specific activation of mammalian Hox promoters in …genesdev.cshlp.org/content/6/4/591.full.pdfspecific activation of mammalian Hox promoters in mosaic transgenic zebrafish Monte

specific activation of mammalian Hox promoters in mosaic transgenic zebrafish Monte Westerf ie ld / Jeremy W e g n e r / Beatrice G. Jegal ian/ Eddy M. D e R o b e r t i s / and Andreas W. Puschel*'^

'Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 USA; ^Department of Biological Chemistry, University of California, Los Angeles, California 90024 USA; ^Max-Planck Institut fiir Biophysikalische Chemie, Abteilung Molekulare Zellbiologie, D-3400 Gottingen, Germany

Homeo box-containing genes {Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgene expression, and examined the activities of two mammalian Hox genes, mouse Hox-1.1 and human HOX-3.3. We found that these Hox promoters are activated in specific regions and tissues of developing zebrafish embryos and that this specificity depends upon the same regulatory elements within the promoters that specify the spatial expression of these genes in mice. Our results suggest that the promoter activities have been remarkably conserved from fish to mammals. To study the regulation of Hox expression in the developing nervous system, we analyzed the promoter activities in spt-1 mutants that have a mesodermal deficiency. Our results suggest that interactions, probably with the paraxial mesoderm, differentially regulate the activities of Hox promoters in the developing nervous system.

[Key Words: Homeo box; embryogenesis; spinal cord; sclerotome; mesoderm; in situ hybridization]

Received December 18, 1991; revised version accepted January 23, 1992.

A central problem of developmental neurobiology is to understand how cells become specified to form particular neurons. Initially, the ectoderm is induced, by signals from the mesoderm, to form the nervous system (Spe-mann 1938; Cairns and Saunders 1954). Recent studies have shown that particular regions of the mesoderm can induce region-specific gene expression in the developing neuroectoderm (Frohman et al. 1990; Hemmati-Brivan-lou et al. 1990). It is still unclear, however, what changes are induced in cells of the neuroectoderm and how these changes regulate the specification of neuronal fates.

One way that cells become specified is through the selective expression of transcriptional regulators. Homeo box genes, including Hox genes, may serve this function during development of the vertebrate nervous system, because they are expressed in particular regions of the embryo (for reviews, see Holland and Hogan 1988; Kessel and Gruss 1990) and they are thought to code for DNA-binding proteins (Gehring et al. 1990) that may act as transcription factors (Levine and Hoey 1988; Wright et al. 1989; Johnson and Krasnow 1990; Kessel and Gruss 1990). We studied the induction of homeo box genes in mosaically transgenic zebrafish by analyzing the promoter activities of two Hox genes that are expressed in the anterior spinal cord of mammals. We found that activation of one but not the other promoter was affected by a mutation that acts on a subset of mesodermal cells. Our results suggest that interactions with mesodermal

cells may differentially activate particular Hox gene promoters in the neuroectoderm.

Results

Expression of transgenes in developing embryos

To study the regulation of Hox promoters, we injected recombinant DNA fragments, containing fusions of the mouse Hox-1.1 or the human HOX-3.3 promoter to a marker gene, p-galactosidase (p-gal), into one-cell zebrafish embryos and assayed for p-gal expression in live or fixed animals after ~ 1 day of development. In many of the injected embryos, one to several hundred cells expressed the transgene (average 10 ± 5, n = 59 embryos), although the average number of expressing cells varied with different transgene constructs (numbers are given in the figure legends). Figure 1 shows an embryo in which the HOX-3.3 transgene was expressed in a specific subset of spinal cord sensory neurons. The putative endogenous zebrafish Hox-3.3 protein, which can be detected with an antibody derived from the Xenopus ho-molog (XlHbox-1) of the HOX-3.3 homeoprotein, is known to be expressed in this particular type of neuron in the same region of the spinal cord (Molven et al. 1990).

Distribution of injected genes

The cells expressing the transgene were mixed together with nonexpressing cells in a variegated manner; ubiq-

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Westerfield et al.

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Figure 1. The human HOX-3.3 promoter directs expression of a marker gene, P-gal, in Uve transgenic zebrafish. The 3-gal activity was detected in two Rohon-Beard neurons (identified according to Grunwald et al. 1988) in the dorsal region of trunk spinal cord segments 5 and 7 of a live, 24-h (h = hours after fertilization at 28.5°C) embryo using a fluorescent substrate {A) and in the same two neurons after fixation using a galactoside substrate (B). The histological procedures resulted in shrinkage; thus, the embryo in B appears somewhat smaller. Expression of p-gal was observed in 62 of 75 similarly injected embryos. Side view, anterior to the left, dorsal to the top. Bar, 25 i.m.

uitous expression throughout or within a given region of the embryo w^as never observed. Most frequently, expressing cells were grouped together in the same region of the embryo. The variegated expression pattern could have occurred for several reasons, for example, differences in activation of the promoters in various cells (Stuart et al. 1990) or an uneven distribution of the transgene within the injected embryos. To examine the second possibility, we injected fluorescently labeled DNA and watched its subsequent movement in the embryo (Fig. 2). Immediately after injection, the labeled DNA formed a spherical bolus that became parceled among a subset of the cellular progeny of the injected cell.

The mosaic distribution of the injected DNA could explain the variegated expression pattern. However, we know from previous analysis of transgenic zebrafish (Stuart et al. 1988) that injected DNA is replicated during early cleavage stages. Our labeling technique allowed us to follow only the injected (labeled) DNA, and the trans-gene may have been more widely expressed as a result of diffusion of the replicated (unlabeled) DNA or the gene product during early developmental stages when the blastomeres are still connected by cytoplasmic bridges (Kimmel and Law 1985). Moreover, the transgene may have been present in some cells at a low concentration that we were unable to detect with fluorescence optics. We examined these possibilities by fixing some of the embryos and staining for (3-gal activity. In some cases, the p-gal expression, detected in fixed embryos, was dis

tributed more widely than the injected DNA. Taken together, these results suggest that expression is limited not only because some cells do not activate the promoter but also by an uneven distribution of the transgene within the embryo. Replication of the injected DNA may broaden this distribution.

Cell-specific activation of Hox promoters

The Hox promoters were specifically activated in particular subsets of cells in the early embryos. By comparison, a viral promoter, cytomegalovirus (CMV), was activated about equally well in all of the tissues examined. To demonstrate this, we analyzed 24-h (hours after fertilization at 28.5°C) embryos, which had been injected with one of three different promoter constructs, and counted the numbers and types of labeled cells. Figure 3 shows that the Hox-1.1 and HOX-3.3 transgenes were expressed in similar distributions of tissues, which differed from that of the CMV transgene. Both Hox promoters were activated most frequently in mesodermal or neuroectodermal tissues, with <15% of the expressing cells appearing in other tissues. The expressing mesodermal cells were primarily mesenchymal, and their ventral medial positions within the somite were appropriate for the putative sclerotome. Only occasional muscle, skin, or notochord cells expressed these constructs. Within the spinal cord, the HOX-3.3 promoter was activated primarily in more dorsally located cells than was the Hox-1.1 promoter.

Position-specific activation of Hox promoters

The mouse Hox-1.1 promoter was activated in specific cell types in anterior segments of the embryo. This was demonstrated by superimposing the expression patterns observed in many injected embryos. As illustrated in Figure 4A, the majority of expressing cells were located in trunk segments, although a few cells in the heads of three embryos expressed the Hox-1.1 transgene. Expression within the nervous system was strongest in anterior segments, which correspond to somites 3-10. The anterior border was approximately at the level of the fin bud, near the boundary between somites 2 and 3. The posterior extent of expression was less distinct; occasionally, cells in the nervous system posterior to somite 15 expressed the transgene. This pattern corresponds approximately to the region where Hox-1.1 is expressed in the neural tube of mice, beginning near the border between C3 and C4 and fading out gradually at lower lumbar levels (Mahon et al. 1988; Dressier and Gruss 1989; Piischel et al. 1990). These results suggest that the mammalian promoter functions in zebrafish and is activated within cells and regions in the fish that correspond to those in which the promotor is activated in mice.

We examined whether the regulatory elements of the Hox-1.1 promoter, identified previously in mice (Piischel et al. 1991), are also responsible for the restricted transgene expression in the fish. Deletion of 130 bp (element B), at the 5' end of the coding sequence adjacent to the

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Hox promoter activation in transgenic zebrafish

Figure 2. Injected DNA is compartmentalized. A single embryo is shown at four developmental stages after injection of labeled (red) DNA (pUSVCAT) at the one-cell stage. [A] The fluorescently labeled, injected DNA was contained in one cell at the two-cell stage. (B) After two subsequent cell divisions, two of the eight cells in the embryo contained the injected DNA. These cells were among the progeny of the labeled cell shown in A. (C) By the 128-cell stage, approximately 8 cells contained the labeled DNA. (D) A small cluster of cells contained the fluorescent DNA by the late blastula stage. As the labeled cells moved apart later during gastrulation, they became difficult to resolve with fluorescence microscopy. The embryos are oriented with the animal pole up. Bar, 100 |xm in A-C and 150 fxm in D.

3-gal gene, removed the posterior expression border and resulted in p-gal expression in the posterior end of the embryo (Fig. 4B). Deletion of 1.94 kbp at the 5' end of the promoter produced a pattern of expression indistinguishable from the full-length promoter. However, deletion of an additional 108 bp (element A) eliminated the anterior border of expression completely (Fig. 4C). Each of these deletions altered the expression pattern in zebrafish, as reported previously for the mouse (Piischel et al. 1991). This comparison demonstrates that the same elements, of ^100-130 bp each, are required for the spatial regulation of transgene expression in these two species.

Less extensive analysis of the HOX-3.3 promoter demonstrated a similar specificity. Expression was observed most often in trunk segments (Fig. 5A), although we occasionally found cells in the head and tail that expressed the transgene. The anterior borders of p-gal expression,

as defined by expressing cells from several embryos, were quite distinct. In the periphery, expressing cells anterior of the border between somites 4 and 5 were virtually never seen, with the exception of the fin bud. In the nervous system, expression extended anteriorly to the junction between the hindbrain and the spinal cord. Deletion of the 5.2 kbp of human sequences reduced the levels of expression and eliminated the region-specific activity of the promoter; expressing cells were found in the head and tail.

The anterior borders of transgene expression in the nervous system and periphery correspond quite well to the borders of expression of the putative endogenous gene. In a previous study, Molven et al. (1990) examined the distribution of the endogenous putative gene product, as indicated by staining with the XlHbox-I antibody. In the periphery, antibody labeling began sharply

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Notochord Other

Figure 3. The Hox-1.1 and HOX-3.3 promoters are specifically activated in the nervous system and the sclerotome. The percentage of the total number of expressing cells in each category is plotted for the promoters: Hox-1.1 (m61acZ4 and m61acZlI|, solid bars; HOX3.3p-gal, open bars; CMV-IEP, hatched bars. Nervous system cells were identified on the basis of their locations within the spinal cord or brain. Cells of the sclerotome were identified by their close packing, rounded nuclei, and ventral medial positions in the somites. Muscle cells were identified by their elongated morphologies and striations. Skin cells were the most superficial, flattened cells of the embryo. Expressing cells were counted as notochord if they were in or associated with the notochord. Cells that could not be placed into one of these five categories or that were ambiguous to identify were classified as other. The data came from 132 cells in 31 animals for m6lacZ4 and m61acZll, 175 cells in 10 animals for HOX3.3p-gal, and 148 cells in 18 animals for CMV-lEP.

at the border between somites 4 and 5 and extended posteriorly for several segments with no clear posterior border. Similarly, antibody labeling within the spinal cord started at segment 2 and extended posteriorly for at least six or seven segments. Thus, the anterior borders of transgene and endogenous gene expression match almost exactly in the somites but are slightly displaced in the nervous system, with transgene expression starting one to two segments more anteriorly. In posterior regions, the transgene is expressed much more extensively than the endogenous gene in both tissues.

Hox promoter activation in spt-1 mutants

It has been suggested that Hox gene expression may be regulated by interactions between the mesoderm and the neuroectoderm (DeRobertis et al. 1989). We explored the role of interactions between these two tissues by examining the activities of Hox promoters in the spt-1 (bl04) zebrafish mutant, in which mesodermal derivatives in trunk segments are deficient (Kimmel et al. 1989). In spt-1 embryos, precursors of somitic muscle cells fail to converge into trunk segments, although some other me-sodermally derived cells, including those of the notochord, develop normally (Ho and Kane 1990). The mutation acts cell autonomously on mesodermal precursors but not on precursors of the nervous system (Ho and Kane 1990; Eisen and Pike 1991).

We found virtually no expression of the Hox-1.1 transgene in either the neuroectoderm or the mesoderm of trunk segments affected by the mutation, whereas expression was observed in more posterior segments as in wild types (Fig. 5C,D). Additionally, an abnormal accumulation of Hox-2.1-positive cells appeared in the "spade tail" (Kimmel et al. 1989), the region to which mutant trunk mesodermal cells migrate abnormally. With the Hox-1.1 promoter construct, which lacks both the A and B elements (Fig. 3C), cells in the head and tail of spt-1 embryos expressed (i-gal as in wild types. However, the trunk segments affected by the mutat ion lacked expressing cells (data not shown).

In contrast, the HOX-3.3 promoter, which is activated in some of the same trunk segments as Hox-1.1 in wild types, was appropriately induced in both neuroectoderm and mesoderm of the regions affected by the spt-1 mutation (Fig. 5A,B). These results suggest that the mutation affects the activation of one, but not the other, ho-meo box promoter.

Discussion

In this study we analyzed the expression of a marker gene, (J-galactosidase, under the control of mammalian homeo box gene promoters, injected into developing zebrafish embryos to learn how well their functions have been conserved between fish and mice and to analyze their activation during neural induction. Our results indicate that the promoters are specifically activated in particular cell types in particular positions within the embryo and that Hox promoter activities within the nervous system may be regulated by interactions with the mesoderm.

We assayed the specificity of promoter activation by analyzing the patterns of expression in injected embryos, rather than in permanent germ-line transformants. The main reason for this choice was the ease and speed of analysis (Stuart et al. 1988, 1990; Gulp et al. 1991). Although we do not yet know definitively whether expression of these genes will be the same in stable transgenic lines, several observations suggest that the patterns we observed are regulated specifically by the activities of the promoters. First, the expression patterns were highly reproducible in many hundreds of embryos in dozens of different experiments (Figs. 4 and 5). Second, the patterns were dependent upon the specific DNA sequences of the promoters; different promoters or different forms of a given promoter produced reproducibly different expression patterns (Figs. 3 and 4). Third, in the case of the HOX-3.3 promoter, the pattern of transgene expression (Fig. 5A) could be compared more directly to expression of the endogenous gene; many, although not all (as discussed below), aspects of the expression pattern were reproduced by the transgene.

Specificity of Hox promoter activity

The expression of p-gal is specifically regulated by the Hox promoters. Both the Hox-1.1 and the HOX-3.3 pro-





Hox promoter activation in transgenic zebrafish

Mouse Zebrafish

Hox 1.1 promoter B-gal

H H

lAl P

r~p"

Figure 4. Two domains of the Hox-1.1 promoter are required for region-specific activation of the transgene. {A) The 3.64 kbp of promoter sequence (m6lacZ4 and m61acZll), including components A, P, and B [right], was activated primarily in spinal cord and somite cells of anterior trunk segments [middle]. [B] Deletion of 130 bp 5' from the Hox-1.1 ATG, component B [right], allowed activation of the promoter (m6lacZl and m61acZlA) in posterior regions of the embryo [middle]. (C) Deletion of 108 bp at the 5' end, component A [right], eliminated the anterior border of expression and allowed activation of the promoter (m6lacZlB) in the head [middle]. Summary diagrams of the expression domains of these three promoters in transgenic mice are shown at left as reported previously (Piischel et al. 1991). No apparent differences were observed between m61acZ4 and m61acZll expression. The expression patterns were superimposed from all of the injected embryos that expressed each transgene. In A, the borders of the expression domain were defined by expressing cells from more than one embryo. The number of embryos that expressed the transgene was 53 of 96 injected [A], 38 of 60 injected [B], and 56 of 67 injected (C). Bar, 150 ^m for the zebrafish and 650 |xm for the mice.

moters direct expression primarily to nervous system and sclerotome cells within particular regions along the anterior-posterior axis. These patterns arc very reminiscent of the expression patterns of the endogenous genes in mammals; both the Hox-1.1 (Mahon et al. 1988; Piischel et al. 1990) and HOX-3.3 (Simeone et al. 1987; Oliver et al. 1988; Sharpe et al. 1988) genes are expressed in the spinal cord and sclerotome of mice. Moreover, they are distinctly different than the pattern observed after injection of the viral promoter CMV (Fig. 3). The CMV promoter was activated in many tissues, similar to our previous results with SV40/RSV (Rous sarcoma virus) promoters in transgenic zebrafish (Stuart et al. 1990).

The p-gal expression pattern directed by the HOX-3.3 promoter could be compared directly to labeling with an antibody directed against the endogenous putative gene product (Molven et al. 1990). Some aspects of the two patterns were nearly identical, whereas others were not. For example, the anterior borders of the two expression domains corresponded quite well, both within the nervous system and in the somites. On the other hand, transgene-expressing cells were observed in posterior segments that were unlabeled with the antibody. Additionally, we seldom saw expression of the transgene in the notochord (Fig. 3), a tissue that is recognized by the antibody (Molven et al. 1990). The difference between

the two patterns in posterior segments may indicate that p-gal transgene detection, which is more sensitive than in situ hybridization (Piischel et al. 1991; Schughart et al. 1991), may also be more sensitive than antibody labeling. The absence of transgene expression in the notochord may indicate a difference between the human and fish promoters, because expression of HOX-3.3 in the notochord of other species has not been reported. Alternatively, these differences may indicate that zebrafish fail to induce completely normal activity of the human promoter or that the promoter sequences may be incomplete or may function correctly only in the context of the intact HOX-3 cluster (Gaunt and Singh 1990). In Xeno-pus, expression of two gene products from the HOX-3.3 homolog is regulated by promoters separated by 9 kbp (Cho et al. 1988). These possibilities can be tested with additional constructs and by direct comparisons between the promoter sequences of the zebrafish and mouse genes.

The spatial expression of p-gal is regulated by distinct elements within these Hox promoters. Deletion of two elements from the Hox-1.1 promoter eliminated the spatial regulation of expression (Fig. 4). One element (B), near the 3 ' end of the promoter, limits expression in posterior segments, and the other (A), farther from the coding region, is required for the anterior border of ex-





Westerfield et al.

HOX 3.3 / B-gal Hox 1.1 /B-gal

Q .

Figure 5. The Hox-1.1, but not the HOX-3.3, promoter is activated abnormally in the spinal cord of spt-1 embryos. [A] The HOX-3.3 promoter was activated in anterior trunk segments of transgenic zebrafish. [B] The HOX-3.3 promoter was activated similarly in spt-1 mutants. The pattern of expression was not significantly different from that observed in wild-type embryos [A], except that there were more expressing cells in the tail. (C] The Hox-1.1 promoter was activated in phenotypically wild-type (wild-type and heterozygous spt-1) embryos. (D) Hox-1.1 promoter activity was disrupted in spt-1 homozygous mutant embryos. Very few cells expressed the transgene in trunk segments, whereas expression in the spade tail (Kimmel et al. 1989) was seen frequently. The injected promoter sequences were from HOX3.3^-gal in A and B, and m6lacZ4 or m6lacZl 1 in C and D. The expression patterns were superimposed from the 46 expressing embryos of 58 injected embryos {A), 37 expressing of 45 injected [B], 34 expressing of 101 injected (C), and 36 expressing of 90 injected (D). After deletion of the HOX-3.3 promoter, only 6 of 59 injected embryos expressed the transgene. Bar, 150 \x.m.

pression. Because these same elements regulate the spatial pattern of expression of Hox-1.1 in mice (Puschel et al. 1990, 1991), we suggest that their functions have been evolutionally conserved from fish to mammals. Direct comparisons of these promoter sequences, however, must await isolation and nucleotide analysis of the homologous fish gene. Our results suggest further that the network of developmental control genes has been conserved to the extent that the mouse promoter functions in the fish as it does in the mouse.

Induction of Hox promoter activity

We found that the two Hox promoters, Hox-1.1 and HOX-3.3, were differentially affected by the spt-1 mutation. The HOX-3.3 p-gal expression pattern in homozygous spt-1 mutants was virtually the same as in wild types. These results are consistent with previous studies showing that the zebrafish HOX-3.3 protein is expressed approximately between the normal anterior and posterior boundaries in both neuroectoderm and mesoderm of spt-1 mutants (Molven et al. 1990).

In contrast, activation of the Hox-1.1 promoter was altered by the mutation; neither somitic nor spinal cord cells in the affected segments expressed p-gal. The expression pattern of endogenous Hox-1.1 in zebrafish is unknown, but it is possible that it includes a subset of mesodermal cells that are affected by the mutation. They could either be cells different from those expressing HOX-3.3 or they could be the same cells differentially affected, in terms of Hox promoter activity, by the mutation. In either case, this would account for the absence of Hox-1.1 transgene expression in the mutant

somites. Previous studies demonstrated that the deficiency of cells in the trunk of spt-1 embryos results from abnormal migration of mesodermal precursors into the tail (Kimmel et al. 1989). The cells ectopically expressing the Hox-1.1 transgene in the tail may be the cells that migrate abnormally. Ectopic cells in the tails of spt-1 mutants sometimes express fates inappropriate for their origins (Kimmel et al. 1989); however the activity of the Hox-1.1 promoter in these cells would suggest that at least some of their properties, that is, their segmental identities as indicated by an active Hox-1.1 promoter, are unchanged.

The absence of Hox-1.1 promoter activity in trunk segments of the spt-1 spinal cord is probably the result of the deficient paraxial mesoderm. Alternatively, it could be the result of a direct effect of the mutation on the neuroectoderm. This latter possibility seems less likely because genetic mosaic analyses of spt-1 embryos suggest that aspects of the mutant phenotype are expressed autonomously in paraxial mesoderm (Ho and Kane 1990) but not in the neuroectoderm; mutant neuronal precursors (Ho and Kane 1990) and neurons can develop normally when transplanted to wild-type hosts (Eisen and Pike 1991). Moreover, the segmental patterning of the spinal cord is altered in concert with changes in the somites produced either by the spt-1 mutat ion or by surgical ablation (Eisen and Pike 1991). Thus, we suggest that interactions with the paraxial mesoderm, which are disrupted in spt-1 mutants^ normally induce the Hox-1.1 promoter activity that we observe in the trunk spinal cord.

These interactions appear to be specific, because the HOX-3.3 promoter, whose activity in the mesoderm is





Hox pTomoter activation in transgenic zebiafish

unaffected by the spt-1 mutation, is also activated in the spt-1 trunk spinal cord. In the mouse spinal cord (Piischel et al. 1991), Hox-1.1 is expressed primarily in ventrally located cells, whereas HOX-3.3 is expressed more dorsally in both mammals (Simeone et al. 1987) and zebrafish (Molven et al. 1990). Yamada et al. (1991) have suggested that the dorsal fate is a constitutive spinal cord program that is modified by ventralizing signals originating from the notochord and floor plate. Our results are consistent with this notion and suggest further that normal mesoderm is required for promoting a ventral fate, activation of the Hox-1.1 promoter, spt-1 is the first example of a mutation that differentially affects Hox promoters. Differential activation of specific Hox genes may be a mechanism that leads to the specification of neuronal diversity.

Materials and methods

Animals

Zebrafish embryos were obtained by natural crosses. Wild-type embryos were from the Oregon AB line. Homozygous spt-1 mutant embryos were obtained from crosses between heterozy-gotes. Mutants were identified by their phenotypes at 20 h, and phenotypically wild-type siblings (homozygotes and heterozy-gotes) served as controls. Developmental stages arc expressed as hours after fertilization at 28.5°C (h). For viewing p-gal activity in vivo, embryos were anesthetized by immersion in tricaine (Westerfield 1989).

Gene constructs

HOX3.3p-gal was constructed using a Notl site located in the homeo box of the human HOX-3.3 gene. It contains 5.2 kbp of human DNA, including 3.6 kbp 5' of the transcription start site of promoter II (Cho et al. 1988), fused to a p-gal reporter containing a nuclear localization signal. The Hox-1.1 constructs have been described previously (Piischel et al. 1990, 1991). The pUSVCAT is from Stuart et al. (1990), and CMV-IEP is from MacGregor and Caskey (1989). The fusion constructs were separated from vector sequences and purified by sucrose gradient centrifugation followed by phenol-chloroform extraction and precipitation in ethanol. The precipitates were resuspended at 50 ng/|xl in 250 mM KCl and 0.1% phenol red.

Injections and analysis of expression

Approximately 300 pi of DNA solution was injected into one-cell embryos with a glass micropipette (Stuart et al. 1990). After 20-24 h, the embryos were soaked in 20 ji.M ImaGene lacZ substrate (Molecular Probes) for 30 min at 28.5°C, rinsed, and viewed with fluorescence optics using a low-light video camera (SIT-57, General Electric) and an image processor. Development appeared to continue normally after treatment. To confirm that the fluorescence signal was specifically the result of p-gal activity, some embryos were then fixed in 4% paraformaldehyde, 0.2% glutaraldehyde, 4% sucrose, 0.15 mM CaClj, and 0.1 M sodium phosphate (pH 7.3) for 30 min at 4°C. The embryos were then rinsed in 0.1 M sodium phosphate, incubated in 4% 5-bromo-4-chloro-indoxyl-p-D-galactoside, 150mMNaCl, 1 mM MgClj, 1.5 mM K4[Fe3(CN)6l, 1.5 mMK3[Fe2(CN)6], in 5 mM sodium phosphate buffer (pH 7.3), at 37°C for 3 hr, rinsed again, and mounted in glycerol. Individual expressing cells were

counted and identified according to their locations and morphologies using Nomarski optics. In almost every case (>99% of 1480 expressing cells), the (3-gal activity detected with the ga-lactosidase substrate was limited to the nucleus. Embryos that were obviously malformed were excluded from the analyses.

Visualization of injected DNA

Plasmid DNA was labeled by mixing it with 1% ethidium bromide. Unbound dye was separated by passing the mixture over a Sephadex G-50 (Pharmacia) column. The labeled DNA was then injected into embryos, and the embryos were viewed with bright-field and fluorescence optics. After contrast enhancement and coloring, the bright-field and fluorescence images were superimposed using an image processor.

Acknowledgments

The ImaGene lacZ substrate was a generous gift from R. Hau-gland of Molecular Probes. We thank C. Kimmel for helpful comments on the manuscript. This work was supported by the National Institutes of Health and the McKnight Foundation. A.W.P. was supported by a grant from the Bundesministerium fiir Forschung and Technologic (to P. Gruss).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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10.1101/gad.6.4.591Access the most recent version at doi: 6:1992, Genes Dev.

M Westerfield, J Wegner, B G Jegalian, et al. transgenic zebrafish.Specific activation of mammalian Hox promoters in mosaic

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