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