhibited GATA4 mediated activation by 48 T 9% and 42 T 9%, plasmids for murine GATA4 (columns 2–5) and zebrafish fog1 (column 3), respectively (Fig. 3B, columns 3, 4, and 5). In all cases, the fog2a (column 4), or fog2b (column 5) were included. Forty-eight hours post- repression observed was statistically significant (P < 0.0001, transfection, cells were harvested and assayed for hGH. Results are reported as T P = 0.0002, and P = 0.001, respectively). Taken together, the mean SEM (n = 12). these observations suggest that the three genes identified here are the zebrafish homologues of the murine FOG genes. the zebrafish fog genes, we used whole-mount in situ hybridization as shown in Fig. 4.LikemurineFOG-1, Expression of the fog genes in the developing zebrafish embryo zebrafish fog1 is expressed in the developing heart starting at 24 h post-fertilization (hpf) and continuing through 72 hpf Murine FOG-1 is expressed in the adult mouse in the spleen, (arrows, Fig. 4). Expression of fog1 was also detected in the liver, gonads, and at lower levels in the heart, brain, and lungs developing brain (arrowheads, Fig. 4), along the developing (Katz et al., 2002; Tsang et al., 1997). Within the developing spinal cord, and in the site of blood production in the early heart, murine FOG-1 is expressed in the endocardial cushions zebrafish embryo, the intermediate cell mass (asterisk, Fig. 4). of the outflow tract and atrioventricular canal at embryonic day Zebrafish fog2a and fog2b are expressed in the mid-brain, but 12.5. Murine FOG-2 is expressed in the adult heart, brain, and in contrast to the expression pattern of murine FOG-2, gonads as well as at low levels in the lung and liver (Lu et al., expression of fog2a or fog2b could not be detected in the 1999; Svensson et al., 1999; Tevosian et al., 1999). In early zebrafish heart at 24 or 48 hpf (Fig. 4). Thus, of the three fog mouse development, murine FOG-2 is first expressed in the genes in the zebrafish genome, only one, fog1, is expressed in looped heart tube at embryonic day 8.5 as well as the septum the heart during early cardiac development and might therefore transversum. To determine the pattern of expression of each of be expected to play a role in early heart morphogenesis. 488 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493

Fig. 4. Expression of Fog genes during zebrafish development. Whole-mount in situ hybridization using digoxigenin-labeled antisense cRNA fog1, fog2a, and fog2b probes on zebrafish embryos from 19 hpf to 72 hpf. An asterisk in the top left panel indicates intermediate cell mass expression of fog1. In all panels, arrows indicate heart staining; arrowheads indicate brain staining.

Morpholinos against fog1 inhibit cardiac looping lacking all of its zinc fingers. To test the effectiveness of these morpholinos, we used the RT-PCR with primers located in As discussed above, the FOG genes in mice are critical for flanking exons (exons 3 and 5 for fog1 and fog2a, exons 4 and normal hematopoiesis and cardiac development. To ascertain if 6 for fog2b) on RNA derived from zebrafish embryos injected the fog genes in zebrafish are also important for cardiac with these morpholinos. The results shown in Fig. 5B indicate development, we used antisense morpholinos to reduce Fog that all three morpholinos were effective in disrupting splicing protein levels during early zebrafish development. We designed of their target message. morpholinos against the translation initiation site in each fog To determine the role of Fog proteins in zebrafish mRNA and tested their effectiveness using an in vitro development, we injected each morpholino into one-cell translation reaction for each fog message in the absence or stage zebrafish embryos and allowed these embryos to presence of morpholino (Fig. 5A). As can be seen, for each develop for 48 h. Embryos injected with the control message, the specific morpholino effectively inhibited protein morpholino or a morpholino directed against the translation production, while the morpholinos to the other fog mRNAs or initiation site of fog2a or fog2b, the exon 4 splice donor site a 5-base mismatch control had no effect. These results of fog2a, or the splice donor site of exon 5 of fog2b showed demonstrate that these morpholinos are specific and effective no apparent phenotype (n > 100). In contrast, by 48 h post- at blocking Fog protein expression in vitro. We also designed injection, 75% (n = 260) of the embryos injected with the morpholinos specific to each fog message to block their proper morpholino against the fog1 translation initiation site showed splicing. These morpholinos were designed to the splice donor a pericardial effusion and blood pooling just below the atrium site of exon 4 (fog1 and fog2a) or the splice donor site of exon (arrow, Fig. 5C). We also observed a decrease in the number 5(fog2b) such that they would produce aberrant splicing of the of circulating erythrocytes within these morphants, suggesting primary transcript resulting in the deletion of the targeted exon. a defect in erythropoiesis. Similar results were obtained with Such a deletion would be predicted to lead to a frame shift in a morpholino directed against the splice donor site of exon 4 the resulting message, producing a truncated Fog protein for fog1 (data not shown). To characterize the cardiac defect R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493 489 further in the fog1 morphants, we used whole-mount in situ hybridization with a probe against cardiac myosin light chain (cmlc2) to visualize the developing atrium and ventricle (Fig. 5C). At this stage in development, the heart has undergone looping in greater than 94% of uninjected embryos (n =55) or embryos injected with a 5-base mismatch control

Fig. 6. Fog1 morphants have a hypoplastic ventricular wall. Gross morphology of a 4.5 days post-fertilization (a and d) or 7 days post-fertilization (b and e) embryo uninjected (a, b) or injected with an anti-fog1 morpholino at the single- cell stage (d, e). Note the extensive pericardial effusion in the Fog1 morphant. Below, longitudinal sections through the heart of an uninjected embryo (c) or a fog1 morphant (f) at 7 days post-fertilization. The ventricle is indicated by ‘‘v’’, the atrium by ‘‘a’’. Scale bar is 50 Am.

morpholino (n = 51). The same was seen in morphants of fog2a and fog2b. However, in 75% (n = 70) of embryos injected with the anti-fog1 morpholino, looping did not occur. Instead, the heart remained as a linear tube (Fig. 5C). This tube was able to beat and drive the circulation but not as effectively as a wild-type heart as evidenced by the blood pooling seen in these embryos. These results demonstrate that fog1 is required for normal cardiac looping during zebrafish cardiac development. Although heart looping was perturbed, morphants were able to survive for at least 7 days post-fertilization. At 4.5 days post- fertilization, an extensive pericardial effusion was readily

Fig. 5. Fog1 is required for cardiac looping. Anti-fog morpholinos inhibit translation or splicing of fog mRNA. In panel A, in vitro transcription and translation of Fog1 (top panel), Fog2a (middle panel), or Fog2b (bottom panel) in the absence (column 1) or presence of gene-specific morpholino (columns 2–4) or mismatch control (column 5). In panel B, RT-PCR of RNA from 48 hpf zebrafish embryos uninjected (À) or injected (+) with morpholinos directed against the exon 4 splice donor site of fog1 (left panel), fog2a (middle panel), or the exon 5 splice donor site of fog2b (right panel) using primers from flanking exons. Arrowheads indicate the correctly spliced product; asterisks indicate aberrantly spliced products. Developmental defects in morpholino injected embryos. In panel C, morpholinos directed against fog1 (a and b), fog2a (c and d), fog2b (e and f) or a mismatch control (g and h) were injected into single-cell zebrafish embryos and embryos photographed 48 h post- fertilization (a, c, e, g). Arrows indicate a pericardial effusion and blood pooling in the atrium. Panels on the right (b, d, f, h) are in situ hybridizations using the cmlc2 probe on 48 hpf morphants. 490 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493

The FOG repression motif is required to rescue cardiac looping in fog1 morphants

Since there is significant sequence divergence between murine and zebrafish fog1 at the morpholino target site, the fog1 translation initiation site morpholino should not be able to block translation of murine FOG-1 mRNA. To determine if murine FOG-1 could rescue the phenotype seen in the fog1 morphant, we co-injected the fog1 translation initiation site morpholino with mRNA encoding murine FOG-1 into zebra- fish embryos (Fig. 8). As before, a majority of the embryos (71%, n = 142) injected with the fog1 morpholino developed a pericardial effusion. Co-injection of mRNA encoding murine FOG-1 resulted in a rescue of this phenotype, with only 26% (n = 151) of the co-injected embryos developing pericardial edema and failing to undergo cardiac looping. These results provide further evidence that the phenotype seen in the fog1 morphants is caused by the depletion of the Fog1 protein in these embryos. Our previous work on the important functional domains of FOG proteins has demonstrated that the N-termini of both FOG-1 and FOG-2 contain a conserved domain that mediates transcriptional repression, the FOG repression motif (Lin et Fig. 7. in fog1 morphants. Whole-mount in situ hybridizations al., 2004). Transient transfections into murine fibroblasts have were performed using probes specific for tbx5 (left column) or tbx20 (right column) on 48 hpf wild-type embryos (rows 1 and 3) or fog1 morphants (rows 2 and 4). Arrows indicate heart expression of each of these genes. apparent (Fig. 6, compare panels a and d). By 7 days post- fertilization, these embryos had impressive edema (Fig. 6, compare panels b and e), consistent with cardiac insufficiency. Histologic sections through the heart revealed a dilated, thin- walled ventricle and a dilated atrium (Fig. 6, compare panels c and f). Unlike mice deficient in FOG-2, in fog1 morphant embryos, hyperplastic atrioventricular or outflow tract valves were not present. This analysis suggests that the edema seen in these embryos was due to poor cardiac output from a ventricle with hypoplastic walls. Several zebrafish genes have also been shown to be required for cardiac looping. The heartstrings mutation in the transcrip- tion factor tbx5 generates a fish with a heart tube that fails to loop (Garrity et al., 2002). Further, morpholinos have been used to reduce Tbx5 levels during development and have produced morphants with a linear heart tube (Ahn et al., 2002; Garrity et al., 2002). Similar work targeting tbx20 has also resulted in the inhibition of cardiac looping. In addition, tbx5 is down- regulated in the heart tube of the tbx20 morphant, suggesting that both of these factors lie in the same transcriptional pathway (Szeto et al., 2002). To determine if other genes known to be involved in cardiac looping were altered in the fog1 morphants, we performed whole-mount in situ hybridization of wild-type and fog1 morphants using probes against tbx5 and tbx20 (Fig. 7). Our results indicate no significant difference in expression Fig. 8. Expression of murine FOG-1 rescues the fog1 morphant phenotype. patterns of these genes. This result suggests that either fog1 is a Shown above are 48hpf zebrafish embryos uninjected (a), injected with an anti- fog1 morpholino (b), injected with an anti-fog1 morpholino and mRNA downstream target of these genes, or that fog1 may regulate encoding murine FOG-1 (c), or injected with an anti-fog1 morpholino and cardiac looping in zebrafish through an alternative pathway to mRNA encoding an N-terminal truncation of murine FOG-1 (DFOG-1) (d). those of tbx5 and tbx20. Atrial and yolk sac blood pooling is indicated by the arrows. R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493 491 demonstrated that FOG proteins with deletions of this domain significance of these regions is currently unclear, their high are unable to repress GATA4-mediated promoter activation. degree of conservation suggests that they might be important As indicated in Figs. 1 and 2, this domain is also present in for Fog function. The elucidation of the functional significance all of the zebrafish Fog proteins, providing further evidence of these domains is an ongoing area of investigation within the to support the importance of this domain. To test the laboratory. functional significance of this domain for the regulation of The most striking phenotype seen in the fog1 morphants cardiac looping in zebrafish, we co-injected mRNA encoding was the extensive pericardial edema most likely secondary to murine FOG-1 lacking its N-terminal repression domain (Fig. a hypoplastic ventricular wall and an inhibition of cardiac 8, DFOG-1). This mRNA was almost completely unable to looping. This phenotype could be rescued by overexpression rescue the fog1 morphant phenotype, with 61% (n = 57) of of murine FOG-1 but not by a truncated version of FOG-1 the injected embryos developing a pericardial effusion. Taken lacking the FOG repression motif. This observation is the together, these results demonstrate the importance of the first in vivo demonstration of the functional significance of FOG repression motif for proper cardiac development and the FOG repression motif and highlights the importance of suggest that the mechanism by which Fog proteins mediate this domain for the function of FOG proteins during proper cardiac looping in zebrafish is via transcriptional cardiogenesis. repression. The ventricular wall hypoplasia in the fog1 morphants is similar to what has been seen in FOG-2-deficient mice, Discussion suggesting that FOG proteins are required for myocardial proliferation in both species. In contrast, the complete looping To improve our understanding of Fog protein function and defect seen in fog1 morphants has not been previously provide further insights into the molecular basis of congenital appreciated in FOG-1 or FOG-2-deficient mice. FOG-2- heart disease, we describe in this report the cloning and deficient mice are characterized by a mis-alignment of their characterization of three fog genes from zebrafish. Given the cardiac inflow and outflow tracts, having a double outlet right evidence for a whole genome duplication in the evolution of ventricle and a common AV valve (Svensson et al., 2000b; teleost fish (Amores et al., 1998; Hoegg et al., 2004), it is not Tevosian et al., 2000). Disruption of the murine FOG-1 gene in surprising that we identified two FOG-2 paralogues in endothelium results in a similar phenotype (Katz et al., 2003). zebrafish. On the other hand, only one gene homologous to Both of these cardiac phenotypes can be thought of as a partial murine FOG-1 was identified in the zebrafish genome, looping defect, as cardiac looping is clearly initiated, but is suggesting that either the second fog1 paralogue was lost incomplete due to a failure to properly align the inflow and during evolution, or that it lies in a region of the genome where outflow tracts with the developing ventricles. In contrast to sequencing is not yet complete. mice, the results in this report demonstrate that zebrafish hearts A comparison of the primary amino acid sequences of the only express one fog family member, fog1, and the knockdown Fog proteins from zebrafish and mice identified a number of of Fog1 results in a complete looping defect, as the heart tube domains that show a high degree of sequence conservation. remains as a linear structure. Since both FOG-1 and FOG-2 are One such domain is the CtBP interaction motif (Figs. 1 and 2, expressed in the murine heart tube during looping (Katz et al., orange shading). Conservation of this sequence suggests that 2003; Svensson et al., 1999), it is possible that murine FOG Fog1, Fog2a, and Fog2b are likely able to bind to CtBP as has proteins serve a redundant role in initiating looping but are been shown for their murine homologues (Deconinck et al., unable to compensate for each other at later stages of looping, 2000; Holmes et al., 1999; Svensson et al., 2000a; Turner and resulting in partial looping in FOG-1 or FOG-2-deficient Crossley, 1998). The Fog repression motif is also present in all murine hearts. In the zebrafish system, there is no redundancy zebrafish Fog proteins (Figs. 1 and 2, red shading). As shown in fog expression, and as a result, looping can be completely in this report, zebrafish Fog proteins function as transcriptional inhibited by knockdown of Fog1. Alternatively, the differences co-repressors, as all three repressed GATA4 mediated transac- in FOG’s role in cardiac development in these two systems tivation of the ANF promoter in vitro (Fig. 3B). This may represent a true divergence of FOG function in vertebrate repression is likely mediated at least in part by the FOG heart morphogenesis. repression motif, as has been shown for murine FOG-1 and FOG-2 (Lin et al., 2004; Svensson et al., 2000a). Murine FOG Acknowledgments proteins have also been shown to function as transcriptional co-activators in certain cell and promoter contexts (Jia and The authors would like to thank Dae-gwon Ahn and Takimoto, 2003; Letting et al., 2004; Tsang et al., 1997), so it Deborah Yelon for their technical expertise and insightful is possible that zebrafish Fog proteins may also function as discussions. This work was supported by NIH HL071063, transcriptional co-activators in specific cell and promoter AHA 0230395 and a grant from the Schweppe Foundation. contexts. In addition to these conserved domains, there are also two other regions of high similarity between murine FOG References proteins and their zebrafish homologues, one located N- terminal to zinc finger 1 and the other N-terminal to zinc Ahn, D.G., Ruvinsky, I., Oates, A.C., Silver, L.M., Ho, R.K., 2000. tbx20, finger 5 (Figs. 1 and 2, yellow shading). While the functional a new vertebrate T-box gene expressed in the cranial motor neurons 492 R.Z. Walton et al. / Developmental Biology 289 (2006) 482–493

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