The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo

Brian R. Keegan1,*, Jessica L. Feldman1,*, Diana H. Lee1, David S. Koos2, Robert K. Ho2, Didier Y. R. Stainier3 and Deborah Yelon1,{dagger}

1 Developmental Genetics Program and Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA
2 Department of Molecular Biology, Princeton University, Princeton, NJ, USA
3 Department of Biochemistry and Biophysics and Programs in Developmental Biology, Genetics, and Human Genetics, University of California, San Francisco, San Francisco, CA, USA
* These authors contributed equally to the work

{dagger}Author for correspondence (e-mail: yelon{at}saturn.med.nyu.edu)

Accepted 9 January 2002


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development.

Supplemental figure available on-line

Key words: Heart, Spt6, Spt5, Transcription elongation, Real-time RT-PCR, hsp70, Zebrafish


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryonic differentiation depends upon tissue-specific gene expression programs, created by temporally and spatially regulated transcription. Production of specific mRNAs can be stimulated or repressed via regulation of transcription initiation. Transcript production can also be controlled through a rate-limiting step of transcription elongation (Lis, 1998Go). For example, heat shock response genes, such as hsp70, are constitutively occupied by a RNA polymerase II (Pol II) complex that is paused proximal to the promoter after transcription initiation (Rougvie and Lis, 1988Go; Rasmussen and Lis, 1993Go). Transcription elongation is inhibited until heat shock stimulation occurs, at which time the paused Pol II becomes hyperphosphorylated and transcript synthesis proceeds. Several factors have been implicated in the stimulation or repression of transcription elongation (Conaway et al., 2000Go; Winston, 2001Go; Yamaguchi et al., 2001Go; Zorio and Bentley, 2001Go), but their precise regulatory roles during development remain elusive.

Genetic analysis in yeast provided the first indication that the factors Spt4, Spt5 and Spt6 could regulate transcription elongation (Winston et al., 1984Go; Swanson and Winston, 1992Go; Bortvin and Winston, 1996Go; Hartzog et al., 1998Go). Biochemical studies demonstrated that Spt4 and Spt5 form a physical complex, known as DSIF (DRB sensitivity inducing factor), that interacts with Pol II (Hartzog et al., 1998Go; Wada et al., 1998Go). The DSIF complex is involved with both stimulation and repression of transcription elongation in vitro. For example, in an in vitro assay with limiting nucleotide concentrations, addition of DSIF stimulates elongation; by contrast, under different experimental conditions, DSIF is required for the repression of elongation by the inhibitor DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole) (Wada et al., 1998Go).

Specific in vivo contexts may involve either the stimulatory function or the repressive function of the Spt4/Spt5 complex. During HIV infection, Tat-dependent transactivation of viral transcription requires DSIF for the progress of transcription elongation (Wu-Baer et al., 1998Go; Garber and Jones, 1999Go). A stimulatory role for the Spt4/Spt5 complex has also been suggested by the observation that Spt5 is located at sites of active transcription on Drosophila polytene chromosomes and is recruited to heat shock loci upon heat shock stimulation (Andrulis et al., 2000Go; Kaplan et al., 2000Go). Repressive functions of the Spt4/Spt5 complex may also be important in vivo. A zebrafish mutation (foggym806) that inhibits the development of dopaminergic neurons has been shown to alter one amino acid (V1012D) in the C terminus of the zebrafish Spt5 protein (Guo et al., 2000Go). Biochemical analysis of the foggym806 mutant Spt5 protein indicates that its repressive function is reduced, but its stimulatory function is unaffected, suggesting that Spt4/Spt5-mediated repression of transcription elongation regulates neuronal development. Although these studies suggest that the Spt4/Spt5 complex has multiple important roles in vivo, the developmental consequences of a complete loss of function of Spt4 or Spt5 have not yet been analyzed.

Spt6 is often considered together with Spt4 and Spt5, as mutations of all three genes cause similar phenotypes in yeast (Winston et al., 1984Go; Swanson and Winston, 1992Go; Hartzog et al., 1998Go). However, the biochemical properties of Spt6 are distinct from those of Spt4 and Spt5. Spt6 is not tightly bound to the Spt4/Spt5 complex (Hartzog et al., 1998Go), and, unlike Spt4 and Spt5, Spt6 binds to histones, particularly histone H3 (Bortvin and Winston, 1996Go). Furthermore, Spt6 can assemble nucleosomes on a plasmid template in vitro, and yeast spt6 mutations can cause alterations in chromatin structure similar to those caused by mutations in histone genes (Bortvin and Winston, 1996Go). Altogether, these data suggest that Spt6 could regulate transcription by modulating chromatin assembly.

The roles of Spt6 during transcription elongation have not been studied in as much detail as the roles of the Spt4/Spt5 complex. Like Spt4 and Spt5, Spt6 may stimulate transcription elongation: in Drosophila, Spt6 colocalizes with Spt5 at sites of active transcription and is recruited to heat shock loci upon heat shock stimulation (Andrulis et al., 2000Go; Kaplan et al., 2000Go). Spt6 may also function to repress transcriptional progress: in yeast, mutation of spt6 suppresses the repression of SUC2 transcription caused by a snf5 mutation (Bortvin and Winston, 1996Go). Thus, Spt6 could potentially play either a stimulatory or a repressive role during development. In the C. elegans embryo, emb-5, which encodes a Spt6-like protein, interacts genetically with the Notch pathway (Hubbard et al., 1996Go). Furthermore, EMB-5 can interact physically with Notch, and so EMB-5 may facilitate transcriptional responses to Notch signaling (Hubbard et al., 1996Go). Overall, the exact functions of Spt6 in vivo, and how they relate to the functions of the Spt4/Spt5 complex, are not yet clear.

Direct comparison of the effects of disabling the Spt4/Spt5 complex and the effects of disabling Spt6 could clarify the roles of these factors. We describe a class of zebrafish mutations (pandora, sk8 and s30) that cause similar embryonic phenotypes. We demonstrate that the pandora gene encodes a zebrafish Spt6 protein, and that the sk8 and s30 mutations are null alleles of the foggy/spt5 locus. Furthermore, using both a heat-inducible reporter transgene and quantitative real-time RT-PCR, we show that both Spt6 and Spt5 are necessary for efficient kinetics of the transcriptional response to heat shock stimulation. These data provide the first genetic evidence for parallel stimulatory functions of Spt6 and Spt5 in metazoan transcription. Furthermore, this study demonstrates the importance of the precise control of transcriptional kinetics for proper embryogenesis.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Zebrafish and embryos
Zebrafish and embryos were maintained at 28°C and staged as previously described (Westerfield, 1995Go). panm313, fogs30 and fogsk8 are recessive embryonic lethal mutations that segregate in a Mendelian fashion. Mutant embryos were generated by mating adult heterozygotes or double heterozygotes. The sk8 mutation was identified during a screen for ethylnitrosourea-induced mutations that disrupt cardiogenesis (D. H. L., A. Schier, and D. Y., unpublished). The s30 mutation arose spontaneously in the Princeton University zebrafish colony and was identified during routine intercrosses. sk8 and s30 fail to complement each other: when crossing a sk8 heterozygote to a s30 heterozygote, 24.4% (133/545) of the progeny display the mutant phenotype.

In situ hybridization
Whole-mount in situ hybridization was carried out as previously described (Yelon et al., 1999Go). An antisense spt6 probe was generated from a 1534 bp fragment of spt6 cDNA (beginning at nucleotide 2425). An antisense spt5 probe was generated from a full-length spt5 EST (fb16g03; http://zfish.wustl.edu).

Photography
Embryos were viewed with Zeiss Axioplan and Zeiss M2Bio microscopes and photographed with a Zeiss Axiocam digital camera. Images were processed using Zeiss Axiovision and Adobe Photoshop software.

Linkage analysis
To map mutations, heterozygous females were bred to wild-type males from the polymorphic strain Wik, and the progeny of these crosses were raised to adulthood. Heterozygous females from the new lines were used to generate gynogenetic haploids or diploids (Westerfield, 1995Go). Genomic DNA preps, bulk segregant analysis, and half-tetrad analysis were performed as previously described (Talbot and Schier, 1999Go; Yelon et al., 2000Go). Markers for linkage analysis were identified on current maps of the zebrafish genome:

http://zebrafish.mgh.harvard.edu/mapping/ssr_map_index.html; http://zebrafish.stanford.edu/genome/Frontpage.html; http://zfish.wustl.edu; http://wwwmap.tuebingen.mpg.de/; http://mgchd1.nichd.nih.gov:8000/zfrh/current.html; and http://134.174.23.167/zonrhmapper/Maps.htm.

Fragment length polymorphisms were detected on agarose gels and single-stranded conformational polymorphisms (SSCP) were detected on acrylamide gels (Shimoda et al., 1999Go; Fornzler et al., 1998Go). SSCP PCR primers were 5'-TTTCTGAAGATGTTCACCGTCTAA-3' and 5'-GTTTTGTTTTCCAGTTCAAATCCT-3' for the 3' UTR of spt6, and 5'-GCTTCTGGCAGAGACACACC-3' and 5'-ACAACTAAGCAGTTAATCATTCTGAGG-3' for the pan mutation.

Cloning of zebrafish spt6 cDNA
Total RNA was extracted from wild-type embryos using Trizol (Life Technologies). Reverse transcription and 5' RACE were performed using the SMART RACE cDNA amplification system (Clontech). RACE primers were based on the fj42h11.y1 EST sequence. PCR products were sequenced using an ABI Prism 377 apparatus. Sequence analysis was performed using NCBI BLAST algorithms and Lasergene (DNASTAR) software. Sequence was confirmed in at least three independent amplifications of each region of cDNA. The GenBank Accession Number for zebrafish spt6 is AF421378.

Identification of mutations in cDNA and genomic DNA
cDNA and genomic DNA were generated from wild-type embryos and mutants as previously described (Yelon et al., 2000Go). Regions of cDNA and genomic DNA were PCR amplified and sequenced, and sequences were confirmed in at least three independent amplifications. PCR primers used were for the mis-spliced region of spt6 (Fig. 2A) (5'-GCTTCTGGCAGAGACACACC-3' and 5'-AGCCGTCCAGAACTTCAATGT-3'), the 5' region of spt5 (Fig. 3A) (5'-GGAAGAACAGGGAAGTGTAGCCGGC-3' and 5'-CATCCAAGATGAAGCCACCATGTCG-3'), the 3' region of spt5 (Fig. 3A) (5'-CGTGAAGAGGAATTCCATTGTTAT-3' and 5'-ACCTTTAATGTGCTCAGTCAG-3') and the mis-spliced region of spt5 (Fig. 3B) (5'-GATGACATCACACAGCAACAATTGC-3' and 5'-ACAAGTGAATTTGCTGTAGGTTTG-3').



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Fig. 2. The panm313 mutation causes mis-splicing of spt6 mRNA and truncation of Spt6 protein. (A) spt6 cDNA from pan mutants at 30 hpf contains a 92 nucleotide insertion. PCR primers that flank an 808 bp fragment of wild-type spt6 cDNA amplify a 900 bp fragment from pan mutant cDNA. This larger fragment represents the major splice isoform of spt6 in pan mutants, although we have infrequently detected trace amounts of normally spliced cDNA that could represent a low level of maintained maternal mRNA or a low level of normal splicing of zygotic mRNA. (B) Comparison of wild-type and pan mutant spt6 cDNA. Labeled regions in the wild-type ORF are proposed to encode discrete protein domains (see D below). The 92 nucleotide insertion in pan mutant cDNA is indicated by a loop, and the location of the in-frame stop codon is marked with an asterisk. (C) Genomic sequence of exon-intron boundaries flanking the mutated intron. The insertion in pan mutant cDNA corresponds to a 92 bp intron. pan genomic DNA contains a point mutation (boxed) in the second nucleotide of this intron. This mutation prevents correct splicing at this junction in pan mutants. The in-frame stop codon is underlined and marked with an asterisk. (D) Predicted protein structure for wild-type Spt6 protein (1726 amino acids) and truncated pan mutant Spt6 protein (829 amino acids). The acidic region, S1 RNA binding domain and SH2 domain are indicated. The insertion of intronic sequence in pan mutant cDNA is predicted to result in the addition of nine mis-sense amino acids before reaching a premature in-frame stop codon. Therefore, the pan mutant Spt6 protein would lack both the S1 RNA binding domain and SH2 domain.

 


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Fig. 3. The s30 and sk8 mutations disrupt the foggy/spt5 locus. (A) The s30 mutation deletes spt5. The first and second lanes demonstrate that a 145 bp fragment of the 5' end of spt5 genomic DNA can not be amplified by PCR from s30 genomic DNA. The third and fourth lanes demonstrate a similar result for a 172 bp fragment of the 3' end of spt5 genomic DNA. Similar results were obtained for all regions of spt5 genomic DNA (data not shown). A control fragment (225 bp) can be amplified from all samples. (B) spt5 cDNA from sk8 mutants is missing 31 bp. PCR primers that flank a 310 bp fragment of wild-type spt5 cDNA amplify a 279 bp fragment from sk8 mutant cDNA. This smaller fragment represents the major splice isoform of spt5 in sk8 mutants at 24 hpf, although we have infrequently detected trace amounts of normally spliced cDNA that could represent a low level of maintained maternal mRNA or a low level of normally spliced mutant mRNA. (C) Comparison of wild-type and sk8 mutant spt5 cDNA. Labeled regions are proposed to encode discrete protein features (see E). The location of the missing nucleotides is indicated following nucleotide 520 in sk8 mutant cDNA. (D) Structure of spt5 genomic DNA indicates that the missing bases correspond to a 31 bp exon, flanked by a 88 bp intron and a 103 bp intron. In wild-type embryos, this region is spliced normally and the 31 bp exon is located between nucleotides 520 and 552 in spt5 cDNA. In sk8 mutants, incorrect splicing results in the omission of the exon and both introns from spt5 cDNA. sk8 genomic DNA contains a point mutation (boxed) in the first nucleotide of the intron following the 31 bp exon. (E) Predicted protein structure for wild-type Spt5 protein (1084 amino acids) and truncated sk8 mutant Spt5 protein (184 amino acids). The acidic region, KOW motifs, and hexapeptide repeats are indicated (Guo et al., 2000Go). The omission of an exon in sk8 mutant cDNA creates a frameshift that is predicted to result in the addition of 10 mis-sense amino acids after residue 174 before reaching a premature in-frame stop codon. Therefore, the sk8 mutant Spt5 protein would lack all KOW motifs and hexapeptide repeats.

 
Microinjection of mRNAs and morpholinos
Expression constructs were generated by subcloning either the spt6 or spt5 ORF into pCS2. The spt6m313 mutant construct was generated by replacing a 375 bp fragment of pCS2-spt6 with a 466 bp fragment amplified from panm313 cDNA. To generate the spt5sk8 mutant construct, the first 609 bp of the ORF in pCS2-spt5 were replaced with the first 578 bp of the ORF amplified from fogsk8 cDNA. Capped synthetic mRNA was generated from expression constructs using the SP6 mMessage mMachine system (Ambion). The spt6 morpholino sequence was: 5'-CCTCGCTCTCGATGAAGTCAGACAT-3' (GeneTools). Embryos were microinjected at the one-cell or two-cell stage with 50-100 pg of mRNA or 3 ng of morpholino.

Genotyping
Embryos were genotyped after in situ hybridization, mRNA injection or heat shock treatment as necessary. Genomic DNA was extracted from individual embryos as previously described (Yelon et al., 2000Go). pan+/+, panm313/+ and panm313/m313 embryos could be distinguished by a SSCP polymorphism generated by the panm313 point mutation. fogs30/s30 embryos could be detected using multiplex PCR to amplify a region of spt5 genomic DNA and a positive control region of genomic DNA simultaneously (Fig. 3A). PCR primers included those described above and positive control primers from the 3' UTR of EST fj20e05 (http://zfish.wustl.edu): 5'-CCATCATCAAGTTCCCCTTG-3' and 5'-GCAACATCCCATGCATCATA-3'. PCR primers used to detect the hsp70-egfp transgene were 5'-TCCGGAGCCACCATGGTGAGCAAGGGCGAGG-3' and 5'-GGTCGGGGTAGCGGCTGAAGCACTGCACGCC-3'.

Analysis of hsp70-egfp transgenics
Fish carrying the stably integrated hsp70-egfp reporter transgene (Halloran et al., 2000Go) were bred to fogs30;pan double heterozygotes. The progeny were raised and adult fish heterozygous for the hsp70-egfp transgene and either or both of fogs30 and pan were identified. These fish were mated with heterozygotes for either or both of fogs30 and pan. The resulting embryos were heat shocked via transfer to embryo medium prewarmed to 37°C. After 1 hour, embryos were cooled to 28°C. Embryos were photographed 30 minutes after the completion of heat shock. Embryonic genotypes were confirmed after photography.

Analysis of hsp70 induction using real-time RT-PCR
Embryos were grown until 18.5 hpf (19-somite stage) and heat shocked as described above. From each clutch, a set of three mutants and a set of three wild-type siblings were flash-frozen at each time point (0, 10, 20, 30, 40 and 60 minutes after initiation of heat shock). Total RNA was extracted using Trizol and treated with DNAse. First-strand cDNA was reverse transcribed using oligo-dT primers and Superscript reverse transcriptase (Life Technologies). For amplification of hsp70 and ß-actin, PCR primers used were 5'-CAACAACCTGCTGGGCAAA-3' and 5'-GCGTCGATGTCGAAGGTCA-3' for hsp70 (AF006007); and 5'-CGAGCAGGAGATGGGAACC-3' and 5'-CAACGGAAACGCTCATTGC-3' for ß-actin (AF057040). Primers were tested using conventional PCR and shown to amplify a single band of approximately 100 bp without production of primer dimers. Real-time PCR, which quantifies the amount of product after each cycle, was carried out on each cDNA sample using an i-cycler (BioRad). Each reaction (30 µl) contained 3 µl of cDNA, 15 µl of 2xSYBR Green Master Mix (ABI) and primers at a final concentration of 100 ng/µl. Reactions were heated to 50°C for 2 minutes followed by 95°C for 10 minutes. Subsequently, the reactions proceeded through 50 cycles at 95°C for 10 seconds and 60°C for 1 minute. The fluorescence level (a quantification of product) was determined after each cycle, allowing the detection of the log phase of amplification. i-cycler software was used to define the cycle in which each sample attained the threshold value of fluorescence. The average ß-actin threshold cycle value was very consistent between preparations. Mean ß-actin threshold cycle value and standard deviation for each genotype including all time points were 22.96±0.94 (wild type); 22.79±0.50 (pan); 22.31±0.53 (fog); and 22.42±0.80 (fog;pan). To account for small variations between preparations, the hsp70 threshold cycle value was compared with the corresponding ß-actin threshold cycle value for each sample. Furthermore, the level of hsp70 transcript detected was compared with the baseline level detected at time zero to indicate the degree of hsp70 induction. The equation used to calculate the relative amount of hsp70 induction was:



(1)

where HX is the hsp70 threshold cycle value at time X, AX is the ß-actin threshold cycle value at time X, H0 is the hsp70 threshold cycle value at time 0, and A0 is the ß-actin threshold cycle value at time 0. According to this equation, hsp70 levels at time 0 were set to a value of 1. hsp70 levels relative to ß-actin at time 0 were very consistent between genotypes. Specifically, the average values (with standard deviation) of 2–(H0–A0) were: 0.136±0.010 (wild type); 0.143±0.021 (pan); 0.140±0.015 (fog); 0.144±0.025 (fog;pan). For both hsp70 and ß-actin, no-RT controls and water controls gave similar high threshold cycle values, demonstrating that contamination contributed to less than 0.1% of quantified product.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pandora, sk8 and s30 mutations cause similar embryonic defects
The pandoram313 (pan) mutation was originally identified during a large-scale screen for mutations affecting zebrafish embryogenesis (Driever et al., 1996Go). Embryos that are homozygous for the pan mutation exhibit several developmental defects, including reduced pigmentation, limited tail growth and small ears (Fig. 1A,B) (Malicki et al., 1996Go). Additionally, pan mutants have a reduced number of myocardial cells, with an especially significant reduction in the number of ventricular myocardial cells (Stainier et al., 1996Go; Yelon et al., 1999Go). As a result, pan mutants fail to establish circulation and exhibit pericardial edema (Fig. 1A,B).



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Fig. 1. Morphological and molecular defects are similar among a class of zebrafish mutants. (A-E) Lateral views of live embryos at 36 hours post-fertilization (hpf), anterior towards the left. All embryos are depicted at the same magnification. Compared with wild-type embryos (A), pan (B), sk8 (C) and s30 (D) mutants exhibit reduced pigmentation, short tails, small ears and pericardial edema. s30;pan double mutants (E) exhibit a more extreme phenotype, including a shorter tail and neural cell death. (F-U) Whole-mount in situ hybridization indicates expression of gata4 (F-I), nkx2.5 (J-M) or cmlc2 (N-U); dorsal views of embryos, anterior towards the top. At 13 hpf (eight-somite stage), expression of gata4 and nkx2.5 are comparable in wild-type embryos (F,J), pan mutants (G,K) and s30 mutants (H,L); however, expression is reduced in s30;pan double mutants (I,M). At 16.5 hpf (15-somite stage), wild-type embryos (N) exhibit robust expression of cmlc2 in differentiating myocardiocytes, but pan mutants (O), s30 mutants (P) and s30;pan double mutants (Q) all lack cmlc2 expression. By 26 hpf, wild-type embryos (R) form a cmlc2-expressing heart tube, and pan mutants (S) generate a small and variable number of disorganized myocardiocytes (Yelon et al., 1999Go), but cmlc2 remains undetectable in s30 mutants (T) and s30;pan double mutants (U).

 
We identified two new mutations, called sk8 and s30, that are phenotypically similar to pan (Fig. 1C,D). Like pan, sk8 and s30 cause several abnormalities, including pigmentation, tail, ear and cardiac defects. sk8 and s30 fail to complement each other (see Materials and Methods), indicating that these mutations affect the same locus. sk8 homozygotes, s30 homozygotes and sk8/s30 trans heterozygotes display similarly potent phenotypes (data not shown); hereafter, only the s30 mutant phenotype will be depicted.

While morphological aspects of the pan and s30 mutant phenotypes are very obvious by 36 hours post-fertilization (hpf) (Fig. 1A-D), mutants appear morphologically normal until mid-somitogenesis (17-18 hpf), when they begin to display subtly shorter tails than their wild-type siblings (data not shown). To further investigate the earliest requirements for pan and s30, we focused our attention on the cardiac phenotype. Myocardial cells arise from bilateral populations of anterior lateral plate mesoderm that express gata4 and nkx2.5 (Serbedzija et al., 1998Go; Chen and Fishman, 1996Go). Initial expression of gata4 and nkx2.5 appears normal in pan mutants and s30 mutants (Fig. 1F-H,J-L). However, terminal myocardial differentiation is dramatically inhibited: pan mutants and s30 mutants fail to express the myocardial gene cmlc2 (Yelon et al., 1999Go) at 16.5 hpf (Fig. 1N-P). s30 mutants never generate significant numbers of myocardial cells, but pan mutants can generate a small number of disorganized myocardiocytes by 26 hpf (Fig. 1R-T) (Yelon et al., 1999Go). These phenotypes suggest that the pan and s30 gene products are both required for the normal differentiation of precardiac mesoderm into myocardial tissue.

To investigate the genetic relationship between pan and s30, we examined embryos homozygous for both mutations. s30;pan double mutants exhibit a more extreme phenotype than either pan or s30 mutants (Fig. 1E). Notably, double mutants have a greater reduction in tail length and a significant amount of neural cell death. In addition, s30;pan double mutants exhibit a more dramatic cardiac phenotype than either pan or s30 mutants. Specifically, s30;pan double mutants exhibit reduced expression of gata4 and nkx2.5 (Fig. 1I,M). Predictably, s30;pan double mutants fail to generate any differentiated myocardial tissue (Fig. 1Q,U). Overall, the pan, s30 and s30;pan mutant phenotypes suggest that the pan and s30 gene products have related functions and that they cooperate during several developmental processes.

Identification of candidate genes on the zebrafish genome map
In order to identify the genes encoded by the pan and s30 loci, we mapped both mutations using half-tetrad analysis and bulk segregant analysis (Talbot and Schier, 1999Go). We mapped pan to a <1 cM interval between the SSLP markers Z21106 (two recombinants in 505 meioses) and Z10567 (12 recombinants in 2282 meioses) on zebrafish linkage group 21. We mapped s30 to a <1 cM interval between the SSLP markers Z7871 (four recombinants in 587 meioses) and Z20993 (one recombinant in 587 meioses) on zebrafish linkage group 15.

Hypothesizing that pan and s30 play similar roles, we examined the pan and s30 intervals on current maps of the zebrafish genome (see Materials and Methods), focusing on mapped genes that might have similar functions. In the s30 interval, we found the previously described zebrafish gene foggy/spt5, predicted to encode a transcription elongation factor (Guo et al., 2000Go). In the pan interval, we identified an EST (fj42h11; http://zfish.wustl.edu) that is predicted to encode a partial protein with similarity to Spt6, another transcription elongation factor. We then pursued spt6 and spt5 as candidates for the genes affected by the pan and s30 mutations, respectively.

The pan locus encodes Spt6
Using a polymorphism located within the 3' UTR of the fj42h11 EST, we determined that fj42h11 was tightly linked to the pan mutation (0 recombinants in 1652 meioses). We then used 5' RACE to clone a complete cDNA corresponding to fj42h11. Sequence analysis revealed a 5178 bp open reading frame (ORF) (Fig. 2B), predicted to encode a protein similar to the human, mouse, Drosophila, C. elegans and yeast Spt6 proteins (Table 1). All of these Spt6 homologs have similar amino acid sequences, including an acidic N-terminal region, a S1-type RNA binding domain, and a SH2 domain (Table 1, Fig. 2D). Based on these sequence comparisons, we hereafter refer to this zebrafish gene as spt6.


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Table 1.
 
Examination of spt6 cDNA sequence from pan mutants revealed that the pan mutant cDNA is larger than the wild-type cDNA, owing to an insertion of 92 nucleotides following nucleotide 2460 (Fig. 2A,B). Analysis of spt6 genomic DNA indicated that this insertion corresponds to the intron that is normally spliced out between nucleotides 2460 and 2461 of the wild-type spt6 mRNA (Fig. 2C). In pan mutant cDNA and genomic DNA, we detected a T-to-A transversion in the second nucleotide of this intron (Fig. 2C). This point mutation is tightly linked to the pan mutant phenotype (0 recombinants in 2282 meioses). A conserved T is found in the second intronic nucleotide of all eukaryotic splice donor sites (Padgett et al., 1986Go). Thus, the pan mutation is predicted to inhibit splicing at this junction, consistent with the sequence of the pan mutant spt6 cDNA. Owing to an in-frame stop codon located within the insertion, the pan mutant spt6 cDNA is predicted to produce a truncated Spt6 protein that is unlikely to retain normal function (Fig. 2C,D). To confirm that reduction of Spt6 activity causes the pan mutant phenotype, we inhibited Spt6 translation in vivo using an antisense morpholino (Summerton and Weller 1997Go; Nasevicius and Ekker, 2000Go) directed against spt6. Injection of the anti-spt6 morpholino produced a phenocopy of the pan mutation (see on-line supplemental data). These data indicate that the pan locus encodes Spt6.

The s30 and sk8 mutations are null alleles of foggy/spt5
Similar to our analysis of spt6 as a candidate gene for pan, we looked for mutations in spt5 cDNA and genomic DNA in s30 and sk8 mutants. PCR analysis of genomic DNA indicated that the entire spt5 gene is deleted in s30 mutants (Fig. 3A). This deletion is tightly linked to the s30 mutant phenotype (0 recombinants in 587 meioses). Thus, s30 is a deficiency allele of foggy/spt5, but the deletion may also remove neighboring genomic DNA. By contrast, the sk8 mutation causes a lesion specific to spt5. Examination of spt5 cDNA sequence from sk8 mutants revealed that the sk8 mutant cDNA is smaller than the wild-type cDNA, with 31 nucleotides missing after nucleotide 520 (Fig. 3B,C). Analysis of spt5 genomic DNA indicated that this omission corresponds to a 31 bp exon (Fig. 3D). In sk8 mutant genomic DNA, we detected a point mutation (G-to-A transition) in the first nucleotide of the intron following this small exon (Fig. 3D). This mutation affects a nucleotide that is conserved in all splice donor sites (Padgett et al., 1986Go). Thus, splicing is inhibited at the mutated junction, evidently leading to the removal of an entire exon. This splicing error produces a frameshift in the sk8 mutant spt5 cDNA, and, owing to an in-frame stop codon 30 nucleotides downstream of the frameshift, this cDNA is predicted to produce a truncated Spt5 protein (Fig. 3E). The predicted sk8 mutant Spt5 protein lacks all KOW motifs (predicted to bind to Pol II) and hexapeptide repeats (predicted to be phosphorylation sites), and is unlikely to retain normal function. These genetic data indicate that the s30 and sk8 mutations are alleles of the previously described foggy (fog) locus (Guo et al., 2000Go). Furthermore, as fogs30 is a deficiency and fogsk8/s30 trans heterozygotes are phenotypically identical to fogs30/s30 homozygotes (data not shown), both fogs30 and fogsk8 appear to be null alleles. By contrast, the previously identified hypomorphic allele fogm806 is a mis-sense mutation that alters a residue in the C-terminus of Spt5 (V1012D) and leaves significant aspects of Spt5 function intact (Guo et al., 2000Go).

Overexpression of spt6 and spt5 can rescue respective mutant phenotypes
To provide additional confirmation of the genes disrupted by the pan, s30 and sk8 mutations, we examined the effects of overexpressing spt6 and spt5. Injection of synthetic spt6 mRNA at the one-cell stage can rescue the pan mutant phenotype (Fig. 4A-H; Table 2). Similarly, injection of synthetic spt5 mRNA can rescue the fog mutant phenotype (Fig. 4I-P; Table 2). In both cases, overexpression of synthetic mRNA restores normal pigmentation, tail length, ear formation and cardiac function to mutants without affecting the development of wild-type siblings (Fig. 4; Table 2). Examination of cmlc2 expression confirmed that myocardial differentiation occurs efficiently in rescued embryos (Fig. 4E-H,M-P). Injection of spt6m313 mutant mRNA fails to rescue the pan mutant phenotype, and injection of spt5sk8 mutant mRNA fails to rescue the fog mutant phenotype, verifying that the mutant gene products are nonfunctional (Table 2). Injection of spt5 mRNA can not rescue pan mutants, and injection of spt6 mRNA can not rescue fog mutants (Table 2). Altogether, these data confirm that inactivation of Spt6 and Spt5 are responsible for the pan and fog mutant phenotypes, respectively.



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Fig. 4. spt6 and spt5 mRNA rescue the defects in pan and fogs30 mutants, respectively. Uninjected embryos (A,B,E,F,I,J,M,N) are compared with embryos that were injected with synthetic mRNA, either spt6 (C,D,G,H) or spt5 (K,L,O,P). All embryonic genotypes were confirmed following photography (see Materials and Methods). Genotypes are: wild-type embryos (A,C,E,G,I,K,M,O); pan mutants (B,D,F,H); and fogs30 mutants (J,L,N,P). (A-D,I-L) Lateral views of live embryos at 36 hpf, anterior towards the left. Injection of wild-type mRNA rescues pigmentation, tail length, ear formation and cardiac function in mutants, and does not affect wild-type siblings. Rescued embryos survive for 4-5 days post-fertilization. (E-H,M-P) Whole-mount in situ hybridization indicates expression of cmlc2 at 16.5-17.5 hpf; dorsal views of embryos, anterior towards the top. Injection of wild-type mRNA rescues the timely and robust expression of cmlc2 expression in mutants and does not affect expression in wild-type siblings.

 

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Table 2.
 
spt6 and spt5 are expressed maternally and zygotically
Previous studies of spt5 expression in zebrafish indicated its broad distribution throughout the embryo from 10 hpf onwards (Guo et al., 2000Go). We extended this analysis by comparing the expression patterns of spt6 and spt5. At early stages, both transcripts are supplied maternally and found throughout the blastoderm (Fig. 5A,D). After the initiation of zygotic transcription, the expression of both genes remains ubiquitous (Fig. 5B,E). By 24 hpf, spt6 and spt5 expression levels are still broad, but are highest in the embryonic brain (Fig. 5C,F).



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Fig. 5. spt6 and spt5 are expressed maternally and zygotically throughout the zebrafish embryo. Whole-mount in situ hybridization, comparing the expression patterns of spt6 (A-C) and spt5 (D-F). Wild-type embryos at 2.75 hpf (512-cell) (A) or 2.25 hpf (128-cell) (D) demonstrate that both spt6 (A) and spt5 (D) are expressed throughout the blastoderm before the initiation of zygotic transcription and are therefore maternally supplied. (A,D) Lateral views, with the animal pole at the top. Wild-type embryos at 16.5 hpf (B) or 10 hpf (E) demonstrate that spt6 (B) and spt5 (E) are expressed throughout the embryo after the initiation of zygotic transcription. Inset in E depicts a fogs30 mutant embryo at 10 hpf. spt5 mRNA is barely detectable in fogs30 mutants at this stage, in keeping with the deletion of spt5 in fogs30 mutant genomic DNA. (B,E) Lateral views, anterior towards the top. Wild-type embryos at 24 hpf (C) or 26 hpf (F) demonstrate that spt6 (C) and spt5 (F) are still broadly expressed, with highest levels in the embryonic brain. (C,F) Lateral views, anterior towards the left.

 
Zygotic expression of spt5 is not detectable in fogs30 mutants, in accordance with the deletion of spt5 from fogs30 genomic DNA (Fig. 5E, inset). The expression pattern of spt5 in fogs30 mutants indicates that most maternally provided spt5 mRNA is degraded by 10 hpf (Fig. 5E). Expression of spt5 and spt6 in both fogsk8 mutants and pan mutants is similar to expression in wild-type siblings (data not shown).

Spt6 and Spt5 are required to promote transcriptional response to heat shock
Having established that pan and fog mutants lack zygotic supplies of Spt6 and Spt5, we were provided with an opportunity to test the roles of these factors during transcription in vivo. First, we generated wild-type and mutant embryos that were heterozygous for a hsp70-egfp reporter transgene, a stably integrated construct in which egfp expression is controlled by the zebrafish hsp70 promoter (Halloran et al., 2000Go). When maintained at normal temperature, transgenic wild-type embryos, pan mutants, fogs30 mutants and fogs30;pan double mutants do not produce detectable levels of GFP (Fig. 6A-D), although consistent autofluorescence is visible in the embryonic yolk. We administered a 1 hour heat shock to these embryos beginning at 18.5 hpf, a stage at which we expected minimal maternal contribution of Spt6 or Spt5. After heat shock stimulation, wild-type embryos produce high levels of GFP, but pan mutants and fogs30 mutants produce only low and less uniform levels of GFP (Fig. 6E-G,I-K). fogs30;pan double mutants are more severely affected and do not produce any detectable GFP (Fig. 6H,L). Thus, heat shock response appears to be inefficient in the absence of Spt6 or Spt5 and undetectable in the absence of both proteins.



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Fig. 6. Spt6 and Spt5 are required for an efficient heat shock response. (A-L) Lateral views of live embryos at 20 hpf (22-somite stage), anterior towards the left. Green fluorescence indicates production of GFP after a 1 hour heat shock and a 0.5 hour recovery period. Autofluorescence (yellow-green) is also visible in embryonic yolks. All photographs were taken with the same exposure time, and embryonic genotypes were confirmed after photography (see Materials and Methods). Genotypes are wild-type embryos (A,E,I); pan mutants (B,F,J); fogs30 mutants (C,G,K); and fogs30;pan double mutants (D,H,L). All embryos are heterozygous for the hsp70-egfp transgene (Halloran et al., 2000Go). Embryos maintained at normal temperature do not produce detectable GFP (A-D). After heat shock, pan and fog mutants produce less GFP than wild-type embryos, and double mutants do not produce any detectable GFP (E-H, with higher magnification views of the tail in I-L). Similar results were obtained when heat shock was administered from 15-16 hpf, before mutant phenotypes are morphologically apparent (data not shown). (M) Spt6 and Spt5 are required for efficient induction of hsp70 expression during a 1 hour heat shock. Graph of induction of endogenous hsp70 expression, detected by real-time RT-PCR. (See Materials and Methods for details of technique and data processing.) Wild-type embryos (black) induce significantly higher levels of hsp70 mRNA with more rapid kinetics than pan mutants (green) or fogs30 mutants (blue). hsp70 induction is further inhibited in fogs30;pan double mutants (red). Each data point represents the average degree of induction of hsp70 expression at a particular time point, relative to the low, but detectable, levels of hsp70 expression at time zero. Standard deviation from the mean is indicated by error bars. To account for small variances in RNA extraction and cDNA synthesis, levels of hsp70 expression were normalized relative to levels of stable ß-actin expression (see Materials and Methods).

 
Inhibition of GFP production in hsp70-egfp transgenic embryos could be the result of diminished transcription; alternatively, GFP production could be inhibited post-transcriptionally through modification of transcript stability, translation or protein processing/folding. To determine whether absence of Spt6 and/or Spt5 interferes with the transcriptional response to heat shock, we examined the levels of endogenous hsp70 mRNA directly, using real-time RT-PCR (see Materials and Methods). We administered a 1 hour heat shock to embryos beginning at 18.5 hpf, the earliest stage at which mutants can be reliably distinguished from their wild-type siblings by subtle morphological criteria. Within 10 minutes of heat shock stimulation, wild-type embryos express 25 times more hsp70 than they do before heat shock (Fig. 6M). Wild-type expression of hsp70 peaks at nearly 75 times the basal expression level after 40 minutes of heat shock, in accordance with previous descriptions of the kinetics of hsp70 induction in zebrafish (Lele et al., 1997Go). In contrast to the dynamics of wild-type expression, induction of hsp70 expression in pan mutants or fogs30 mutants is slow and weak (Fig. 6M). After 10 minutes of heat shock stimulation, pan or fogs30 mutants express only 15 times more hsp70 than before heat shock. Additionally, in pan or fogs30 mutants, the level of hsp70 induction does not rise more than 21 times over baseline during the course of a 1 hour heat shock. The sensitivity of the real-time RT-PCR assay also revealed that fogs30;pan double mutants can generate a weak response to heat shock stimulation, although induction of hsp70 expression in double mutants is more severely inhibited than in single mutants (Fig. 6M). Therefore, efficient kinetics of the transcriptional response to heat shock depends upon the individual and coordinated functions of Spt6 and Spt5.


    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The zebrafish panm313, fogsk8 and fogs30 mutations provide the first opportunity to compare the effects of spt6 and spt5 loss-of-function in metazoans. These pan and fog mutations cause similar embryonic defects, including inhibition of myocardial differentiation. Furthermore, fog;pan double mutants have enhanced abnormalities, including earlier defects in the formation of precardiac mesoderm. We have observed the same functional relationship between pan and fog during the transcriptional response to heat shock stimulation: pan mutants and fog mutants exhibit similar difficulties with efficient induction of hsp70, and fog;pan double mutants exhibit more extreme inhibition of hsp70 induction. Altogether, our genetic analysis indicates that Spt6 and Spt5 play essential roles of comparable importance during development, controlling multiple aspects of differentiation through stimulation of gene expression.

Spt6 and Spt5 promote transcription during heat shock response
Previous studies in Drosophila have established that Spt6 and Spt5 colocalize with hyperphosphorylated Pol II at transcriptionally active loci on polytene chromosomes (Andrulis et al., 2000Go; Kaplan et al., 2000Go). In particular, both Spt6 and Spt5 are recruited to heat shock genes, including hsp70, shortly after the initiation of heat shock. These data suggested that Spt6 and Spt5 could function to promote transcription elongation at heat shock loci. Our data provide genetic evidence in support of this model by demonstrating that the absence of Spt6 or Spt5 significantly affects the efficacy and kinetics of hsp70 induction.

Our analysis cannot resolve whether Spt6 and Spt5 act directly at the hsp70 promoter, nor can it resolve whether Spt6 and Spt5 act at the level of transcription elongation per se. It is possible that Spt6 and Spt5 regulate hsp70 mRNA stability, or that they are responsible for generating other factors required to manifest a heat shock response. However, the zebrafish hsp70 promoter has significant sequence similarity to characterized heat shock promoters in Drosophila (Halloran et al., 2000Go), and it is therefore reasonable to expect that Spt6 and Spt5 would be present at the zebrafish hsp70 promoter and could function there directly. In any case, our data clearly indicate that Spt6 and Spt5 act to promote gene expression, possibly via regulation of transcription elongation.

Spt5 and Spt6 seem to be equivalently important for transcriptional efficiency, but their biochemical properties are very different. We have shown that Spt5 can not rescue pan mutants and that Spt6 can not rescue fog mutants, confirming that these factors can not substitute for each other functionally, as previously demonstrated in yeast (Swanson and Winston, 1992Go). Thus, Spt5 and Spt6 are likely to play distinct biochemical roles that both contribute to transcriptional progress. Removal of either Spt5 or Spt6 cripples transcriptional efficiency, and removal of both Spt5 and Spt6 compounds the transcriptional defects. Even so, hsp70 induction is not completely eliminated in fog;pan double mutants. The low levels of hsp70 in double mutants may be facilitated by residual supplies of maternal Pan or Fog or contributions from other elongation factors, including additional Spt5 or Spt6 homologs. However, there are currently no indications of other zebrafish genes similar to spt6 or spt5 in EST or genomic sequence databases (J. L. F., B. R. K. and D. Y., unpublished).

Previous studies have indicated that the Spt4/Spt5 complex can repress transcription elongation in vitro (Wada et al., 1998Go). Furthermore, colocalization of Spt5 with paused, hypophosphorylated Pol II at heat shock loci in Drosophila has suggested that Spt5 could be involved with polymerase pausing (Andrulis et al., 2000Go; Kaplan et al., 2000Go). We do not detect a loss of repression (i.e. constitutive activation) of hsp70 expression in fog or pan mutants. However, detection of constitutive expression in our RT-PCR assay depends upon the successful production of full-length transcripts. Therefore, evidence in favor of a repressive function of Spt5 could be masked by the requirement for a stimulatory function of Spt5.

Spt6 and Spt5 promote transcription during development
The embryonic phenotypes of pan and fog mutants indicate that the loss of either Spt6 or Spt5 can cause similar developmental defects. Extrapolating from the activities of Spt6 and Spt5 during hsp70 induction, it is likely that the pan and fog mutant phenotypes represent the cumulative effects of inefficient transcription at multiple embryonic loci. For example, myocardial development in zebrafish requires the activity of the transcription factors Gata5 and Hand2 (Reiter et al., 1999Go; Yelon et al., 2000Go). In pan and fog mutants, it is likely that the genes regulated by Gata5, Hand2 and other myocardial transcription factors are inefficiently expressed; thus, terminal myocardial differentiation is inhibited, although precardiac mesoderm forms relatively normally. In fog;pan double mutants, transcriptional efficiency is further compromised; thus, myocardial development is affected at an earlier stage, during the formation or maintenance of precardiac mesoderm. Similar models can be proposed for other aspects of the mutant phenotypes; however, not every tissue seems to be equally affected by a loss of Spt6 and/or Spt5. For example, fog;pan double mutants exhibit normal lens induction at 19.5 hpf and normal touch responses at 24 hpf, even though they are significantly affected in other aspects of differentiation at these stages (B. R. K. and D. Y., unpublished).

Because of the maternal contribution of Spt6 and Spt5, it is difficult for our studies to resolve whether Spt6 and Spt5 are universally required to promote transcription at all zebrafish promoters. Although pan and fog mutants are significantly abnormal, more severe phenotypes, particularly developmental arrest before gastrulation, can be generated by treating zebrafish embryos with the Pol II inhibitor {alpha}-amanitin (Kane et al., 1996Go) or by morpholino-mediated inhibition of TBP function (Muller et al., 2001Go). Both of these treatments affect zygotic transcription from its beginning in the embryo. Therefore, we expect that maternally supplied Spt6 and Spt5 permit normal transcription at early stages in pan and fog mutants, and that the observed phenotypes reflect the roles of zygotic gene products after maternal supplies have been depleted in particular tissues. Further study will be required to determine whether any specific promoters vary in their requirements for Spt6 and Spt5.

Previous studies of the fogm806 hypomorphic allele have indicated specific roles for the repressive activity of Spt5 during neuronal development in zebrafish (Guo et al., 2000Go). In vitro assays have indicated that the fogm806 mutant Spt5 protein has limited repressive function, but retains its stimulatory function. fogm806 mutants exhibit a significant deficit of dopaminergic neurons at 28 hpf, but have normal body shape, tail length, ear development and heart tube formation, although cardiovascular function appears impaired after 30 hpf (Guo et al., 1999Go; Guo et al., 2000Go). By contrast, the mutant phenotypes caused by the null alleles fogs30 and fogsk8 are substantially more dramatic and appear earlier than the fogm806 mutant phenotype. Based on these phenotypic comparisons and our demonstration of the stimulatory role of Spt5 during hsp70 induction, we propose that the stimulatory role of Spt5 is more widely employed, in a broader range of embryonic tissues, than is its repressive role. This model is consistent with colocalization studies in Drosophila that demonstrate preferential association of Spt5 with hyperphosphorylated Pol II rather than paused Pol II (Andrulis et al., 2000Go; Kaplan et al., 2000Go).

A system for study of transcription elongation during development
Future studies of zebrafish pan and fog mutants will allow a more detailed analysis of the precise roles of Spt6 and Spt5 during embryogenesis. In particular, our phenotypic rescue assay will allow detailed structure-function analysis of Spt6 and Spt5 in vivo, facilitating correlation of proposed functional domains (Yamaguchi et al., 1999Go; Ivanov et al., 2000Go) with specific roles during embryogenesis. Furthermore, ongoing genetic screens are likely to identify additional mutations in the pan/fog phenotypic class. We have already identified a new mutation in this class (sk11) that is phenotypically indistinguishable from fogs30 or fogsk8 and that complements both pan and fog (D. H. L., A. Schier, and D. Y., unpublished). Thus, the gene disrupted by sk11 is likely to be another important component of the transcriptional apparatus, perhaps even a novel factor. Altogether, the zebrafish embryo provides unique opportunities for the analysis of transcriptional regulation during embryogenesis.


    ACKNOWLEDGMENTS
 
We thank several people for their contributions to this project, particularly Benson Lu for mapping pan between Z10567 and Z21106. We also thank C. Ott for initial work on pan; S. Zimmerman, T. Bruno, A. Navarro and S. Waldron for excellent fish care; J. Malicki for pan heterozygotes; J. Kuwada for hsp70-egfp transgenic fish; R. Yang and B. Baysa for sequencing; N. Tanese, J. Treisman and members of the Schier and Yelon laboratories for helpful discussions; D. Littman, J. Lafaille, M. Lafaille and A. Wensky for help with real-time RT-PCR; and A. Schier for exceptional support and generosity. B. R. K. and D. H. L. are supported by the MSTP of NYU School of Medicine. This work was supported by a Burroughs Wellcome Fund Career Award and a New York City Council Speaker’s Fund for Biomedical Research Award to D. Y.


    REFERENCES
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Andrulis, E. D., Guzman, E., Doring, P., Werner, J. and Lis, J. T. (2000). High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 14, 2635-2649.[Abstract/Free Full Text]

Bortvin, A. and Winston, F. (1996). Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272, 1473-1476.[Abstract]

Chen, J.-N. and Fishman, M. C. (1996). Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation. Development 122, 3809-3816.[Abstract/Free Full Text]

Conaway, J. W., Shilatifard, A., Dvir, A. and Conaway, R. C. (2000). Control of elongation by RNA polymerase II. Trends Biochem. Sci. 25, 375-380.[Medline]

Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y. R., Zwartkruis, F., Abdelilah, S., Rangini, Z. et al. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46.[Abstract/Free Full Text]

Fornzler, D., Her, H., Knapik, E. W., Clark, M., Lehrach, H., Postlethwait, J. H., Zon, L. I. and Beier, D. R. (1998). Gene mapping in zebrafish using single-strand conformation polymorphism analysis. Genomics 51, 216-222.[Medline]

Garber, M. E. and Jones, K. A. (1999). HIV-1 Tat: coping with negative elongation factors. Curr. Opin. Immunol. 11, 460-465.[Medline]

Guo, S., Wilson, S. W., Cooke, S., Chitnis, A. B., Driever, W. and Rosenthal, A. (1999). Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons. Dev. Biol. 208, 473-487.[Medline]

Guo, S., Yamaguchi, Y., Schilbach, S., Wada, T., Lee, J., Goddard, A., French, D., Handa, H. and Rosenthal, A. (2000). A regulator of transcriptional elongation controls vertebrate neuronal development. Nature 408, 366-369.[Medline]

Halloran, M. C., Sato-Maeda, M., Warren, J. T., Su, F., Lele, Z., Krone, P. H., Kuwada, J. Y. and Shoji, W. (2000). Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127, 1953-1960.[Abstract/Free Full Text]

Hartzog, G. A., Wada, T., Handa, H. and Winston, F. (1998). Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12, 357-369.[Abstract/Free Full Text]

Hubbard, E. J., Dong, Q. and Greenwald, I. (1996). Evidence for physical and functional association between EMB-5 and LIN-12 in Caenorhabditis elegans. Science 273, 112-115.[Abstract]

Ivanov, D., Kwak, Y. T., Guo, J. and Gaynor, R. B. (2000). Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20, 2970-2983.[Abstract/Free Full Text]

Kane, D. A., Hammerschmidt, M., Mullins, M. C., Maischein, H. M., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P. et al. (1996). The zebrafish epiboly mutants. Development 123, 47-55.[Abstract/Free Full Text]

Kaplan, C. D., Morris, J. R., Wu, C. and Winston, F. (2000). Spt5 and Spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 14, 2623-2634.[Abstract/Free Full Text]

Lele, Z., Engel, S. and Krone, P. H. (1997). hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos. Dev. Genet. 21, 123-133.[Medline]

Lis, J. (1998). Promoter-associated pausing in promoter architecture and postinitiation transcriptional regulation. Cold Spring Harb. Symp. Quant. Biol. 63, 347-356.[Medline]

Malicki, J., Schier, A. F., Solnica-Krezel, L., Stemple, D. L., Neuhauss, S. C., Stainier, D. Y., Abdelilah, S., Rangini, Z., Zwartkruis, F. and Driever, W. (1996). Mutations affecting development of the zebrafish ear. Development 123, 275-283.[Abstract/Free Full Text]

Muller, F., Lakatos, L., Dantonel, J., Strahle, U. and Tora, L. (2001). TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Curr. Biol. 11, 282-287.[Medline]

Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216-220.[Medline]

Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S. and Sharp, P. A. (1986). Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55, 1119-1150.[Medline]

Rasmussen, E. B. and Lis, J. T. (1993). In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci. USA 90, 7923-7927.[Abstract/Free Full Text]

Reiter, J. F., Alexander, J., Rodaway, A., Yelon, D., Patient, R., Holder, N. and Stainier, D. Y. R. (1999). Gata5 is required for the development of the heart and endoderm in zebrafish. Genes Dev. 13, 2983-2995.[Abstract/Free Full Text]

Rougvie, A. E. and Lis, J. T. (1988). The RNA polymerase II molecule at the 5' end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell 54, 795-804.[Medline]

Serbedzija, G. N., Chen, J. N. and Fishman, M. C. (1998). Regulation in the heart field of zebrafish. Development 125, 1095-1101.[Abstract/Free Full Text]

Shimoda, N., Knapik, E. W., Ziniti, J., Sim, C., Yamada, E., Kaplan, S., Jackson, D., de Sauvage, F., Jacob, H. and Fishman, M. C. (1999). Zebrafish genetic map with 2000 microsatellite markers. Genomics 58, 219-232.[Medline]

Stainier, D. Y. R., Fouquet, B., Chen, J. N., Warren, K. S., Weinstein, B. M., Meiler, S. E., Mohideen, M. A., Neuhauss, S. C., Solnica-Krezel, L., Schier, A. F. et al. (1996). Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 123, 285-292.[Abstract/Free Full Text]

Summerton, J. and Weller, D. (1997). Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187-195.[Medline]

Swanson, M. S. and Winston, F. (1992). SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. Genetics 132, 325-336.[Abstract/Free Full Text]

Talbot, W. S. and Schier, A. F. (1999). Positional cloning of mutated zebrafish genes. Methods Cell Biol. 60, 259-286.[Medline]

Wada, T., Takagi, T., Yamaguchi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yano, K., Hartzog, G. A., Winston, F., Buratowski, S. and Handa, H. (1998). DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343-356.[Abstract/Free Full Text]

Westerfield, M. (1995). The Zebrafish Book. Eugene, OR: University of Oregon Press.

Winston, F. (2001). Control of eukaryotic transcription elongation. Genome Biol. 2, 1006.1-1006.3.

Winston, F., Chaleff, D. T., Valent, B. and Fink, G. R. (1984). Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 107, 179-197.[Abstract/Free Full Text]

Wu-Baer, F., Lane, W. S. and Gaynor, R. B. (1998). Role of the human homolog of the yeast transcription factor SPT5 in HIV- 1 Tat-activation. J. Mol. Biol. 277, 179-197.[Medline]

Yamaguchi, Y., Wada, T., Watanabe, D., Takagi, T., Hasegawa, J. and Handa, H. (1999). Structure and function of the human transcription elongation factor DSIF. J. Biol. Chem. 274, 8085-8092.[Abstract/Free Full Text]

Yamaguchi, Y., Narita, T., Inukai, N., Wada, T. and Handa, H. (2001). SPT genes: key players in the regulation of transcription, chromatin structure and other cellular processes. J. Biochem. 129, 185-191.[Abstract]

Yelon, D., Horne, S. A. and Stainier, D. Y. (1999). Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev. Biol. 214, 23-37.[Medline]

Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K., Silver, L. M. and Stainier, D. Y. R. (2000). The bHLH transcription factor Hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development 127, 2573-2582.[Abstract/Free Full Text]

Zorio, D. A. and Bentley, D. L. (2001). Transcription elongation: the ‘Foggy’ is lifting. Curr. Biol. 11, R144-R146.[Medline]