NXT2 is required for embryonic heart development in zebrafish
BMC Developmental Biology volume 5, Article number: 7 (2005)
NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.
A zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2.
NXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate species.
In a living eukaryotic cell, the nuclear envelope is a critical barrier between the nucleus and the cytoplasm, which prevents macromolecules from freely exchanging between the two compartments. The eukaryotic cells have developed multiple pathways designed to transport various types of macromolecules through specialized channels that are incorporated within the nuclear envelope. There are four major components involved in nucleocytoplasmic transport, including cargos, receptors, nuclear pore complex (NPC), and Ran [1–3]. Cargos are proteins, RNAs or ribonucleoprotein (RNP) particles that contain either nuclear localization signals destined for import into the nucleus from the cytoplasm or nuclear export signals destined for export from the nucleus to the cytoplasm [4–6].
NXF (nuclear export factor) family members (NXF 1 to 6) play essential roles in RNA exporting out of the nucleus [7, 8] through NPC. The structure of the founding member, NXF1 (also called TAP), has modular organization consisting of four major domains: a noncanonical RNP-type RNA-binding domain (RBD), four leucine-rich repeats (LRRs), an internal domain sharing significant sequence homology with the nuclear transport factor 2 (the NTF2-like domain) and a C-terminal ubiquitin associated (UBA)-like domain. The RBD domain and LRRs are responsible for recognition and binding of RNAs through RNP and CTE (constitutive transport element-a cis-acting RNA sequence). The UBA-like domain interacts with multiple nuleoporins of NPC. The NTF2-like domain heterodimerizes with another protein, NXT1 [9, 10].
NXT1 was initially identified from human based on its sequence homology to NTF2 (~26%) [11, 12] and association with the RNA exporting factor NXF1/TAP . Earlier studies suggested that NXT1 was necessary for Crm1-mediated nuclear export of proteins , in which NXT1 directly binds to export receptor Crm1 and RanGTP (a form of Ran binding GTP), and efficiently stimulates the release of cargos in the cytoplasm after translocation through NPC . However, recent experiments demonstrated that NXT1 appears to exclusively participate, independently of Ran and Crm1, in TAP-mediated nuclear export of RNAs. This finding has been widely confirmed in yeasts, Drosophila, C. elegans and vertebrates [7, 9, 10, 16–18]. It was shown that formation of a NXF1/NXT heterodimer via NTF2-like domain is required for the nuclear export of RNAs in a wide range of eukaryotic organisms [10, 19, 20]. Recently, another member, NXT2 (also called p15-2), was identified to be able to substitute NXT1 in forming NXF1/NXT heterodimers . NXT2 has two alternative splicing variants of transcripts encoding p15-2a and p15-2b proteins that differ in the first exon . To date, the function of NXT2 variants has not been elucidated.
We previously generated cloned zebrafish from long-term cultured fibroblast cells infected with a retrovirus expressing GFP reporter gene . We bred the GFP transgene to homozygosity and found that one of the cloned fish lines bore a recessive embryonic lethal mutation, suggesting that viral insertion is the cause of the cardiac defect. More interestingly, the mutant embryos have reduced circulation, cardiac edema and aberrant heart valve formation after three days of development. Sectioning of homozygous mutant embryos revealed that the ventricular wall is thinner and the space between the myocardium and endocardium is enlarged. Molecular analysis of these mutants revealed that NXT2 was disrupted by the viral insertion. Whole-mount RNA in situ hybridization detected NXT2 expression in the heart as well as other tissues. RT-PCR analysis suggested that NXT2 has multiple alternative mRNA splicing forms and intriguingly, one of them is significantly reduced in mutant embryos. Morpholino knockdown of this specific splicing form reproduced the mutant phenotypes. Taken together, our data demonstrate that NXT2 has a critical role in the morphogenetic integrity of the embryonic heart in zebrafish, and provide the first functional study of NXT2 in a vertebrate animal species.
Identification and characterization of a cloned zebrafish exhibiting heart defects
Cloned zebrafish were obtained by nuclear transfer  from long-term cultured cells that were infected with retrovirus  containing a GFP reporter gene driven by the Xenopus ef1 alpha promoter. It has been shown that retroviral integration can cause mutations in zebrafish . We therefore bred all of the cloned fish lines to homozygous for GFP to screen for abnormality. One of these lines exhibited specific heart defects. The mutant phenotype co-segregated with proviral GFP expression (n > 2000), suggesting that the heart defects were caused by disruption of a functional gene by retroviral insertion. The mutant embryos were first distinguishable from their wild-type siblings by slightly dilated heart, slow heart rate, circulation reduction and mild pericardial edema after 3 days of development (Fig 1A, B, D and 1G). At this stage, cardiac function assays showed that the atrium and ventricle both contracted rhythmically in a normal atrio-venticular sequence. But, the heartbeat rates of mutants (134.3 ± 11.1 per minute, n = 72) were statistically significantly (P < 0.01) slower than that of their wild-type siblings (154.7 ± 8.7 per minute, n = 42) and the significant difference remained at 4 days post fertilization (dpf) (mutant 134 ± 12.8 per minute, n = 37 vs wild type 169 ± 4.7 per minute, n = 23; P < 0.01). Furthermore, while the wild-type embryos (Fig 1E) develop a narrow heart with forward blood flow through the embryo, both cardiac chambers are dilated in the enlarged pericardial sac (Fig 1C and 1H) and blood cells regurgitated between ventricle and atrium, resulting in either pooling the blood cells in two heart chambers or peripheral veins of mutant embryos at 4–5 dpf (Fig 1F, H and 1I). By 5 dpf, the mutant hearts are stretched to a long tubular structure (Fig 1F). Pericardial edema became prominent, circulation was completely terminated and severe edema appeared in the whole abdominal cavity, leading to necrosis in fin, muscle, skin and other tissues (data not shown).
The reduced circulation and regurgitation defects may be the results of abnormalities in cardiac contractibility, differentiation of cardiomyocytes or the formation of cardiac valve. To further investigate these possibilities, we analyzed histological sections of wild type and mutant embryos. Normally, by 2 dpf, the ventricular myocardial cells thicken and become the primary pumping force of the heart. We found that in 4 dpf mutant embryos, the ventricular wall remains thin and the space between myocardium and endocardium was diminished in the ventricle but enlarged in the atrium compared to that of wild type embryos (Fig 1J and 1K). In addition, we frequently observed mutant samples lacking cardiac valve structure. In 5 dpf mutant embryos, the ventricular wall remained thinner and cardiac chambers were severely dilated (data not shown). These data suggested that myocardial structures were disturbed in mutant embryos during early development.
Various heart chamber-specific gene expressions demarcate vertebrate heart compartments and play an important role in regulating critical steps of chamber formation. To understand whether the identified mutation affects formation of specific chambers and morphogenetic movements of dynamic cardiomyocycte, expression of chamber-specific genes including Vmhc, Amhc and Cmlc2 was examined. As shown in Fig 1L–Q, no difference was detected in expressions of Amhc (L and O), Vmhc (M and P) and Cmlc2 (N and Q) between wild-type (L, M and N) and mutant (O, P and Q) embryos at 3 dpf (same results were obtained at 4 and 5 dpf, data not shown), suggesting that the mutated gene is not required for the initial chamber-specific differentiation of cardiomyocytes, in which Amhc, Cmlc2 and Vmhc play important roles [24–27].
Epithelial-to-mesenchymal transition (EMT) plays a critical role in atrio-ventricular valve formation. In the zebrafish, this process begins at 33 hpf, when the myocardial cells at the atrio-ventricular boundary induce the adjacent endocardial cells to undergo EMT[28, 29]. Bmp4 and versican (cspg2) are genes involved in cardiac valve formation. Coincidently, their cardiac expressions become restricted to the myocardial cells at the valve forming region at the same time as the formation of the prevalvular cushions, suggesting that they are thereby markers for the differentiation of the myocardial cells in the valve forming region and misexpression of these genes may indicate defects in valve formation[27, 28]. As shown in Figure 1 (R-W), expression of versican and bmp4 in mutant embryos (Fig 1U, 1V, and 1W) was broadly up-regulated and expanded throughout the two heart chambers at 3 dpf at which stage their expression should be restricted to the atrioventricular boundary as in wild-type embryos (Fig 1R, S and 1T). Meanwhile, no difference of versican expression (Fig 1S and 1V) was detected in the otoliths of developing ears in wild-type and mutant embryos [30, 31], suggesting that the abnormal versican expression pattern is due to heart defects observed in these mutant embryos other than general developmental delay.
NXT2 is responsible for heart defects
Of the 2000 analyzed mutated embryos, the heart defect always co-segregate with the proviral GFP expression, suggesting that the viral insertion causes the observed heart phenotypes. By analyzing the genomic DNA by Southern blot, we found that there is a single viral insertion in this fish line (Fig 2A and 2B). A 1071 bp genomic sequence flanking the viral insertion was isolated by inverse PCR and used to search EST databases, leading to the identification of a candidate gene encoding a 143 amino acid protein that shares high homology with that of NXT1 and/or NXT2 from human, mouse, rat, Drosophila and C. elegans. By comparing genomic structures of NXT1 and NXT2 genes from these species, we noticed that NXT1 was encoded by a single exon whereas NXT2 gene contains multiple exons, which is similar to our candidate gene (Fig 2C). Based on similarities in protein sequences (Fig 2D) and genomic organizations, we concluded that our candidate gene represents a homolog of NXT2 rather than NXT1and named it zNXT2.
To determine the expression pattern of zNXT2, we carried out RNA whole mount in situ hybridization with its sense and antisense RNA probes. It was found that zNXT2 was ubiquitously expressed during early embryogenesis but clearly detectable in heart at 2 dpf (Fig 2E).
zNXT2a is the alternatively spliced transcript required for heart function
Search for zNXT2 homologues revealed that one copy of NXT gene exists in Drosophila, C. elegans and mouse whereas two copies are present in rat (rNXT1 and rNXT2) and human (hNXT1 and hNXT2) genomes, respectively (Fig 3A). In humans, two alternative splicing variants of NXT2 (NXT2a and NXT2b) were identified in EST database, which were further confirmed by cloning and sequencing the corresponding cDNAs . In order to determine if zNXT2 also has multiple alternative RNA transcripts, 5' RACE analysis was performed  and the PCR products were sequenced. From 19 sequences obtained, 6 represented the original zNXT2 encoding 143 amino acid protein as mentioned above (now named as zNXT2a), 2 lacked the third exon, and 11 did not have the first two exons and part of third exon. The last variant, now named zNXT2b, appeared to be transcribed from the third exon and started at the putative codon GTG (Fig 3A). The same observation has also been made in humans in that the second splicing variant NXT2b starts from the putative initiation codon GTG . The putative zNXT2b protein contains most of the functional domains of zNXT2a, since they share the last two identical exons.
To determine which transcript is responsible for the mutant phenotype we compared zNXT2a and zNXT2b mRNA expression level in mutant and wild type embryos. Two sets of primers, NTf/NTr for the first and the fourth exon to detect zNXT2a and NXTf/NXTmr for the fourth and the fifth exon to detect both zNXT2a and zNXT2b, were designed for RT-PCR analysis (Fig 3). As shown in Fig 3B, zNXT2a expression level was dramatically reduced in homozygous mutant embryos (indicated by an asterisk) compared to their wild-type or heterozygous siblings whereas no difference of mRNA levels was observed when both zNXT2a and zNXT2b were detected using NXTf/NXTmr primers. Taken together, these data suggested that loss of zNXT2a transcripts might be responsible for this mutant phenotype.
Blocking zNXT2a activity by morphlino phenocopies the mutant heart defects
Morpholino technology is a powerful tool for blocking translation of specific mRNA transcripts and has been extensively used in zebrafish [33–35]. To further verify that zNXT2a was responsible for the observed heart defects, an antisense morpholino oligo specific to this transcript was injected in wild type embryos at one to four-cell stage. Analysis of more than 500 embryos from multiple injected clutches showed that approximately 80% of injected embryos developed the same heart defects as that in the virus-induced mutant embryos (Fig 4A–H), including pericardial edema, slightly dilated heart, slow heart rate and reduced circulation. Mock-injections have no effect on heart development in control embryos (data not shown).
The ability to use morpholinos to reproduce zNXT2a mutant phenotypes allowed us to confirm cardiac defects observed in living mutant embryos (Fig 1) through using live transgenic zebrafish embryos expressing GFP in specific cell lineages. First, cardiac myosin light chain 2 (CMLC2) promoter driving GFP transgenic zebrafish  embryos were used to determine development of two heart chambers. As shown in Fig 4, heart chambers of morpholino injected embryos at 3 dpf exhibited pericardial edema and dilated heart compared with that of wild-type embryos (Fig 4A, B, C and 4D). In 4–5 dpf morpholino injected embryos, cardiac chambers became abnormally stretched and relative positions of two chambers were abnormal (Fig 4F and 4H) whereas wild-type embryos had heart chambers in a narrowed pericardial sac (Fig 4E and 4G). This finding is consistent with the previous results described based on viral insertional mutated embryos (Fig 1).
We further examined endocardial morphology at the atrioventricular valve region using receptor tyrosine kinase flk-1 promoter driving RFP transgenic zebrafish embryos. Previously, tie-2 GFP transgenic zebrafish has been used to address this issue. Similar to tie-2, flk-1 also plays a crucial and unique role in endothelial cell differentiation and vascular morphogenesis, including vessels and heart endocardium [36, 37]. In uninjected wild-type embryos, endocardial cells cluster at the atrioventrivular boundary and form a ring shape structure at the valve forming region (Fig 4I). However, more than 70% of the zNXT2a morpholino injected flk-1-RFP transgenic embryos (n = 129) failed to form this cluster (Fig 4J), suggesting that zNXT2 is required for this early endocardial morphogenetic event. Together with RNA whole mount in situ data of bmp4 and versican, these results indicate that cardiomyocyte differentiation and thickening of the endocardium at the atrioventricular boundary in the mutant embryos were significantly impaired, resulting in abnormal valve formation.
Ventricular myocardial thickening and atrioventricular valve are impaired by disruption of zNXT2a
In zebrafish, between 24 and 48 hpf, the linear heart tube gradually bends at the boundary of two chambers to create an S-shaped loop. Meanwhile, clustering of endocardial cells as a ring structure at the atrioventrocular boundary by 33–37 hpf initiates the first step of the formation of cardiac valve [27, 28, 31]. While the heart is pumping, differentiation and morphogenesis continue, which includes remodeling of the cardiac valve to completion, finally building on a linear heart tube to produce an adult heart.
Sectioning of zNXT2a homozygous mutant embryos revealed that the ventricular wall is thinner as early as 3 dpf (data not shown, but see Fig 1 for 4 dpf data). Decreased cell proliferation and/or increased apoptosis may cause such a defect. Since the thinner myocardial wall appears very early during development we prefer a model that the under-developed ventricular wall is due to a deficiency in myocardial cell proliferation. Alternatively, this could result from abnormally tight intercalation of ventricular cells with the same numbers of cells in the mutant embryos as previously described . In regard to the atrioventricular valve defect, it has been reported that endothelial shear stress (blood flow) plays a critical role in endocardial cushion by using special surgical experiments in zebrafish embryos . In contrast, other researchers  argued that reduction in myocardial function is primarily responsible for the defect in atrioventricular cushion development, which was observed in sih (no early heartbeat) and cfk (cardiac dilation and no early blood flow) mutant embryos. In zNXT2 mutant embryos, the earliest observable defects are reduced heart rate, dilated cardiac chambers and a thin ventricular myocardial wall. Heart morphology and chamber specification ascertained by expression of Amhc, Vmhc and Cmlc2 are all normal at this stage in mutant embryos (Fig 1 and 4, data not shown by 2 dpf). Later on from 3–4 dpf, the atrioventricular valve was aberrant and the relative position of atrium and ventricle was perturbed, as revealed by sectioning analysis, expression of versican and bmp4 (Fig 1) and morpholino knockdown in CMLC2-GFP and flk-GFP transgenic embryos (Fig 4), respectively. Additionally, injection of a higher dose (approximately 10 ng per embryo) of zNXT2a morpholino into embryos causes defective endocardial cushion formation as early as 2 dpf, as illustrated by a failure of endocardial clustering at atrioventricular boundary (data not shown). This indicates that valve malformation resulted by interruption of zNXT2a might be either a primary defect or a defect due to disturbed function of chambers at later stage. In addition, our data implies that the mutant cardiac valve malformation is either an independent event or caused by lacking of myocardial cell proliferation or differentiation. The later situation will be consistent with the observation by Bartman et al (2004). Nonetheless, our findings establish that zNXT2 has a critical role in driving cardiac valve formation to final completion.
zNXT2a is the mRNA transcript variant responsible for heart defects
As in humans, zNXT2 has multiple alternative splicing mRNA transcripts with two distinct start sites (ATG and GTG) (Fig 3A). It is possible that different zNXT2 splicing variants are differentially expressed in different tissues. Moreover, zNXT2b starting from GTG may represent a major transcript because of its higher copy numbers in the amplified cDNA pools (11 out of 19 sequenced samples) whereas zNXT2a starting from ATG might be enriched in heart. The biological significance of such a variation in NXT2 transcription has not been addressed. Our studies demonstrated that the alternatively spliced transcripts might have specific functions in different tissues during embryonic development and organogenesis. Specifically, zNXT2a appears to be required for heart development in zebrafish. Viral insertion in the intron immediately upstream of the exon containing ATG caused specific knockdown of zNXT2a transcription in mutant embryos whereas other transcripts such as zNXT2b appear normal. Injection of a morpholino against zNXT2a can phenocopy heart defects, confirming that zNXT2a protein is the main factor required for zebrafish heart development. Furthermore, when the morpholino dose was increased, more injected embryos developed pericardial edema one day earlier than the insertional mutants of zNXT2 (data not shown). This suggests that our zNXT2a insertional mutant may be a hypomorphic allele due to insertion of the viral vector into a non-coding region of the locus. With morpholino data it is clear that zNXT2a is directly implicated in heart development that requires its sufficient expression activity.
Nuclear transport factors are implicated in tissue-specific biological functions
Nuclear protein or RNA transporting has previously been regarded as a general house keeping function for all biological systems. However, increasing evidence suggest that this can be a highly regulated process for specific biological functions with specific RNA targets. In Drosophila, studies demonstrated that homolog of NTF2 (DNTF2) is required for nuclear translocation of the Rel proteins Dorsal, Dif and Relish into the nucleus. These three Rel proteins are activated in response to immune challenges and imported into the nucleus upon microbial challenges, which further induce and regulate transcriptions of anti-microbial peptides attacin, cecropin, defensin, metchnikowin, diptericin and drosomycin. Disruption of DNTF2 leads to impairment of nuclear import of the Rel proteins and thus immune response is impaired in NTF mutants compared with wild type [41, 42]. NXF5, a member of the nuclear export factor NXF family, binds to RNAs as well as to NXT members for RNA nuclear export. In a male patient with a syndromic form of mental retardation, a nonfunctional NXF5 was identified, which is split by the breakpoint situated in the 5' UTR between exons 1 and 2 of NXF5. This suggests that interruption of the gene NXF5 might result in the disease phenotypes associated with syndromic mental retardation including short stature, general muscle wasting, and facial dysmorphism by deranging the export or transport of specific mRNAs [43, 44].
Formation of an NXT1 (or NXT2) and TAP (NXF1) heterodimer is critically required for mRNA export from the nucleus via TAP/NXT1 pathway [9, 10, 13]. Earlier experiments demonstrated that NXT1 is essential in Crm1-mediated nuclear export of proteins, in which NXT1 directly binds to export receptor Crm1 and RanGTP, and improves the release of cargos into the cytoplasm after translocation through the NPC . Very recently, it has been reported that CRM1 is involved in export of specific subsets of cellular RNAs, which include tra-2 mRNA participating in sex cell fate determination in C. elegans and IFN-a1 mRNA generated upon viral infection to human cells [45, 46]. However, little is known about how their cofactor NXT1 (or NXT2) plays a role in a biological pathway. Our finding provides the first evidence that an alternative splicing form of nuclear transport factor NXT2 is specifically implicated in heart development of zebrafish. For future studies, identification of specific RNA targets and elucidation of the molecular mechanism underlying NXT2-mediated exporting of these targets, either mRNA or micro RNA, in heart should be the focus. In addition, this study might provide the basis for some inheritable cardiac valve diseases in human. It should be a reasonable step to determine genetic linkage relationship between a NXT2 locus and defined cardiac diseases.
We demonstrated that disruption of NXT2 gene, a member of NXT (nuclear export factor) family, is responsible for heart-specific defects in a mutant zebrafish generated by a retroviral insertion introduced into the genome by animal cloning technology. Our study suggests that NXT2 has critical roles required for myocardial cell differentiation and formation of cardiac valve at the atrioventricular boundary. Since NXT2 is highly conserved between fish and man it is conceivable that it may also play a role in mammalian heart development and might be involved in cardiac diseases.
Mutant identification and characterization
We initially obtained 14 cloned zebrafish lines by nuclear transfer . After breeding each line to homozygosity for the retroviral insertion that was introduced into the zebrafish donor nuclei as a traceable GFP marker, we identified one line with embryonic heart defects that co-segregate with the GFP expression of the viral construct.
Adult zebrafish and embryos were maintained and staged as previously described . Homozygous mutant embryos were imaged using an Axiocam imaging system (Zeiss). For histological analysis, fixed embryos were dehydrated, embedded in plastic (JB-4, Polysciences Inc.), sectioned at 8 μm and stained with Toludine Blue.
Identification of NXT2 as the candidate gene
Two rounds of inverse PCR were performed to isolate genomic DNA fragments immediately flanking the viral insertion based on the retroviral sequence . For the first inverse PCR, genomic DNA from heterozygous mutant fish was restricted with Dra I. This generated an approximately 3.8 kb proviral-genome-containing genomic DNA fragment as revealed by Southern blot hybridization with a specific viral probe (Fig 2A and 2B). Using Geneclean III kit (Bio 101, Vista, California), a pool of genomic DNA containing this fragment was purified from the gel, then ligated into circular form and finally used as inverse PCR templates. Two sets of primers specific to viral -sequences were designed as follows:
HT1:5'-AATCCCGGACGAGCCCCCAAATGAAAGA-3' and HT3: 5'-ATAGAGTACGAGCCATAGTTAAAATAAA-3' (outer primers);
HP2: 5'-TTTCTTTGTTCCTGACCTTGATC-3' and 1 HP: 5'-AACCCCTCACTCGGCGCGCCAG-3' (inner primers). Templates were amplified for two rounds. The final PCR products were subcloned into the pCR2.1 vector (Invitrogen, Life Technologies) for sequencing. The obtained sequence was used for designing new primers for additional inverse PCR. Similarly, heterozygous genomic DNA was also digested with Hpa I, which generated a 4.3 kb fragment containing retroviral genome and a flanking sequence for PCR amplifications. Again, two rounds of amplification were carried out for the second inverse PCR, in which a set of primers (HP2: 5'-TTTCTTTGTTCCTGACCTTGATC-3'; 2 HP: 5'- TCGTGGTCTCGCTGTTCCTTGG-3') against the retroviral LTR region was used to enrich for DNA templates. Then the second round of PCR amplification was performed from the enriched templates with another set of primers HP2 and F2 (5'-GTGCATGGATAAGAAAAGACGGGTAACG-3', which is against the zebrafish genomic sequence flanking the proviral insertional locus) (Fig 2A and 2B). A total of 1071 bp genomic sequence was obtained and used to search for EST and genomic databases of zebrafish.
To identify the mRNA coding sequence, the 1071 bp genomic flanking sequence was used to BLAST search against the zebrafish EST database. An EST (BI325953) was identified that shares identical sequence to the inverse PCR product and contains an open reading frame of 143 amino acids. Highly similar protein sequences from different species including human, mouse, rat, Drosophila and C. elegans were retrieved from cDNA databases by performing protein sequence similarity searches using the deduced zebrafish amino acid sequence. Multiple sequence alignments were constructed by using the Jotun Hein Algorithm Method (DNA Star Software). To predict the genomic structures of NXT homologues in various organisms, the BLASTN program for genomic sequences was employed based on cDNA and /or EST sequences.
A morpholino was designed against ATG region of zebrafish NXT2 (5'-TCCACCGTTAAAGACATGACTGGTC-3', Gene Tools, LLC, Corrallis, OR). It was injected into one-cell to four-cell stage embryos at 3 μg/μl (2 nl per embryo) in 1 X Danieau solution as described by other researchers . Different stages of injected embryos were imaged under a regular bright filter on a Zeiss microscope. Mock-injections using a morpholino targeting a pancreas-specific gene  or a morpholino with a random sequence at 4–6 ng per embryo was performed as controls.
Identification of alternative splicing variants of zebrafish NXT2
To identify alternative splicing transcripts of NXT2, FirstChoice RLM-RACE Kit (Catalog #1700, Ambion) was employed to specifically amplify the 5' intact region of zNXT2 mRNA through its first strand cDNA using two pairs of 5' adaptor primers (outer primer: 5'-GCTGATGGCGATGAATGAACACTG-3'; inner primer: 5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3') and 3' specific primers (NXTmr – outer primer: 5'- AAGCTCAGTTTGCCCAGTCT-3'; NTr – inner primer: 5'-TGGCAGTCAAGAGTTTGCAC-3') against the internal exons of the gene. RLM-RACE strategy is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band for a specific gene after PCR . Total RNA isolated from 3-day wild-type embryos was used for 5' RACE experiments.
Detection of mRNA transcripts by RT-PCR
To compare relative expression levels of NXT2 transcripts, total RNA samples were isolated from homozygous mutant embryos (-/-), their wild-type (+/+) and GFP positive normal (+/-) sibling embryos, respectively, which were sorted out based on GFP expression and heart phenotypes. For measuring mRNA levels, equal amounts of total RNA isolated from three types of embryos were subjected to first strand cDNA synthesis by reverse transcription under the same condition using M-MLV Reverse Transcriptase (Gibco, Invitrogen). And then, equal amounts of cDNA product were used for PCR amplification of targeted cDNA. Amplification of another cDNA, ef1 alpha, was used as a control. The following specific primers against zNXT2 coding sequences were designed across introns for this purpose: NTf, 5'-GACGGATGCTGGATTTGTTT-3'and NTr, 5'-TGGCAGTCAAGAGTTTGCAC-3'; NXTf, 5'-GAAACGCAGTCACTGGTCAA-3' and NXTrm, 5'-AAGCTCAGTTTGCCCAGTCT-3'. ef1 alpha primers were zEF1a-1: 5'-TCACCCTGGGAGTGAAACAGC-3' and zEF1a-2: 5'-ACTTGCAGGCGATGTGAGCAG-3'. Different cycle numbers (20, 25, and 29) of quantitative PCR amplification were employed to determine a threshold that produced detectable products on gel using the following program: 94°C, 3 minutes; 94°C 30 seconds, 60°C 40 seconds, 72°C 1.5 minutes; 72°C 10 minutes.
Whole-mount RNA in situ hybridization
Sense and antisense digoxigenin-labeled RNA probes were generated from cDNA clones of the zebrafish NXT2, amhc, vmhc, versican, cmlc2 and bmp4 genes using a DIG/Genius 4 RNA Labeling kit (Boehringer Mannheim). Whole-mount RNA in situ hybridization conditions were as described by Jowett . Images were obtained using an AxioCam digital camera (Zeiss) on Stemi SV11 Apo (Zeiss) dissection microscope.
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We thank Xiaodong Shu for helpful discussion and are grateful to Huidong Pan for taking care of our fish. This work was supported by grants from NIH to SL (R01 DK65638) and to Chen (R01HD41367).
HH contributed to all aspects of the experimental data and drafting the manuscript. BZ performed some RNA in situ hybridization experiments. PAH carried out sectioning of mutants. JC provided discussions and edited the manuscript. SL designed the study, drafted and finalized the manuscript. All authors read and approved the final manuscript.
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Huang, H., Zhang, B., Hartenstein, P.A. et al. NXT2 is required for embryonic heart development in zebrafish. BMC Dev Biol 5, 7 (2005). https://doi.org/10.1186/1471-213X-5-7