©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Presence of isl-1-related LIM Domain Homeobox Genes in Teleost and Their Similar Patterns of Expression in Brain and Spinal Cord (*)

(Received for publication, July 7, 1994; and in revised form, November 18, 1994)

Zhiyuan Gong (1) (2)(§) Chi-chung Hui (1) Choy L. Hew (1) (2)

From the  (1)Research Institute, Hospital for Sick Children, Toronto and the (2)Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario M5G 1L5, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Three novel LIM domain homeobox cDNAs encoding proteins structurally related to the Isl-1 protein were isolated from a chinook salmon pituitary cDNA library. Southern blot analysis of genomic DNA indicate that they are derived from three distinct genes, designated as isl-2a, isl-2b, and isl-3 genes. Nucleotide sequence analysis of reverse transcriptase-polymerase chain reaction amplified products reveal that the isl gene family contains two members (a and b) each of both isl-1 and isl-2 genes, and one member of isl-3 gene in the two tetraploid salmonid species, chinook salmon and rainbow trout, and only one member each of isl-1, isl-2, and isl-3 genes in the diploid zebrafish. The expression of the three isl genes in the rainbow trout were studied by reverse transcriptase-polymerase chain reaction analysis of embryonic and adult RNAs, and by in situ hybridization analysis of 8-week-old hatchlings. The transcripts of all three genes could be detected as early as 4 weeks postfertilization (the eye stage) and increased dramatically in 5-week-old embryos. In the adult, the three isl mRNAs appear to be differentially distributed in various tissues. The level of isl-1 mRNA is generally higher than those of isl-2 and isl-3 mRNAs. In situ hybridization analysis indicates that the transcripts of all three genes are localized in subsets of neurons in the brain and spinal cord. In the retina, isl-1 mRNA could be found in both the ganglion and inner nuclear layers while isl-2 and isl-3 mRNAs could only be detected in the ganglion layer. High level of isl-1 mRNA could also be found in mid-gut and interrenal organ where endocrine cells are densely populated. Based on these observations, we speculate that the three structurally related isl genes may play similar roles in cell determination and differentiation in the developing nervous system.


INTRODUCTION

Isl-1 is a homeodomain-containing transcription factor initially identified in rats as an insulin enhancer binding protein (Karlsson et al., 1990). Subsequent investigations indicate that its expression is not limited to pancreatic cells, but also widely distributed in many neurons in the brain, spinal cord, and peripheral nervous system, and in some endocrine cells in the pituitary and thyroid (Thor et al., 1991; Dong et al., 1991). Recently, expression of Isl-1 in cell types of non-neuroendocrine lineage was also reported (Wang and Drucker, 1994). In chick embryos, Isl-1 is expressed in motor neurons immediately after their final mitosis and is the earliest molecular marker of developing motor neurons (Ericson et al., 1992). In zebrafish embryos, Isl-1 expression is initiated in many primary neurons in the brain and spinal cord at the end of gastrulation (Korzh et al., 1993; Inoue et al., 1994). Despite these studies, the role of Isl-1 remains largely unknown. The broad expression of isl-1 gene in many neurons and endocrine cells during embryogenesis and adult development suggests that it may be involved in both differentiation and maintenance of these cells.

Isl-1 belongs to a group of homeobox proteins which contain, in addition to a homeodomain, two tandem repeated Cys-His motifs, termed as LIM domains. The acronym LIM is derived from three prototype homeobox genes: lin-11, Isl-1 and mec-3 (Freyd et al., 1990; Karlsson et al., 1990), which all encode proteins containing the conserved Cys-His motifs. Both lin-11 and mec-3 were isolated from genetic analysis of Caenorhabditis elegans. lin-11 is involved in the control of cell identities in a subset of vulval cells (Ferguson et al., 1987) and mec-3 controls the terminal differentiation of touch receptor neurons (Way and Chafie, 1989). The LIM domains are also presented in some non-homeobox proteins characterized from both animals and plants (Birkenmeier and Gordon, 1986; McGuire et al., 1989; Boehm et al., 1991; Hempe and Cousins, 1991; Sadler et al., 1992; Wang et al., 1992; Baltz et al., 1992; Mizuno et al., 1994). The function of LIM domains is not clear. It has been proposed that they may be involved in specific DNA-protein or protein-protein interaction during transcriptional regulation (Freyd et al., 1990; Karlsson et al., 1990). The primary structure of LIM domains resembles that of zinc fingers, the well characterized DNA binding motifs. The LIM domains have been shown to contain zinc (Li et al., 1991; Michelsen et al., 1993; Archer et al., 1994; Kosa et al., 1994) but there is no evidence that they are involved in DNA binding. Recently, the LIM domains of rat Isl-1 have been shown to inhibit DNA binding by the homeodomain (Sanchez-Garcia et al., 1993). Our data indicate that the LIM domain from an Isl-1-related protein has no specific or nonspecific DNA binding activity (Gong and Hew, 1994). Therefore, it is likely that the LIM domain is involved in some protein-protein interaction. Consistent with this notion, a synergistic stimulation of the activity of an insulin gene promoter by a LIM domain homeodomain protein Lmx-1 and a basic helix-loop-helix protein has been reported and the LIM domains of Lmx-1 cannot be functionally replaced by the LIM domains of Isl-1 for stimulation (German et al., 1992). Recently, specific protein-protein interaction has been reported for two cytoskeletal LIM proteins, chicken CRP and zyxin (Sadler et al., 1992).

Thus far, many members of the LIM domain homeobox gene family have been characterized from several species and they all appear to be expressed in subsets of neurons and other cell types. These genes include xlim-1 and xlim-3 in Xenopus (Taira et al., 1992; 1993), apterous in Drosophila (Bourgouin et al., 1992; Cohen et al., 1992), lmx-1 in the hamster (German et al., 1992), and LH-2 in the rat (Xu et al., 1993). We report here the characterization of several new LIM domain homeobox cDNAs encoding proteins similar but distinct from Isl-1. Sequence analysis of RT-PCR (^2)products from the chinook salmon, rainbow trout, and zebrafish clearly indicate the existence of an isl-1-related gene family consisted of isl-1, isl-2, and isl-3 genes. All three genes are active during embryogenesis and in a variety of adult tissues. In situ hybridization indicated that they are expressed in similar sets of neurons in the brain and spinal cord. Some difference in the expression of the three genes are also noted and their functional importance is discussed.


EXPERIMENTAL PROCEDURES

Materials

The chinook salmon (Oncorhynchus tschawytscha) were collected at the Spring Creek National Hatchery on Columbia River in Washington state. The rainbow trout (Oncorhynchus mykiss) and embryos were collected at the Rainbow Springs Hatchery, Thamesford, Ontario, Canada. The zebrafish (Danio rerio) embryonic poly(A) RNA was kindly provided by Drs. Barbara Jones and Martin Petkovich of Queen's University, Canada.

Construction and Screening of Pituitary cDNA Library

Poly(A) RNA was isolated from several chinook salmon pituitaries using the FastTrack mRNA Isolation Kit (Invitrogen). The pituitary cDNA library was constructed with a Lambda Uni-ZAP XR vector system (Stratagene). Screening for salmon homeobox cDNA clones was carried out by hybridization at a low stringency (55 °C, 0.3 M Na) using a partial homeobox cDNA probe from a randomly tagged clone from a pituitary cDNA library of the winter flounder, Pleuronectes americanus (Gong et al., 1994). Subcloning of cDNA inserts was carried out by in vivo excision in Escherichia coli based on the instruction of Stratagene. Phylogentic analysis was performed using the PHYLIP package.

DNA Sequencing and Sequence Analysis

Double strand DNA sequencing was performed by dideoxynucleotide chain termination using the T7 Sequencing Kit from Pharmacia. Deletions of cDNA clones for DNA sequencing were made based on their restriction maps. DNA and protein sequences were analyzed using DNASIS and PROSIS computer programs (Pharmacia Biotech Inc.), respectively.

Genomic Southern Analysis

Genomic Southern blotting was performed as described previously (Gong and Brandhorst, 1987). When genomic blots were used repeatedly, radioactive probes were removed by washing the blots in boiling 0.1 times SSC (15 mM NaCl and 15 mM sodium citrate) prior to the subsequent round of hybridization. Salmon genomic DNA, isolated from a testis from the chinook salmon, was kindly provided by Dr. F. Xiong. DNA fragments used as hybridization probes were generated by restriction enzyme digestion and purified by electroelution from agarose gels following electrophoretic separation.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNAs from embryos and adult tissues were prepared by acid guanidine thiocyanate-phenol-chloroform extraction as described previously (Gong et al., 1992). The first strand of cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase. A typical reaction mixture (40 µl) contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl(2), 0.1 mg/ml bovine serum albumin, 10 mM dithiothreitol, 0.5 mM of each dNTP, 40 units of RNAsin (Promega), 10 µg of template RNA, and 400 pmol of random hexamer primer (Bethesda Research Laboratories), and 200 units of Moloney murine leukemia virus reverse transcriptase. The reaction was carried out at 37 °C for 50 min. Generally, 2 µl of cDNA (equivalent to 0.5 µg of total RNA) was used for each PCR amplification. PCR was performed in 100 µl of reaction mixture containing 1 times PCR buffer (50 mM KCl, 1.5 mM MgCl(2), 0.1% Triton X-100, and 10 mM Tris, pH 8.8), 0.1 mM of each of the dNTP, 0.1-0.3 µg of each of the oligonucleotide primers, and 2 units of Taq DNA polymerase. The following temperature profile was used for 33 cycles: 92 °C/1 min (denaturation), 55 °C/1.5 min (primer annealing), and 65 °C/1.5 min (extension). After PCR amplification, 20 µl of each sample was electrophoresed on agarose gels, blotted, and hybridized by a gene-specific probe. The validation of the RT-PCR assay was confirmed by the following observations. No detectable Isl signal was present by performing the PCR directly using RNA as the template without reverse transcription. For each batch of cDNA, the quality the cDNAs was justified by amplification of ubiquitously expressed alpha-tubulin mRNAs (the tubulin PCR primers were a gift of Dr. Nancy Sheawood, University of Victoria, Canada). For cloning of the PCR-amplified DNA fragments, the DNA fragments of interest were purified by electroelution from the agarose gel and directly ligated into pT7Blue T-vector (Novagen) based on the manufacturer's instruction.

For quantitation of the level of Isl transcripts, competitive RT-PCR was carried out (Siebert and Larrick, 1992). Homologous DNA competitors were generated by an internal deletion based on the method of Galea and Feinstein(1992). The concentrations of competitor DNAs were determined by staining with ethidium bromide. RT-PCR was performed as described above except for the presence of competitor DNA. Optimal concentrations of competitor DNA used in the assay were determined by addition of different amounts of competitor DNA. RT-PCR products were separated on an agarose gel, blotted, and hybridized with respective isl DNA probes. Quantification of the level of Isl mRNAs was carried out by scintillation counting of the radioactive signals. The number of Isl transcripts in a particular sample was calculated based on the ratio of endogenous signal over the signal from competitor DNA which had been loaded at a know number. Comparison of the relative level of different Isl transcripts were based on the absolute numbers of transcripts in a sample (Table 2).



PCR Primers

In this study, the following PCR primers were used and the specificity of these primers in PCR were confirmed by preliminary PCR experiments using isl-1, -2, and -3 cDNA clones as the template, respectively. 1) Degenerated homeobox left primer HbL (35 mer): 5`-GTGTCTAGAGTN^A/(C)GIACNGTI^T/(C)TNAA^T/(C)GA^G/(A)AA^G/(A)CA-3`, corresponding to sequences at 381-406 in Fig. 3. 2) Degenerated homeobox right primer HbR (35 mer): 5`-GTCGGATCC^G/(A)TC^T/(C)TT^G/(A)CANC^G//(C)TT^G/(A)TT^T/(C)TG^G/(A)AACC-3`, corresponding to sequences immediately after the 3` ends of all isl DNAs in Fig. 3.


Figure 3: Alignment of all available sequences of isl gene family from the chinook salmon (CS), rainbow trout (RT), zebrafish (ZF), and rats (RAT). CSIsl-2a, CSIsl-2b, and CSIsl-3 are excerpted from respective cDNA clones. ZFIsl-1 and RATIsl-1 are based on Inoue et al.(1994) and Karlsson et al.(1990), respectively. All other sequences are based on RT-PCR clones from this study as described in text. The sequences include a part of the LIM domain, the variable region and the homeodomain. Dashes indicate identical nucleotides and asterisks are insertions of gap for maximal alignment. The homeobox region is underlined.



Both HbL and HbR primers include several degenerate nucleotides based on all available sequences for the isl-1 gene family in order to amplify all potentially related homeobox DNA sequences. The smaller letters with back slashes represent the mixed nucleotides incorporated. The letter N denotes all four nucleotides at that position and letter I stands for inosine. The first 9 bases were designed to create a restriction site. 3) isl-1-specific left primer 1L (20-mer): 5`-GGTTTCAGCAAGAATGACTT-3`, corresponding to the zebrafish isl-1 sequence immediately prior to the 5` end of ZFIsl-1 in Fig. 3. 4) isl-1-specific right primer 1R (19-mer): 5`-CCAGACCCGGATGACTCGG-3`, complementary to CS/RTIsl-1 sequences at the 3` ends in Fig. 3. 5) isl-2/-3 specific left primer 2/3La (19-mer): 5`-GAGAC^G/ATGCACTTGCTTCG-3`, corresponding to Isl-2 and -3 sequences immediately prior to the 5` ends in Fig. 3. 6) Another isl-2/-3 left primer 2/3Lb (20-mer): 5`-AACCACGTCCACAAGCAGTC-3`, corresponding to sequences just before the homeodomain at 349-369 in Fig. 3. 7) isl-2-specific right primer 2R (20-mer): 5`-CTCGAATTGGACTGCCTGCC-3`, corresponding to Isl-2a/2b sequences downstream of the homeodomain at amino acid residues of 275-280 (Fig. 1). 8) isl-3-specific right primer 3R (23-mer): 5`-CCCTGTAGACTGAAGATGCTTAC-3`, corresponding to Isl-3 sequences with 4 extra amino acid residues downstream of the homeodomain (Fig. 1).


Figure 1: Sequences of three salmon Isl-1-related proteins (CSIsl-2a, CSIsl-2b, and CSIsl-3) aligned with the zebrafish and rat Isl-1's (ZFIsl-1 and RATIsl-1). The complete sequence of CSIsl-2a is shown. Dashes represent identical amino acid residues and asterisks are insertions of gap for maximal alignment. The Cys-His LIM motifs are boxed and the homeodomain is underlined. 18 amino acid residues are absent for CSIsl-2b due to incompleteness of the cDNA clone. The rat Isl-1 sequence is based on Karlsson et al.(1990) and zebrafish Isl-1 from Inoue et al.(1994).



In Situ Hybridization

In situ hybridization on rainbow trout embryos was carried out according to a protocol on mouse embryos as described previously (Hui and Joyner, 1993). The embryos were fixed in 4% fresh paraformaldehyde in phosphate-buffered saline (0.13 M NaCl, 7 mM Na(2)HPO(4), 3 mM NaH(2)PO(4)) embedded in paraffin wax after dehydration and sectioned to 6 µm. Hybridization and washing conditions were the same as described previously (Hui and Joyner, 1993). Riboprobes were synthesized with [P]UTP using T7 RNA polymerase. For each slide, about 150 µl of hybridization solution (50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10 mM NaPO^4, pH 8.0, 10% dextran sulfate, 1 times Denhardt's, 0.5 mg/ml yeast tRNA, 10 mM dithiothreitol) containing 1.5 times 10^7 cpm of P-labeled riboprobe was applied, and the slides were overlaid with coverslips and incubated at 55 °C overnight in a humid chamber with tissues soaked with 50% formamide and 5 times SSC. After posthybridization washes, the slides were exposed to the emulsion for 3 weeks, developed in Kodak D19, and counter stained with toluidine blue. The riboprobes used in this study were gene specific and transcribed from 3`-untranslated regions. Under our hybridization and wash conditions, there is no cross-hybridization among the three genes (Fig. 2).


Figure 2: Genomic blot analysis of the isl-1 gene family. Chinook salmon sperm DNA prepared from a single individual was cut by HindIII (H) and EcoRI (E), blotted, and hybridized with the isl-1 homeobox probe (panel A); isl-2 homeobox probe (panel B); isl-3 homeobox probe (panels C and D); isl-2a 3`-noncoding probe (panel E), and isl-2b 3`-noncoding probe (panel F), respectively. Blots in panels A and D were washed at the low stringency (55 °C, 0.3 M Na) and all others at the high stringency (65 °C, 0.03 M Na). Molecular weights in kilobase are indicated on the left. 10 µg of digested genomic DNA was loaded in each lane.




RESULTS

Isolation and Sequencing of Salmon Homeobox Genes Similar to isl-1

During the course of our cDNA clone tagging study in teleosts (Gong et al., 1994), similar to the work of Adams et al.(1991), we sequenced several randomly selected cDNA clones from the winter flounder pituitary cDNA library and found that one of the clones contained part of a homeobox sequence. In order to isolate a full-length cDNA clone of the homeobox gene and to study its potential role in the regulation of pituitary gene expression, we constructed a salmon pituitary cDNA library. After screening 600,000 primary clones using the flounder homeobox containing probe at a low stringency (55 °C, 0.3 M Na), 10 positive salmon clones were isolated and characterized. Sequence analysis indicated that they were derived from three distinct genes. The protein sequences deduced from the largest clones of the three genes are shown in Fig. 1. A comparison of the protein sequences of the salmon clones with known homeobox proteins indicated that these clones encode LIM domain homeobox proteins very similar to the rat Isl-1 (Karlsson et al., 1990). Like the rat Isl-1, the salmon proteins also contain two tandemly repeated finger like Cys-His motifs at their N-terminals, a central homeodomain, and a Gln-rich C-terminal. The complete salmon protein sequences are 74-75% identical to that of rat Isl-1 while higher identities (88-95%) are observed among the three salmon clones. The homeodomain and the first Cys-His motif are highly conserved from salmon to rats while the second Cys-His domain is less conserved. The 60 amino acid homeodomains have only one or two conserved amino acid substitutions. There is also a variable region between the second Cys-His motif and the homeodomain.

Presence of a Gene Family Related to isl-1 Gene

Since our cDNA clones share relatively low identity with the rat isl-1 cDNA clone, it is interesting to determine whether these salmon clones represent homologs or orthologs of the rat isl-1 gene. Recently, the zebrafish isl-1 gene has been isolated (Inoue et al., 1994) and its protein product is more homologous (98% identity) to the rat Isl-1 than the protein products deduced from these salmon clones to the rat Isl-1 (74-75% identity). Therefore, the salmon clones likely represent homologous members of a gene family containing the isl-1 gene. To clone a potential salmon isl-1 orthologous gene, an isl-1-specific left primer (1L) was designed based on the isl-1 DNA sequences of the zebrafish and rat. Using primers 1L and a degenerated homeobox right primer HbR, the region including part of the 2nd LIM motif, the most variable region, and the homeobox was amplified and cloned from the pituitary RNA by RT-PCR. Sequencing analysis of an RT-PCR cDNA clone indicated that the encoded protein is nearly identical to zebrafish Isl-1 (CSIsl-1a, Fig. 3). Its deduced amino acid sequence contains only 1 amino acid substitution out of 143 residues over the region and thus it is an authentic salmon isl-1 homolog. The cDNA clones isolated earlier represent novel LIM domain homeobox genes which are structurally similar to the isl-1 gene. Because of the high identities between isl-1 and the newly discovered salmon genes, we named these genes isl-2a, isl-2b, and isl-3 genes.

To further investigate the genomic organization of the isl gene family, genomic Southern blot hybridization was performed. As shown in Fig. 2, the homeobox probes from isl-1, isl-2a, and isl-3 cDNA clones hybridized to different sets of DNA fragments (panels A-C). Under a low stringency wash (0.3 M NaCl, 55 °C), the isl-3 homeobox probe also hybridized the two isl-2a positive fragments but not the isl-1 fragments (panel D). Due to the high sequence identity (95% in coding region) between isl-2a and -2b, the isl-2a homeobox probe likely also hybridized to isl-2b DNA. To confirm this, 3` noncoding probes from the isl-2a and -2b cDNA clones were used separately to hybridize identical genomic blots. As shown in panels E and F, the two probes hybridized to each of the two fragments recognized by the isl-2a homeobox probe, suggesting that the salmon genome contains one copy each of isl-2a and -2b genes. There are only one or two bands hybridized to the isl-3 homeobox probe at high stringency. When a 3`-noncoding probe was used, only one of the HindIII fragments was hybridized even at low stringency; thus, it is likely that HindIII cuts the isl-3 genomic sequence covered by the isl-3 homeobox probe, resulting in two bands of the same gene. By taking account of the fact that only one isl-3 sequence was isolated by library screening and RT-PCR amplification (see below), we tentatively conclude that there is only one copy of isl-3 gene in the genome, although the possibility of the presence of two indistinguishable copies cannot be ruled out. The isl-1 probe hybridized to two distinct DNA fragments (panel A), confirming that isl-2 and -3 genes are two distinct genes. The two isl-1 DNA fragments likely represent the two members of isl-1 genes (a and b, see below). Some minor bands in the isl-1 blot can be aligned to the isl-2 and -3 fragments. Together, these data suggest that there is one copy for each of the isl-1a, isl-1b, isl-2a, isl-2b, and isl-3 genes in the salmon genome.

The salmonid is a pseudotetraploid fish. During evolution its genome duplicated and the resulting DNA sequences have ever since diverged (Ohno et al., 1968). Therefore, most of the single copy gene in a diploid fish species can be found with two copies in salmonid. The strong similarity of isl-2a and -2b genes may be such an example, but the possibility that they are polymorphic alleles remains. To further investigate the number of isl gene families and their evolutionary relationship, we have amplified several isl-2- and isl-3-related sequences by RT-PCR using embryonic RNA from both a closely related tetraploid species, rainbow trout, and a diploid zebrafish. The PCR primers used were 2/3La and HbR. This pair of primers is able to amplify all three salmon genes: isl-2a, isl-2b, and isl-3, but not isl-1. Three distinct sequences were identified from the rainbow trout and two sequences from the zebrafish (Fig. 3). The three rainbow trout sequences are almost identical (99.4-99.8% identity) to the three known sequences from the chinook salmon. This may not be surprising since the rainbow trout (O. mykiss) and the chinook salmon (O. tschawytscha) belong to the same genus. Among the two zebrafish cDNA sequences, one is more similar to the salmon isl-2a and -2b sequences than the isl-3 sequence and the other more similar to the salmon isl-3 sequence than the isl-2a and -2b sequences. Therefore, the two zebrafish sequences are named ZFIsl-2 and ZFIsl-3, respectively. The equal distances of ZFIsl-2 to salmonid isl-2a and -2b sequences supports our notion that the isl-2a and -2b genes had evolved by tetraploidization after the separation of zebrafish and salmonid. The isl-1 sequences were also amplified from the rainbow trout using the isl-1-specific primer 1L and the degenerated homeobox primer HbR. Two rainbow trout isl-1 sequences were identified (RTIsl-1a and RTIsl-1b). The percentages of DNA sequence identity among all the available members of the isl gene family from teleosts and the rat are presented in Table 1. Their possible evolutionary relationship is proposed in Fig. 4. The tentative conclusion from these data is that there are three distinct members in the isl LIM domain homeobox gene family: isl-1, isl-2, and isl-3 genes. Due to the tetraploid nature of the salmonid genome, both the chinook salmon and rainbow trout have two members (a and b) each of the isl-1 and isl-2 genes in their genomes. Only one member of the isl-3 gene was identified in both species, either because the other member was lost during evolution or because the two putative members of the two potential isl-3 genes have indistinguishable sequences in the region examined.




Figure 4: Possible relationship of all available isl sequences based on their similarities listed in Table 1. The phylogenetic tree was constructed manually based on the percentage of sequence identity shown in Table 1and confirmed by the computer program of PHYLIP. For sequence definition, see Fig. 3. Arrows A-E indicate gene duplications or species divergence and are reviewed under ``Discussion.'' A, the ancestor isl gene.



To further demonstrate that there is no more closely related isl sequence in the genome, degenerated homeobox primers HbL and HbR were employed to amplify the homeobox DNAs directly from the genomic DNA of chinook salmon. The amplified DNA fragments were then cloned and sequenced. Five distinct sequences were obtained and matched to the corresponding regions of five distinct cDNA clones (CSIsl-1a, CSIsl-1b, CSIsl-2a, CSIsl-2b, and CSIsl-3) (data not shown). These data indicate that there is no more closely related isl sequence in the genome.

RT-PCR Analysis of the Expression of the Three isl Genes

The expression of isl genes in several stages of embryos and a few adult tissues were examined initially by Northern blot analysis. However, we failed to detect their transcripts probably because of low concentrations of these transcripts in the whole tissues. A more sensitive assay, RT-PCR, was thus carried out. Gene-specific primers were designed as described under ``Experimental Procedures.'' 1L and 1R were used for specific amplification for isl-1 mRNA, 2/3Lb and 2R for isl-2 mRNA, and 2/3Lb and 3R for isl-3 mRNA. The validity and specificity of these primers were confirmed by preliminary PCR experiments using the chinook salmon isl cDNA plasmids as templates. Specific RT-PCR products were further confirmed by Southern blot hybridization using gene-specific probes at a high stringency. The autoradiograms displaying the distributions of isl mRNAs in embryos and a variety of tissues are shown in Fig. 5.


Figure 5: RT-PCR detection of isl mRNAs in a variety of adult tissues and several stages of embryos. Total RNAs were prepared from various tissues and embryos of rainbow trout. RT-PCR was performed using gene specific primers as described under ``Experimental Procedures'' and the PCR products were separated on agarose gels, blotted, and hybridized with gene-specific probes from the cDNA clones. Names of tissues and embryo stages (4-8 weeks) are indicated at tops of each lane. Control lane, PCR was performed without cDNA templates. Primers used are: 1L and 1R for isl-1(a+b) (top panel), 2/3L2 and 2R for isl-2(a+b) (middle panel), and 2/3L2 and 3R for isl-3 (bottom panel). A more accurate representation of the relative levels of these Isl transcripts, which are quantitated by competitive RT-PCR with two sets of experiments, are given in Table 2.



The embryonic activation of the three isl genes appears to be between 4 and 5 weeks postfertilization. In some batches of 4-week-old embryos, we could not detect any of the three isl mRNAs; however, by 5 weeks of embryogenesis, the level of their transcripts appear to be dramatically increased. Therefore, the three isl genes are likely to be temporally co-regulated. The trout embryos at this stage have started eye development and somite formation. This stage is approximately equivalent to the stage of zebrafish embryos (about 12 h postfertilization) in which dramatic increases of isl-1 transcripts and immunoreactivity can be observed (Korzh et al., 1993; Inoue et al., 1994). The expression of the three isl genes were also detected in many adult tissues. isl-1 and isl-2 mRNAs are distributed broadly in almost every tissue examined, including the pituitary, brain, spinal cord, heart, liver, pyloric ceca, gall bladder, intestine, spleen, and kidney. In contrast, the distribution of isl-3 mRNA is more restricted, being detected only in a few tissues such as the pituitary, brain, and spinal cord and faintly in heart. In gills, the tissues containing thyroid cells, isl-1 mRNA is quite abundant while isl-2 and isl-3 mRNAs cannot be detected. Little or none of the Isl mRNAs could be detected in the muscle. In addition, all three isl genes are highly expressed in adult eyes (see Table 2). The relative levels of three isl mRNAs were quantitated by competitive RT-PCR and the results are summarized in Table 2. Generally, the level of isl-1 mRNA is higher than that of isl-3 mRNA and the level of isl-2 mRNA is the lowest of the three genes. These data clearly demonstrate that all the three isl genes are active in both embryos and in a variety of adult tissues.

In Situ Hybridization Analysis of the Expression of the Three isl Genes

To precisely localize the expression of the three isl genes, in situ hybridization on embryo sections was carried out. 8-week-old hatchlings were chosen because by this stage most of the major organs have been developed. Gene-specific riboprobes for the three isl genes were used and under our conditions of hybridization and washing (see ``Experimental Procedures''), no cross-hybridization of the three probes was observed (Fig. 2). Fig. 6shows the expression patterns of the three isl genes on sagittal sections of the embryos. All three isl genes show clear expression in the eyes (also see Fig. 8). isl-1 transcripts are localized in restricted parts of the central nervous system including the ventral parts of the forebrain, hypothalamus, cranial, and spinal ganglia, and spinal cord (Fig. 6, B and E-F). In addition, high levels of isl-1 transcript could also be found in the interrenal organ, a tissue equivalent to the adrenal gland of high vertebrates, and in the mid-gut from which pancreatic cells are derived. In contrast, isl-3 mRNA could only be detected in the hindbrain and spinal cord, but not in the interrenal organ and mid-gut, which is consistent with the results of RT-PCR analysis that the expression of isl-3 gene is more restricted (Fig. 5). isl-2 transcript was only weakly detected in the hindbrain and spinal cord (also see Fig. 7). However, our RT-PCR analysis indicates that isl-2 mRNA is also expressed in several other tissues. It is possible that the level of isl-2 mRNA in these tissues is too low to be detected by in situ hybridization. Similarly, the expression of the isl-1 gene in heart, liver, and spleen may also be too low to be detected by in situ hybridization. These observations are consistent with the results of RT-PCR analysis of the adult tissues, indicating that the three isl genes are highly expressed in tissues densely populated with neurons or endocrine cells.


Figure 6: Expression of the three isl genes. In situ hybridization was performed on sagittal sections of 8-week-old rainbow trout embryos (hatchlings). Slides shown in panels B, C, and D were hybridized with isl-1, -2, and -3 antisense riboprobes, respectively. isl-1 and isl-2 signals detected include both a and b subtypes under the hybridization condition used. Panel A is a bright field picture of B, panels E-H are larger magnifications of the boxed regions shown in A. Abbreviations for panel E: h, hypothalamus; m, metencephalon; mo, medulla oblongata; t, telencephalon. Abbreviations for panel G: at, alimentary tract; h, heart; in, intestine; L, liver; n, notochord; sc, spinal cord; sn, spinal nerves. Bright spots in the skin are hybridization background since similar spots are also presented in the sections hybridized with a sense riboprobe (data not shown).




Figure 8: Comparison of the expression of the three isl genes in retina. Adjacent cross-sections through eyes were hybridized with isl-1 sense riboprobe (B), antisense riboprobes of isl-1 (C), isl-2 (D), and isl-3 (E), respectively. Panel A is the light field picture of C. Abbreviations: g, ganglion layer; out, out nuclear layer; p, pigment cell layer; in, inner nuclear layers. The abrupt boundaries of the labeling of isl-1 and isl-3 transcripts are indicated by arrows in panels C and E. Pigment epithelium and choroid layer absorb riboprobes nonspecifically (panel B). The faint isl-2 signal in the ganglion nuclear layer is likely authentic as compared to the background in the inner and outer nuclear layers (panel D) while the faint isl-3 signal in the inner and outer nuclear layers may be background as compared to that in the lens and other parts (panel E).




Figure 7: Comparison of the expression patterns of the three isl genes in medulla oblongata and spinal cords. Panels A-D, adjacent cross-sections through the medulla oblongata. Panels E-H, adjacent cross-sections through the spinal cord (mid-part of the embryo). These sections were hybridized with isl-1 (B and F), isl-2 (C and G), and isl-3 (D and H) antisense riboprobes, respectively. Panels A and E are bright field pictures of panels B and F. Abbreviations: c, cerebellum; cn, cranial nerves; mo, medulla oblongata; n, notochord; sc, spinal cords; sn spinal nerves.



To further compare the expression patterns of the three genes, the localization of the three isl mRNAs was examined on adjacent transverse sections through the medulla oblongata and the spinal cord (Fig. 7). All three isl mRNAs could be detected in the same two clusters of cells ventrolateral to the neural tube in both medulla oblongata and spinal cord. These cells are likely motor neurons based on the similar location of the motor neurons in the spinal cords of rat, chicken, and zebrafish which also express Isl-1 (Thor et al., 1991; Ericson et al., 1992; Korzh et al., 1993; Inoue et al., 1994). In addition, the three isl mRNAs could also be found in the same cranial and spinal ganglia. The isl-1 mRNA appears to be distributed more broadly than the other two; however, this may be due to the difference of the levels of gene expression and detection limitation of the in situ hybridization. Although it is impossible to conclude whether the three genes are expressed in the same set of cells based on the present study, these observations do suggest that the three genes are expressed in similar types of cells.

In the retina, isl-1 mRNA is presented in both the ganglion layer and the inner nuclear layer including amacrine, bipolar, and horizontal cells, but not in the outer nuclear layer which contains cone and rod photoreceptor cells (Fig. 8C). isl-3 mRNA is limited only to the ganglion layer (Fig. 8D) where isl-2 mRNA could also be faintly detected (Fig. 8E). These data clearly indicate a differential expression of the three isl genes. As indicated by arrows in Fig. 8, C and E, it is interesting to note that there is an abrupt boundary of labeling of isl-1 and isl-3 transcripts at the retinal periphery which contains undifferentiated cells (Wetts et al., 1989; Fermald, 1990), suggesting that isl gene expression is absent in the cells undergoing extensive proliferation. The phenomenon was also observed for another LIM homeobox gene xlim-3 in Xenopus embryos (Taira et al., 1993). This is reminiscent of a previous observation by Ericson et al.(1992) that the Isl-1 immunoreactivity appears immediately after the last mitosis of motor neurons. It is likely here that the isl genes are expressed immediately after the terminal differentiation of certain types of neuroretinal cells. The newly differentiated retinal cells near the retinal periphery appear to have more isl-1 and isl-3 transcripts than other differentiated retinal cells. This is particularly apparent in the nuclear layer where a gradient of isl-1 transcripts can be clearly observed (Fig. 8C). This pattern of expression suggests an important role of isl genes in the early stage of differentiation and maintenance of certain types of neuroretinal cells.


DISCUSSION

Evolution of isl Gene Family

In the present study, three novel LIM domain homeobox genes (isl-2a, isl-2b, and isl-3 genes), which share strong sequence identity with the isl-1 gene, have been identified from the chinook salmon. Together, these genes constitute a novel subfamily of LIM domain homeobox genes, the isl gene family. The pseudotetraploid salmon has two members each of isl-1 (a and b) and isl-2 (a and b) genes and one isl-3 gene. isl-1a and -1b, and isl-2a and -2b genes were likely derived from the same gene by tetraploidization. Consistent with this, the diploid zebrafish has only one member each of isl-1, isl-2, and isl-3 genes.

Based on the sequence comparisons as summarized in Fig. 3and Table 1, the putative evolutionary relationship of these genes is depicted in Fig. 4. An ancestor gene duplicated to give rise to the isl-1 gene and the ancestor isl-2/-3 gene (arrow A). This likely occurred before the divergence of fish and mammals since the rat isl-1 gene is much more homologous to the fish isl-1 genes (81% identity at DNA level) than to isl-2 and -3 genes (68-70%). Therefore, the mammals should have at least one member of isl-1-related genes (isl-2/-3) in their genome if it was not lost during evolution. Consistent with this, we have recently identified an isl-1-related gene in the mouse. (^1)The divergence of isl-2 and isl-3 genes (arrow B) occurred before the divergence of the zebrafish and salmonid species since both the zebrafish and salmon have both genes. Whether the divergence of isl-2 and isl-3 genes occurred before or after the divergence of fish and mammals is unclear. The identity of the zebrafish and rat isl-1 genes at the DNA coding region (81%) is about the same as that of salmon isl-2 and isl-3 (80-81%), although the deduced protein sequences between the two isl-1 genes is more conserved (98%) presumably because of a high selection pressure on the Isl-1 protein. Arrow C indicates the divergence of zebrafish and salmonid and arrow D the tetraploidization of salmonid genome (Ohno et al., 1968). The separation of rainbow trout and chinook salmon occurred after the tetraploidization (arrow E).

Whether there are more genes falling into this isl gene family is not completely clear. However, several observations suggest that no more closely related gene is present in the salmon genome. First of all, only five known isl gene fragments were amplified by PCR using the degenerated homeobox primers, HbL and HbR, from genomic DNA. Second, as shown in Fig. 2, no extra DNA fragment was hybridized to any of the three conserved homeobox probes. Under low stringency, the isl-1 probe hybridized, in addition to the isl-1 fragments, faintly to isl-2 and -3 but not to any other extra fragment; the isl-3 homeobox probe hybridized only to the isl-2 and -3 gene fragments under low stringency. Third, numerous RT-PCR amplifications of isl DNA fragments using degenerated primers from the pituitary and embryonic RNAs did not reveal any additional isl related sequence.

Expression of the isl Gene Family

isl-1 was initially isolated as an insulin gene enhancer binding protein and its mRNA was detected only in the pancreas (Karlsson et al., 1990). Using antisera raised against a recombinant rat Isl-1, Isl-1 immunoreactivity was detected broadly in many neurons and endocrine cells from a variety of tissues including brain, spinal cord, pancreas, pituitary, thyroid, and kidney (Thor et al., 1991; Dong et al., 1991). However, the presence of isl-1-related genes raises an important question about the specificity of Isl-1 immunoreactivity since Isl-1 antisera likely cross-react with other Isl-1-related proteins such as Isl-2 and Isl-3. Consistent with this notion, the Isl-1 antiserum used by Dong et al.(1991) was found to cross-react with the recombinant salmon Isl-2 protein.^2 Therefore, the previous observations on Isl-1 localization by immunocytochemical methods is likely on a combination of all Isl-1-related proteins (Thor et al., 1991; Dong et al., 1991; Ericson et al., 1992; Yamada et al., 1993; Korzh et al., 1993).

In the present study, we have characterized in the rainbow trout the patterns of expression of three closely related isl genes by both RT-PCR and in situ hybridization. Our data indicate that all three isl genes are active in both embryos and in a variety of adult tissues. In adult tissues, the expression of isl-1 genes is widespread in almost all tissues examined, including pituitary, eye, brain, spinal cord, gills (containing thyroid cells), pyloric ceca (containing pancreatic cells), intestine, heart, liver, spleen, and kidney. The isl-2 gene is similarly expressed but at lower levels. In contrast, the expression of the isl-3 gene is more restricted, being detectable only in pituitary, brain, spinal cord, and faintly in heart. The pattern of expression of isl-1 gene in the rainbow trout is in general agreement with the pattern of expression of isl-1 gene in the rat in which the Isl-1 immunoactivity was detected in subsets of neurons and endocrine cells in certain parts of brain, spinal cord, pancreas, pituitary, thyroid, kidney etc. (Thor et al., 1991; Dong et al., 1991). Therefore, the developmental program and the role of isl-1 gene are likely conserved between fish and high vertebrates. The detection of isl gene expression in some tissues such as heart, liver, intestine, and spleen, which have not been reported to express Isl-1 in other systems by means of in situ hybridization and immunocytochemical staining, is likely due to the use of the highly sensitive RT-PCR technique. Quantitative RT-PCR data indicate that level of isl-1 mRNA in these tissues is severalfold lower than that in some highly expressed tissues such as brain and spinal cord (Table 2). Consistent with our data, a low level of isl-1 mRNA was also detected in heart, spleen, and other mouse tissues by an RT-PCR analysis (Dandoy-Dron et al., 1993). The expression of the isl-1 gene in some mammalian cells of non-neuroendocrine origin has also been reported recently (Wang and Drucker, 1994). These observations together suggest that the role of isl-1 may not be limited to neuron and endocrine cell lineages.

In situ hybridization demonstrated that the expression patterns of the three isl genes are similar in developing nervous system. The three isl transcripts are localized in two overlapping clusters of neuron cells ventrolateral to the neural tube in medulla oblongata and spinal cord, and in the same cranial and spinal ganglia, suggesting that the three isl genes may have similar roles in differentiation and maintenance of certain neuronal cell types. However, apparent differences of expression of the three isl genes was also observed. For examples, isl-1 mRNA was found in both inner nuclear layer and ganglion layer while isl-3 mRNA was found only in the ganglion layer. The localization of isl-1 mRNA in brain is more anterior than that of the isl-3 transcript. In addition, isl-1 mRNA, but not isl-3 mRNA, was also found in mid-gut and interrenal organ, consistent with RT-PCR data. Therefore, these differences also indicate some distinctions of the roles of these isl genes.

Predominantly neuronal expression appears to be a common feature for all members of the LIM domain homeobox gene family. Most of the LIM domain homeobox genes are also expressed in some non-neuronal cells. For examples, apterous gene in Drosophila is expressed in selected neurons in both central nervous system and in peripheral nervous and also in some muscle cells (Bourgouin et al., 1992; Cohen et al., 1992); both xlim-1 and xlim-3 in Xenopus are highly expressed in brain tissues and also in some endocrine cells (Taira et al., 1992, 1993); the rat LH-2 gene is predominantly expressed in the brain and also in lymphocytes (Xu et al., 1993). These observations establish the role of LIM homeobox genes in differentiation of distinct classes of neurons. Our in situ hybridization data confirm the expression of the isl-1 gene in subsets of neurons in brain and spinal cord and also in some endocrine cells in mid-gut and interrenal organ (adrenal tissue). isl-2 gene may be similarly expressed, as judged from RT-PCR data. In contrast, isl-3 gene expression is more restricted and may be specific to neurons since both RT-PCR and in situ hybridization failed to detect its expression in many non-neural tissues which express significant amounts of isl-1 and isl-2 mRNAs. The only exception is in pituitary in which the level of isl-3 mRNA is relatively low but significant. However, the teleost pituitary also has a neural part. Therefore, isl-3 gene appears to be the only LIM homeobox gene specifically expressed in the neural cell lineage.

Although the expression patterns of the three isl genes in medulla oblongata and spinal cord have been carefully examined and compared by in situ hybridization on adjacent embryo sections, the question whether their expression is in the same set of neurons or in different sets of neurons in the same region remains unsolved. This question is important to understand the function of the three genes. In zebrafish embryos, the expression of the isl-1 gene is initiated in many primary neurons. Korzh et al.(1993) reported that all three types of primary motor neurons were reactive to the rat Isl-1 antiserum; however, Inoue et al.(1994) found that isl-1 mRNA could only be detected in one of the primary motor neurons. The identification of isl-1-related genes in the present work provides a basis to understand the discrepancy of these observations. It is possible that the primary motor neurons which contain Isl-1 immunoreactivity but not isl-1 mRNA might express other isl-1 related gene products that cross-react with the Isl-1 antiserum. With the isolation of isl-1-related gene clones, this possibility can be examined in the zebrafish. These experiments will be useful to address whether the different isl genes play any role in the determination and differentiation of subtypes of motor neurons.

There is also another possibility that these three isl genes are functionally redundant since they have closely related structures and are expressed in many overlapping regions. Such functional redundancy has been implicated for two similar mouse engrailed genes which are less conserved than the three isl genes; when one of the engrailed genes was knocked out, the other one remained functional for normal development (Joyner et al., 1991). Similarly, functional redundancy has also been explained for the MyoD gene family of myogenic transcriptional regulator (for review, see Weintraub(1993)). Consistent with the functional redundancy, the DNA binding domains of the three Isl proteins have almost identical sequences. The DNA binding sites for the salmon Isl-2 has been determined by binding site selection from a pool of randomly incorporated oligonucleotides (Gong and Hew, 1994). The binding consensus of salmon Isl-2 is consistent with that of rat Isl-1 (Karlsson et al., 1990; Ohlsson et al., 1991; Sanchez-Garcia, 1993); therefore, Isl-2 is likely to interact with the same set of target genes as Isl-1. This may not be surprising since only one amino acid substitution occurs in the homeodomain between the salmon Isl-2 and rat Isl-1. If the functional redundancy is the case for the three Isl proteins, Isl-1 likely plays a major role because its mRNA is distributed more broadly and at higher levels than the other two.

Presence of closely related genes is quite common for transcription factors and regulatory proteins important in development and cell differentiation. Usually, each gene in a family has a distinct program for temporal and spatial expression and thus plays a distinct role by interaction with different sets of target genes or proteins. It has been demonstrated that in many cases the function of such closely related genes are functionally interchangeable. For example, glp-1, a gene coding for a receptor for intercellular signals, can substitute for another related receptor gene, lin-12, in specifying cell fate determination in C. elegans (Fitzgerald et al., 1993). The three structur-ally related paired-box homeobox genes, paired, gooseberry, and gooseberry neuro have also been demonstrated functionally interchangeable (Li and Noll, 1993). These experiments suggest that the distinct functions of similar genes were evolved by acquisition of different cis-regulatory elements in their 5`-upstream promoter regions after gene duplication (Li and Noll, 1993). Differences in gene regulatory region may also be the case for the isl gene family since they are differentially expressed in some tissues or at different levels. However, in contrast to most of the closely related genes, the three isl genes have many similar or overlapping expression domains. Particularly, isl-2 and isl-3 genes seem to be expressed in subdomains of isl-1 expressing cells and at lower levels. This situation may suggest that these two genes have not acquired a new function.


FOOTNOTES

*
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X64883 [GenBank](CSIsl-3), X64884 [GenBank](CSIsl-2b), X64885 [GenBank](CSIsl-2a), U09403 [GenBank](ZFIsl-2), and U09404 [GenBank](ZFIsl-3).

§
To whom correspondence should be addressed: Dept. of Clinical Biochemistry, University of Toronto, 100 College St., Toronto, Ontario M5G 1L5, Canada. Tel: 416-978-8622; Fax: 416-978-8802.

(^2)
Z. Gong, C.-c. Hui, and C. L. Hew, unpublished data.

(^1)
The abbreviation used is: RT-PCR, reverse transcriptase-polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. Alex Joyner for her generous support in in situ hybridization experiments, Richard Kitching and Xiaochun Wang for their assistance in DNA sequencing, Dr. Anderson Wong for his help in analyzing in situ hybridization data, Dr. Harry Elsholtz for his stimulating discussion and supplying two PCR primers HbL and HbR, Drs. Barbara Jones and Martin Petkovich for providing zebrafish embryonic RNA, and Dr. Hitoshi Okamoto for exchanging data prior to publication. PCR primers were synthesized by HSC/ Pharmacia Biotechnology Service Center, Toronto.


REFERENCES

  1. Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Polymeropoulso, M. H., Xiao, H., Merril, C. R., Wu, A., Olde, B., Moreno, R. F., Kerlavage, A. R., McCombie, W. R., and Venter, J. C. (1991) Science 252, 1651-1656 [Medline] [Order article via Infotrieve]
  2. Archer, V. E. V., Breton, J., Sanchez-Garcia, I., Osada, H., Forster, A., Thomson, A. J., and Rabbitts, T. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 316-320 [Abstract]
  3. Baltz, R., Domon, C., Pillay, D. T. N., and Steinmetz, A. (1992) Plant J. 2, 713-721 [CrossRef][Medline] [Order article via Infotrieve]
  4. Birkenmeier, E. H., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2516-2520 [Abstract]
  5. Boehm, T., Foroni, L., Kaneko, Y., Perutz, M. F., and Rabbitts, T. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4367-4371 [Abstract]
  6. Bourgouin, C., Lundgren, S. E., and Thomas, J. B. (1992) Neuron 9, 549-561 [Medline] [Order article via Infotrieve]
  7. Cohen, B., McGuffin, M. E., Pfeifle, C., Segal, D., and Cohen, S. M. (1992) Genes & Dev. 6, 715-729
  8. Dandoy-Dron, F., Deltour, L., Monthoux, E., Bucchini, D., and Jami, J. (1993) Exp. Cell Res. 209, 58-63 [CrossRef][Medline] [Order article via Infotrieve]
  9. Dong, J., Asa, S. L., and Drucker, D. J. (1991) Mol. Endocrinol. 5, 1633-1641 [Abstract]
  10. Ericson, J., Thor, S., Edlund, T., Jessell, T., and Yamada, T. (1992) Science 256, 1555-1560 [Medline] [Order article via Infotrieve]
  11. Fermald, R. D. (1990) in The Visual System of Fish (Douglas, R. H., and Djamgoz, M. B. A., eds) pp. 443-462, Chapman and Hall Ltd., London
  12. Ferguson, E. L., Stermberg, P. W., and Horvitz, H. R. (1987) Nature 326, 260-267
  13. Fitzgerald, K., Wilkinson, H. A., and Greenwald, I. (1993) Development 119, 1019-1027 [Abstract/Free Full Text]
  14. Freyd, G., Kim, S. K., and Horvitz, H. R. (1990) Nature 344, 876-879 [CrossRef][Medline] [Order article via Infotrieve]
  15. Galea, E., and Feinstein, D. L. (1992) PCR Methods Appl. 2, 66-69 [Medline] [Order article via Infotrieve]
  16. German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992) Genes & Dev. 6, 2165-2176
  17. Gong, Z., and Brandhorst, B. P. (1987) Mol. Cell. Biol. 7, 4238-4246 [Medline] [Order article via Infotrieve]
  18. Gong, Z., and Hew, C. L. (1994) Biochemistry 33, 15149-15158 [Medline] [Order article via Infotrieve]
  19. Gong, Z., Fletcher, G. L., and Hew, C. L. (1992) Can. J. Zool. 70, 810-814
  20. Gong, Z., Hu, Z., Gong, Z. Q., Kitching, R., and Hew, C. L. (1994) Marine Mol. Biol. Biotech. 3, 243-251
  21. Hempe, J. M., and Cousins, R. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9671-9674 [Abstract]
  22. Hui, C.-c., and Joyner, A. L. (1993) Nature Genet. 3, 241-246 [Medline] [Order article via Infotrieve]
  23. Inoue, A., Hatta, K., Hotta, Y., and Okamoto, H. (1994) Dev. Dyn. 199, 1-11 [Medline] [Order article via Infotrieve]
  24. Joyner, A. L., Herrup, K., Auerbach, B. A., Davis, C. A., and Rossant, J. (1991) Science 251, 1239-1243 [Medline] [Order article via Infotrieve]
  25. Karlsson, O., Thor, S., Norberg, T., Ohlsson, H., and Edlund, T. (1990) Nature 344, 879-882 [CrossRef][Medline] [Order article via Infotrieve]
  26. Korzh, V., Edlund, T., and Thor, S. (1993) Development 118, 417-425 [Abstract/Free Full Text]
  27. Kosa, J. L., Michelsen, J. W., Louis, H. A., Olsen, J. I., Davis, D. R., Beckerle, M. C., and Winge, D. R. (1994) Biochemistry 33, 468-477 [Medline] [Order article via Infotrieve]
  28. Li, P. M., Reichert, J., Freyd, G., Horvitz, H. R., and Walsh, C. T. (1991) Proc. Natl. Acad. Sci. U. S. A., 88, 9210-9213 [Abstract]
  29. Li, X., and Noll, M. (1993) Nature 367, 83-87
  30. McGuire, E. A., Hockett, R. D., Pollock, K. M., Bartholodi, M. F., O'Brien, S. O., and Korsmeyer, S. J. (1989) Mol. Cell. Biol. 9, 2124-2132 [Medline] [Order article via Infotrieve]
  31. Michelsen, J. W., Schmeichel, K. L., Beckerle, M. C., and Winge, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4404-4408 [Abstract/Free Full Text]
  32. Mizuno, K., Okano, I., Ohashi, K., Numoue, K., Kuma, K., Miyata, Y., and Nakamura, T. (1994) Oncogene 9, 1605-1612 [Medline] [Order article via Infotrieve]
  33. Ohlsson, H., Thor, S., and Edlund, T. (1991) Mol. Endocrinol. 5, 897-904 [Abstract]
  34. Ohno, S., Wolf, U., and Atkin, N. B. (1968) Hereditas 59, 169-187 [Medline] [Order article via Infotrieve]
  35. Sadler, I., Crawford, A. W., Michelsen, J. W., and Beckerle, M. C. (1992) J. Cell Biol. 119, 1573-1587 [Abstract]
  36. Sanchez-Garcia, I., Osada, H., Forster, A., and Rabbitts, T. H. (1993) EMBO J. 12, 4243-4250 [Abstract]
  37. Siebert, P. D., and Larrick, J. W. (1992) Nature 359, 577-558 [Medline] [Order article via Infotrieve]
  38. Taira, M., Jamrich, M., Good, P. J. and Dawid, I. B. (1992) Genes & Dev. 6, 356-366
  39. Taira, M., Hayes, W. P., Otani, H., and Dawid, I. B. (1993) Dev. Biol. 159, 245-256 [CrossRef][Medline] [Order article via Infotrieve]
  40. Thor, S., Ericson, J., Brannstrom, T., and Edlund, T. (1991) Neuron 7, 881-889 [Medline] [Order article via Infotrieve]
  41. Wang, M., and Drucker, D. (1994) Endocrinology 134, 1416-1422 [Abstract]
  42. Wang, X., Lee, G., Liebhaber, S. A., and Cooke, N. E. (1992) J. Biol. Chem. 267, 9176-9184 [Abstract/Free Full Text]
  43. Way, J. C., and Chalfie, M. (1989) Genes & Dev. 3, 1823-1833
  44. Weintraub, H. (1993) Cell 75, 1241-1244 [Medline] [Order article via Infotrieve]
  45. Wetts, R., Serbedzija, G. N., and Fraser, S. E. (1989) Dev. Biol. 136, 254-263 [Medline] [Order article via Infotrieve]
  46. Xu, Y., Baldassare, M., Fisher, P., Rathbun, G., Oltz, E. M., Yancopoulos, G. D., Jessell, T. M., and Alt, F. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 227-231 [Abstract]
  47. Yamada, T., Pfaff, S. L., Edlund, T., and Jessell, T. M. (1993) Cell 73, 673-686 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.