developmental expression of class iii and iv pou domain genes in the zebrafish

JOBNAME: BBRC 218#3 PAGE: 1 SESS: 5 OUTPUT: Fri Apr 12 09:35:25 1996/xypage/worksmart/tsp000/68480f/69

Developmental Expression of Class III and IV POU Domain Genes inthe Zebrafish

Karuna Sampath1 and Gary W. Stuart2

Department of Life Sciences, Indiana State University, Terre Haute, Indiana 47809

Received January 2, 1996

Using redundant primers in a PCR based cloning strategy, POU-domain encoding sequences representing twodifferent class III genes (brain-1.1, brain-1.2) and one class IV gene (brain-3.1) were identified in zebrafish.Using the PCR clones to screen a zebrafish gastrula cDNA library, a third class III POU gene was identified(brain-1.0). RT–PCR assays performed with gene-specific primers indicated that the four POU genes exhibiteddistinct patterns of expression in developing embryos and adult fish. Like their mammalian homologs, the classIII genes were expressed early in development and in the adult brain, while the class IV gene was poorlyexpressed during early development but highly expressed in the adult eye. Since two of the POU PCR cloneswere obtained from genomic DNA, the fish genes may also be similar to their mammalian homologs in beinglargely devoid of introns. © 1996 Academic Press, Inc.

The homeobox codes for a conserved 60 aa DNA-binding domain known as the homeodomain.Homeodomains are present in a diverse array of transcription factors known to play important rolesin the development of a variety of organisms (1). Over thirty subclasses of homeodomains havebeen described (2), and some of these subclasses contain additional conserved functional domains.The POU domain proteins are homeodomain proteins that contain a second conserved region ofabout 90 aa referred to as the POU-specific domain (3). The two domains are typically separatedby a less well conserved linker region of 15–30 aa. POU domains were originally recognized in themammalian transcription factors Oct-1, Oct-2, and Pit-1 and the nematode developmental regula-tor, Unc-86 (4). Additional members of the POU-domain family have subsequently been identifiedin flatworms, echinoderms, and arthropods. Those known to occur in vertebrates are now subdi-vided into at least six classes based primarily on sequence differences within the linker region (5).Early in development, most POU-domain containing genes are widely expressed in the nervous

system. Later in development, a number of these genes are restricted in their expression to subsetsof neural tissues (5). Although POU domain genes can function outside the nervous system (6),many of these genes are generally expected to play important roles in the development of thenervous system and/or in the specification of neuronal cell types (5). In some cases, the requirementfor POU gene function in neural development has already been demonstrated. For example,mutations in theunc-86gene ofC. elegansand thepdmgenes ofDrosophilaalso result in the lossof particular neural cell types (7,8). In humans, natural mutations in thebrain-4 gene were shownto be responsible for one form of profound neural deafness (9).We have used a PCR based approach to identify POU-domain containing genes likely to be

important for neural development in the zebrafish. Screening both a cDNA library and genomicDNA resulted in the identification of several class III and VI POU-domain encoding sequences.The clones derived from genomic DNA represent genes which, like many other class III and VIgenes, lack introns within the POU domain encoding sequences (10). Expression studies indicatethat like their mammalian homologs, the class III genes are expressed early in development, andboth class III and IV genes are expressed in the adult brain. A connection is drawn between the

1 Current address: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.2 To whom correspondence should be addressed. Fax: (812) 237-4480.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS219,565–571 (1996)ARTICLE NO. 0274

5650006-291X/96 $18.00Copyright © 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.


intronless and dispersed nature of some vertebrate POU genes and their likely roles in defining therecently evolved vertebrate brain.

MATERIALS AND METHODS

PCR amplification and cloning.The primers used for the initial amplification of POU sequences from zebrafish (seeFigure 1) were designed from the deduced amino acid sequence of known POU domain genes in various organisms (11).A 20 ng DNA sample derived from either a 19 hr cDNA library (a gift from Dr. Kai Zinn, University of California,Berkeley) or from the zebrafish genome served as the template in 100ml PCR reactions. The reactions were overlaid withmineral oil, incubated at 95°C for 10 min. and at 80°C for 20 min prior to the addition of Taq DNA polymerase. Theremaining program consisted of 35 cycles of denaturation for 1.5 min at 95°C, annealing at 55°C for 1 min and extensionat 72°C for 3 min. A final extension of 10 min at 72°C was included. The major amplification product of 400 bp was purifiedusing GENECLEAN II (Bio 101, La Jolla, CA), digested with Eco RI and Bam HI, and cloned between the Eco RI andBamHI sites of pBluescript II KS+ (Stratagene, La Jolla, CA). Alternatively, the PCR products were cloned directly intothe TA site of pCR II (Invitrogen, San Diego, CA). Plasmids harboring inserts ofz400 bp were used to determine thenucleotide sequence by the dideoxy chain termination method (12). Thebrain-1.2PCR clone was subsequently used in astandard hybridization screen (13) to isolate thebrain-1.0cDNA from a zebrafish 33 hr cDNA library (kindly provided byKai Zinn).Reverse transcription–polymerase chain reaction (RT–PCR).Total RNA from embryos at various stages was prepared

by the guanidine isothiocyanate method (13). A 2.5mg aliquot of each RNA sample was suspended in 15ml of DEPC-treated H2O, denatured at 95°C for 3 min., snap chilled on ice, and used as template in 30ml reverse transcription (RT)reactions (14). The reactions were trace labeled with 0.5mCi [32P]dCTP. Parallel control reactions were run for each RNAsample in which no enzyme was added (RT-). Small aliquots of the RT reactions (2ml) were then used in separate 20mlPCR reactions with primers specific for eachbrain gene. The PCR conditions were as follows: 25 cycles of 1.5 mindenaturation at 95°C, 1 min annealing at 58°C, and extension for 3 min at 72°C. A final extension for 10 min at 72°C wasincluded. Negative control reactions (no template) were also performed. PCR’s with primers specific for zebrafishmaxwereused to determine the quality of the RT reactions. PCR fragments were visualized by autoradiography following electro-phoreses on a 6% polyacrylamide gel.Whole-mount in situ hybridization.Staged zebrafish embryos were dechorionated manually (for embryos younger than

10 hrs) or with Pronase (20mg/ml), washed and fixed overnight in 4% paraformaldehyde (PFA) in 1X Phosphate bufferedsaline (PBS). The embryos were stored in methanol at −20°C for at least 2 hrs. The embryos were rehydrated in amethanol/PBST series (100% methanol, 75% methanol/25% PBST, 50% methanol/50% PBST, 25% methanol/75% PBST,100% PBST) for 15 min each, followed by 2 washes in PBST (1 × PBS, 0.1% Tween-20). Embryos older than 10–12 h weretreated in 10mg/ml proteinase K in PBST for 5–10 min., postfixed in PFA for 20 min., and washed 5 times in PBST.

FIG. 1. PCR amplification of POU-domain encoding sequences from the zebrafish.(A) Degenerate PCR primer pairused to amplify POU gene fragments from a zebrafish cDNA library and zebrafish genomic DNA. Inosine was used inseveral positions to reduce primer complexity.(B) Agarose gel electrophoretic analysis of fragments amplified by PCR withthe degenerate POU primers. Lane 1, lambda Hind III marker; lane 2, PCR amplification products from genomic DNAtemplate. The arrow marks the major product of 400 bp.

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Embryos were then prehybridized in hybridization buffer (50% formamide, 5 × SSC, 0.1% Tween-20, 50mg/ml heparin,500mg/ml tRNA) for at least 2 hrs at 65°C. Digoxigenin labeled sense and antisense RNA probes were synthesized byinvitro transcription using 1mg of linearized plasmids as template (Boehringer Mannheim Biochemicals), and hydrolyzed to200–300 bases using sodium bicarbonate/sodium carbonate (15). The prehybridized embryos were incubated with freshhybridization buffer containing digoxigenin labeled RNA probe at 50–100 ng/200ml buffer and incubated o/n at 65°C.Following hybridization with the RNA probe, the embryos were washed at 65°C for 20 min. each in 100% hybridizationbuffer, 50% hybridization buffer/50% 2 × SSC, and 100% 2 × SSC. This was followed by 2 washes in 0.2 × SSC for 30min. each at 60°C. The embryos were then washed for 5 min. each in 75% 0.2 × SSC/25% PBST, 50% 0.2 × SSC/50%PBST, 25% 0.2 × SSC/75% PBST and 100% PBST. The embryos were incubated for 1 hr at room temperature with shakingin PBST containing 2% sheep serum and 2% goat serum (Sigma) and then with a 1:2000 dilution of preadsorbedanti-digoxigenin antibody for 4 hrs (15). The embryos were subsequently washed and stained with NBT/x-phosphate asdescribed (15). The reaction was stopped in PBST and the embryos were cleared in methyl salicylate or glycerol (16).

RESULTS AND DISCUSSION

Isolation of zebrafish POU-domain sequences.POU-domain encoding cDNA’s were isolatedusing a Polymerase Chain Reaction (PCR) based method. PCR primers were designed to matchsequences encoding two well conserved nonamer peptides FKQRRIKLG and RVWFCNRRQfound within the POU domains of proteins from several different organisms (see methods). Fol-lowing PCR, the products were analyzed via agarose gel electrophoresis. In addition to a few minorbands, the expected 400 bp product was observed when either a 19 hour zebrafish cDNA library(not shown) or zebrafish genomic DNA was used as template (Figure 1). The PCR products werethen cloned directly into phagemid vectors and sequenced (Figure 2).BLAST (NCBI) comparisons performed against the non-redundent nucleotide database allowed

individual PCR clones to be unambiguously identified with respect to their subclass and tentativelymatched with their respective murine homologs (Figure 2). Of the five independent clones derivedfrom genomic DNA, two were found to represent a class III POU-domain gene similar to murineBrain-1 andBrain-2 genes (brain-1.1), and three were found to encode a class IV POU-domainsimilar to the murineBrain-3.1gene (brain-3.1). In addition, the cDNA library template yielded asingle PCR clone representing a different class III gene (brain-1.2).Using the PCR clones described above as probes, two POU-domain encoding cDNA’s were

isolated. One full length clone, obtained from a 33 hr zebrafish embryo library (provided by K.Zinn), was identical to the previously published ZFPOU1 sequence (17). Although not representedin the original PCR clones, this sequence represents the first identified homolog of murineBrain-1,hence it is referred to here asbrain-1.0.A second cDNA matching thebrain-1.1PCR clone wasobtained from a post-somitogenesis stage (20–28 hr) library (provided by D. Grunwald). BLAST(NCBI) comparisons indicate that zebrafishbrain-1.1 is also a close homolog of murineBrain-1;the sequence and expression of this cDNA is the subject of another publication (Sampath andStuart, submitted).Expression of zebrafish class III and IV POU-domain genes.The expression patterns of the

brain-1.0, brain-1.2,andbrain-3.1 genes were determined at various times during development(Figure 3) and in several tissues of the adult fish (Figure 4) using a reverse transcriptase PCR assay

FIG. 2. Comparison of mouse and zebrafish POU domain coding sequences. The zfbrn-1.0 sequence is identical to thatof ZFPOU1 described by Matsuzaki et al. (18). Gaps in the alignment are indicated by ... ; sequence identities are indicatedby ---; m, mouse; zf, zebrafish.


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(RT-PCR). Transcript specific primers were generated by consulting the sequence of each POUgene PCR clone. To maximize the specificity of the assay, at least one of each pair of primers wasdesigned to match a unique sequence either within the highly variable linker region of the target(brain-1.2, brain-3.1) or within the 39 untranslated region of the transcript (brain-1.0). In each case,optimal cycling temperatures were established in mock reactions using cloned templates to ensureexclusive amplification of the target sequence. Primers specific for the zebrafishmaxgene tran-scripts were used as a positive control for verifying equivalent RNA yield and quality. Thezebrafishmaxgene is fairly uniformly expressed throughout development, and produces severaltranscripts of similar size (18).

FIG. 4. Tissue specific expression ofbrain-1.0, brain-1.2andbrain-3.1 in adult zebrafish. Total RNA from variousdissected tissues was converted to first strand cDNA, and subjected to 25 cycles of PCR with gene specific primers.Amplification with primers specific for zebrafish max was used as a control for the cDNA samples. Lane 1, genomic DNA;lane 2, eyes; lane 3, gills; lane 4, ovary; lane 5, brain; lane 6, testes; lane 7, liver.

FIG. 3. Developmental expression of the zebrafishbrain-1.0, brain-1.2,andbrain-3.1transcripts detected by RT–PCR.Total RNA from various developmental stages was converted to first strand cDNA and used in PCR reactions (25 cycles).(A) Gene-specific primer pairs. The first three pairs of primers were used to specifically detect expression of zebrafishbrain-1.0, brain-1.2,andbrain-3.1genes, respectively. The last pair were used to detect the zebrafish max gene transcriptsas a positive control. The expected product sizes forbrain-1.0, brain-1.2,andbrain-3.1were 898 bp, 175 bp, and 191 bp,respectively.(B) Electrophoretic analysis of the RT–PCR products. Lane 1, genomic DNA control; lane 2, 1 cell embryos;lane 3, gastrula (8–10 hrs); lane 4, neurula (10–16 hrs).


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Each of the above POU genes showed distinct temporal expression patterns during embryonicdevelopment (Figure 3). Expression ofbrain-1.0was undetectable in single-cell and gastrula stageembryos (8–10 hrs), but increased greatly in early neurula (12–16 hrs). The expression ofbrain-1.0observed in whole mounts at 18 hrs (see below) and the recovery of abrain-1.0cDNA from a 33hrlibrary (see above) indicates that expression continues into the second day of development. UsingNorthern analysis, Matsuzaki et al. reported a similar developmental expression pattern for ZF-POU/brain-1.0(17). Likebrain-1.0, brain-1.1is first detected during neurulation and continues tobe expressed at high levels in older embryos (Sampath and Stuart, submitted). The expression ofbrain-1.2was first seen at an earlier stage, during gastrulation, and continued into the neurula stage(Figure 3). This temporal pattern of expression is roughly similar to that seen with both theBrain-1and Brain-2 genes of rodents (12). Finally, the zebrafishbrain-3.1 gene showed little or noexpression in single cell, gastrula, or neurula embryos (Figure 3), but was well expressed in 72 hourembryos (not shown). This relatively late expression is similar to that observed forBrain-3.1transcripts in mice, which are first detected at 13 days p.c., an early post-somitogenesis stage (19).Like their mouse homologs, all three zebrafish genes were expressed in the adult brain (Figure

4). While the expression ofbrain-1 appeared to be largely restricted to the brain,brain-1.2andbrain-3.1were clearly expressed in testes as well, andbrain-3.1was well expressed in the eye(Figure 4). By comparison, the three known murinebrain-3genes are also expressed in the eye andthe brain. Expression in the eye is restricted to largely overlapping subsets of retinal ganglion cells,while expression in the brain is found in subsets of cells in the adult midbrain, dorsal root, andtrigeminal ganglia. While several other non-neural tissues were found not to express anybrain-3gene, mouse testes have apparently not yet been tested (20).Pattern of brain-1 expression in the anterior nervous system.The availability of a complete

cDNA for the brain-1.0 transcript made it possible to specifically visualize the spatio-temporalpattern ofbrain-1 gene expression via whole-mount in situ hybridization. In agreement with theresults of RT-PCR, thebrain-1.0 transcript was difficult to detect in gastrula stage embryos, butwas seen along the neural plate at 10 hrs of development (Figure 5). Expression was greatest in theanterior neural plate where the brain forms. By 15 hrs of development (13 somites) the anteriorstaining pattern begins to resolve into an alternating pattern of heavy and light staining reminiscentof the unfolding metameric structure of the nervous system (21). By 18 hrs (18 somites),brain-1.0expression is apparent in the posterior forebrain, in the midbrain, in distinct regions within the

FIG. 5. Whole mountin situ hybridization with thebrn-1.0cDNA. (A) A 10 hr embryo showing hybridization in theanterior neural keel (arrowhead).(B) A 15 hr embryo with periodic staining (arrwheads) in the neural rod.(C) An 18 hrembryo with staining in the posterior forebrain (f), midbrain (m), hindbrain (h), and anterior spinal cord (s). Magnificationis 50×.


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hindbrain, and in the anterior spinal cord. These results are similar to those obtained by Matsuzakiet al. using in situ hybridizations on sectioned material (17).Our results with the whole mount technique allows the alternating nature of thebrain-1.0

expression pattern in the prospective anterior nervous system to be clearly observed, and lendsfurther support to the hypothesis of Matsuzaki et al. that segmentation processes may be partlyresponsible for the observed pattern. Others have also provided evidence that other zebrafish POUgenes are segmentally expressed in the hindbrain (22). Further experiments are required to deter-mine if the vertebratebrain-1 genes or any of the other class III or IV POU genes are involved indefining or elaborating cell types or regional differences within the developing brain and spinalcord.Intronless POU gene evolution and expression.The isolation of POU-domain encoding se-

quences of the predicted size from genomic DNA suggests that these gene are largely or perhapseven completely devoid of introns. Several mammalian class III and IV genes described to date arealso intronless (23) or have few introns (20). Many genes that are part of multigene clusters, likethe histone genes and hox genes, also lack introns. Assuming the “introns late” hypothesis (24) tobe correct, the earliest versions of the hox, POU, and histone genes would have been intronless.Clusters subsequently formed by local duplications of these intronless genes would perhaps tend toremain intronless due to gene conversion, which is more common among clustered genes (25).However, since most class III and IV POU genes appear to be dispersed rather than clustered (26),gene conversion seems unlikely to explain their intron-poor character. An alternative mechanismfor gene duplication involves reverse transcription of mRNA followed by random cDNA insertion.This mechanism initially generates dispersed and intronless copies of genes. POU genes recentlyduplicated by reverse transcription would generally lack or have very few introns. By this account,intronless class III and IV POU sequences may be of relatively recent origin, perhaps even recentenough to show characteristic signs of reverse transcription and cDNA insertion. Short invertedrepeats and a poly dA/T sequence indicative of gene duplication through a cDNA intermediate havein fact been observed flanking the intronless rat class III POU geneTestes-I/SCIP(27). Thehypothesis that intronless versions of POU-domain sequences (mostly class III and IV) are recentlyevolved is also consistent with their likely role in defining cell types within a relatively recentlyevolved structure, the brain.

ACKNOWLEDGMENTS

We thank Angela Pritts and Zhu Zhu for technical assistance. This work was supported by a URC grant from IndianaState University and NIH Grant RO1HD27555 to G.S.

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developmental expression of class iii and iv pou domain genes in the zebrafish

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