Differential Regulation of Hox C6 in the Appendages of Adult Urodeles and Anurans

JMB—MS 594 Cust. Ref. No. CAM 125/94

[SGML] J. Mol. Biol. (1995) 249, 879–889

Differential Regulation of Hox C6 in the Appendages of Adult Urodeles and Anurans Pierre Savard* and Monique Tremblay Unite´ de Me´decine Ge´ne´tique et Mole´culaire, Centre de Recherches du Centre Hospitalier de l’Universite´ Laval, 2705 bld. Laurier Que´bec, G1V 4G2 Canada

*Corresponding author

Morphogenesis and pattern formation are biological processes that rely on the expression of positional determinants to divide the embryo into compartments. Hox genes are among the selector genes that direct the mechanism of positional information. Here we report the molecular structure and pattern of expression of a new Hox C6 transcript in the adult newt. Molecular analysis showed that the gene transcribes a long primary transcript in both limb and tail regeneration territories and subsequently uses maturation events to produce two RNAs that share the same DNA binding domain and differ in their 5' extremity. Both RNAs were found in the limb and showed a proximal-distal gradient of expression. The tail sample showed accumulation of only one Hox C6 transcript. These results suggest that both transcriptional and post-transcriptional regulations are involved in the appendage-specific expression of Hox C6 in the adult newt. Finally, the adult frog, which has lost its regeneration capacity, also post-transcriptionally regulates the expression of Hox C6 in its appendages. Keywords: alternative splicing; limb regeneration; newt; Notophthalmus viridescens; post-transcriptional regulation

Introduction The study of the patterning and development of the vertebrate limb is a fascinating way to investigate morphogenic mechanisms. During limb development, mesenchymal cells are believed to acquire a positional value that may be fixed by local gradients of morphogenetic substances. This value would subsequently be interpreted during morphogenesis and cytodifferentiation to form the different parts of the limb (Wolpert, 1969, 1989; Summerbell et al., 1973). Little is known about the molecular nature of the signaling mechanisms involved in such morphogenetic events. However, vertebrate homeobox genes show a pattern of expression in the limb bud that suggests their participation in the spatial patterning of the anteroposterior (Oliver et al., 1988a,b, 1989; Dolle´ et al., 1989; Izpisu´a-Belmonte et al., 1991; Izpisu´a-Belmonte & Duboule, 1992; Ros et al., 1992), the proximal-distal (Yokouchi et al., 1991; Haack & Gruss, 1993; Peterson et al., 1994) and the dorsoventral axes (Davis et al., 1991). It is therefore Abbreviations used: aa, amino acid(s); ORF, open reading frame; PCR, polymerase chain reaction; RA, retinoic acid; RACE, rapid amplification of cDNA ends; GPX, glutathione peroxidase; BHLD, distal hindlimb blastema; BT, tail blastema; BHLP, proximal hindlimb blastema; RT, reverse transcriptase. 0022–2836/95/250879–11 $08.00/0

conceivable that positional information in limb morphogenesis is encoded by a set of homeobox products. Urodeles, like the newt (Notophthalmus viridescens), can regenerate limbs and tail after amputation. This phenomenon proceeds by the formation of a blastema, a mound of mesenchymatous-like cells, which grows rapidly and undergoes metamorphosis to replace the missing portions of the appendage (for a review, see Wallace, 1981; Stocum, 1984). The process is very precise in that a perfect regenerate will be formed. It has been proposed that regeneration may proceed through reactivation of the embryonic genes involved in limb development (Muneoka & Bryant, 1984). Many homeobox genes are expressed in the adult newt limb and tail and their corresponding regeneration blastemas; they also show an interesting pattern of expression in relation to regeneration (Savard et al., 1988; Brown & Brockes, 1991; Beauchemin & Savard, 1992, 1993; Simon & Tabin, 1993; Beauchemin et al., 1994). We previously described the pattern of expression on NvHBox-1 (Savard et al., 1988). The gene shows extensive aa conservation with XlHBox-1, Hox-3.3, and HOX-3.3 throughout the entire coding region, suggesting that these are newt, frog, mouse, and human versions of the same gene (Simeone et al., 1987; Cho et al., 1988; Sharpe et al., 1988). Subsequent analysis of the genomic locus confirmed that the 7 1995 Academic Press Limited

JMB—MS 594 880 neighboring downstream gene is the newt version of Hox-3.4 (Belleville et al., 1992). In this paper, NvHBox-1 is renamed Hox C6 to fit to the new nomenclature proposed by Scott (1992). We report here an analysis of the cloning and expression of a new form of the newt Hox C6 transcript harboring a new exon 1 that we call Ex. The limb expresses both transcripts (E1-E2 and Ex-E2) at the same level, whereas the tail principally expresses the Ex-E2 form. The level of expression of both transcripts is greater in the proximal regions of the limb compared with the distal regions. The results are discussed in relation to the putative role of Hox C6 in the specification of the regeneration blastema.

Results Hox C6 expression in the appendages of the adult newt The newt Hox C6 is a single gene that shares a high degree of homology with its corresponding

Regulation of Hox C6 in Regeneration

version in other vertebrates. A schematic summary of the cloning in newt genomic and cDNA libraries shows that Hox C6 harbors two exons (Figure 1A). A previous analysis of the molecular structure and pattern of expression of Hox C6 in the adult newt has been reported and a differential splicing of the gene in limb and tail suggested (Savard et al., 1988). A Northern blot of poly(A)+ and poly(A)− RNA from various limb and tail tissues was hybridized serially with Hox C6 probes of exon 1 (E1) or exon 2 (E2) and the normalizing NOR-cDNA (Figure 1B). A single rare 1.8 kb transcript was revealed in both forelimb and hindlimb poly(A)+ RNA samples with the probe E1. A transcript of similar molecular mass was revealed in poly(A)+ RNA samples of forelimb, hindlimb and tail with the E2 probe. We concluded that the transcript expressed in the limbs harbors both the first and second exons, whereas the transcript expressed in the tail has the second but not the first exon, although the size of the transcript is 1.8 kb in both tissues. An attractive model for

Figure 1. Analysis of Hox C6 expression in the limb and tail of the adult newt. A, Schematic representation of the genomic (l02 and l05) and cDNA clones that were isolated. The two genomic clones overlap and the locus harbors the Hox C6 and Hox C5 genes. Two forms of Hox C6 cDNA underlie the genomic clones. Filled boxes represent the homeodomain. Empty boxes represent the exonic regions that have been characterized. Striped box represents the putative exonic sequence identified in this work. E1 stands for exon 1, E2 for exon 2, and Ex for exon X. B, Northern blot hybridization of newt limb and tail poly(A)+ RNA (varying from 1 to 8 mg) and poly(A)− RNA samples (25 mg) hybridized serially with probe of E1, E2, and NOR. Abbreviations: FL, forelimb; T, tail; HL, hindlimb. C, RNase protection analysis of Hox C6 expression. An antisense riboprobe from the newt Hox C6-E1 was hybridized with various RNA fractions of different adult newt tissues; the mixture was subsequently digested with RNase and loaded on a denaturing gel. The protected fragment is 310 bp long. Abbreviations: FL, forelimb; T, tail; L, liver. RNA fractions: total, 25 mg; poly(A)−, 25 mg; poly(A)+, 5 mg. The probes E2, NOR, and actin were used on dot-blot hybridization to normalize the RNA preparations.

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Figure 2. PCR-RACE amplification of Hox C6 in newt tail cDNA and molecular analysis of a new exonic sequence. A, Schematic representation of the cDNA clones obtained after PCR-RACE amplification of newt cDNA. Hox C6 is the PCR product that was obtained with limb cDNA and corresponds to the previously reported Hox C6 E1-E2 (Savard et al., 1988). The first exon is represented by an empty box; the homeodomain, which is part of the second exon, is shown as a filled box. TA + 480, TA + 460, TA + 459, and TA + 145 are four clones obtained with tail cDNA; the first exon is represented with a stippled box. They harbor 480, 460, 459, and 145 bp of new exonic sequences, respectively. B, The new exonic sequence and its conceptual translation product. The arrow at position 481 represents the splice site that join Ex and E2. The beginning of the homeodomain is boxed.

explaining these results is to insert an alternative exon 1 (Ex) into the tail-derived tissue. We loaded different amounts of forelimb, hindlimb, and tail poly(A)+ RNA on the blot to reveal comparable signals with Hox C6 probes (E1 and E2). The band revealed with the NOR probe is representative of the amount of poly(A)+ RNA present in each sample (Figure 1B). We evaluated the amount of transcript harboring E2 in the tail to be about ten times less than that in the forelimb and three to five times less than that in the hindlimb. NOR consists of a fortuitously cloned cDNA fragment that is highly homologous to human DNAbpA and DNAbpB, which encode DNA-binding proteins (Sakura et al., 1988). NOR detects a single abundant 1.9 kb transcript that is ubiquitously expressed except in the brain, where it is dramatically reduced, and the skin, where it is moderately reduced (Beauchemin & Savard, 1992). We further analyzed the expression of Hox C6 by RNase protection of RNA samples from the forelimb, tail, and liver (Figure 1C). The protection of a 310 bp band (specific to Hox C6 E1) was found in RNA samples of limb and tail, and the level of expression was similar in both tissues. On the other hand, liver RNA did not protect any detectable level of Hox C6 probe; the fuzzy signal in the liver total RNA fraction was not specific to Hox C6 because the band was not of the appropriate molecular mass and has not been seen in other protection assays (data not shown). To verify the integrity of each RNA preparation, samples were dot-blotted on a nylon membrane that was then serially hybridized with a Hox C6 E2

probe and the normalizing NOR and actin probes (Figure 1C). The detection of similar levels of Hox C6 E1 RNA in the limb and tail contrasts with the results of the previous Northern blot analysis. One simple explanation is that the Hox C6 transcripts in the tail are of molecular masses and therefore do not compact into a single band on Northern blot. Therefore, the activity level of the Hox C6 transcription unit seems similar in both the limb and tail, whereas the mature 1.8 kb transcript harboring E1 accumulates only in limbs, raising the possibility that post-transcriptional mechanisms may limit expression in the tail. PCR-RACE cloning of new exonic Hox C6 sequences To identify new Hox C6 E1 sequences in newt-tail RNA, we used an E2-specific oligonucleotide to prime cDNA synthesis. Tail cDNAs were then PCR-amplified and cloned (see Materials and Methods). We then analyzed four clones that showed the expected 5' extensions (Figure 2A): TA + 480, TA + 460, TA + 459, and TA + 145 harbor, respectively, 480, 460, 459, and 145 bp of new exon 1 sequence. This new exon is referred to as Ex. The same assay was done with limb cDNA, and the four clones that were obtained harbored sequences of E1-E2, which corresponds to the previously reported Hox C6 transcript (Savard et al., 1988). Figure 2B illustrates the nucleotide and deduced amino acid sequences of Ex. The same splice site, located at position 481, ligates Ex or E1 to E2. Ex

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transcript harboring Ex is also present in the limb; therefore, the signal detected with E2 in limb samples (Figure 1B) may represent the sum of two different mRNAs (E1-E2 and Ex-E2).

PCR amplification analysis of Hox C6 transcripts in limb and tail

Figure 3. Northern blot of poly(A)+ (8 mg) and poly(A)− (25 mg) RNA samples of adult newt tissue. The blot was serially hybridized with Ex and the normalizing probe NOR. Abbreviations: T, tail; FL, forelimb; L, liver.

shows two long ORFs: the first is 69 aa long and continues into E2 for 101 aa in phase with the homeodomain; the putative protein harbors a potential initiator methionine located at position 415 of the cDNA. Its sequence context fits the consensus sequence (-A/GNNATGPu-) established for other eukaryotic translation start sites (Kozak, 1987). The second ORF is 105 aa long and continues into E2 for an additional 279 aa not in phase with the homeodomain. The first methionine in this putative protein is located at the aa 249, and its sequence context does not fit the consensus sequence described by Kozak (1987). We searched for regions conserved in genes from other vertebrates but found no extensive amino acid or nucleotide conservation in the GenBank and EMBL databanks. We aligned the newt Ex DNA sequences with the first exon of another form of the Hox C6 transcript that has been reported in frogs and humans. The Xenopus Hox C6 PR1 mRNA (Cho et al., 1988) shares 58% of its nucleotide sequence with Ex; the degree of homology with the human Hox C6 c8.5111 mRNA (Simeone et al., 1988) is 52%. However, these percentages of homology decrease drastically when the amino acid sequence is used in the comparisons, mainly because of the insertion of many gaps during the alignment of nucleotide sequences. A Northern blot was then hybridized with an Ex probe (Figure 3). A major (1.5 kb) and a minor transcript (1.8 kb) were detected in the Poly(A)+ RNA fractions of both limb and tail, whereas no signal was detected in the liver. The integrity of the RNA samples was verified with the NOR probe. Therefore, detection of a 1.8 kb signal in tail mRNA with the Ex probe could account for the signal detected with E2 in Figure 1B. On the other hand, the 1.8 kb Hox C6

We previously reported (Savard et al., 1988) that Hox C6 is expressed at about a threefold higher level in a proximal forelimb blastema (mid-humerus) than in a distal one (mid-radius and ulna). This observation was discussed in view of a putative function of Hox C6 in the specification of positional information along the proximal-distal axis of the limb. In this work, we found that the limb expresses two types of Hox C6 transcript of similar molecular mass. Is the level of expression of each transcript regulated differently along the proximal-distal axis of the limb? For this analysis, we used a very sensitive detection assay because of the low level of expression of the gene. Moreover, we limited our comparisons to proximal and distal hindlimb blastemas (that is, we sought the same proportion of epidermis and mesenchyme) to avoid comparing body structures of different tissue composition. We primed cDNA synthesis with oligo(dT) and PCR-amplified aliquots with a mixture of oligonucleotides specific to Hox C6 E1-E2, Hox C6 Ex-E2, NvHbox-4, and GPX (Figure 4.) A sample of each reaction was loaded on a gel after various numbers of amplification cycles to ensure that the kinetics of the assay were under control. From this analysis, we concluded that the levels of both transcripts are significantly higher in proximal blastemas compared with distal ones. Moreover, we showed a huge difference in expression of the E1-E2 transcript in the limb and tail, although the same amount of Ex-E2 transcripts was found in both tissues. Therefore, Hox C6 expression is differently regulated in limb and tail tissue and along the proximal-distal axis of the limb. To ensure that the procedure was quantitative we performed the following controls. (1) One mg of mouse RNA (liver) was added to each sample and the reverse transcription product tested for its GPX content; the amount of GPX cDNA was similar in all samples (Figure 4), meaning that the reverse transcription step was comparable in all tubes. (2) Two samples of BHLD (distal hindlimb blastema), one having 1 mg and the other 3 mg of RNA, were done to ensure that the assay was quantitative; the normalizing RNA was NvHBox-4 because its level of expression is not regulated along the proximal-distal axis of the limb, and the amounts of epidermis in a proximal or a distal blastema are believed to be the same (Beauchemin & Savard, 1992); the signal revealed with NvHBox-4 was similar in BT (tail blastema), BHLP (proximal hindlimb blastema), BHLD, and higher in the BHLD (3X) sample; we concluded that there was similar amount of poly(A)+ RNA in BT, BHLP, and BHLD samples and that the

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Figure 4. Quantitative PCR analysis of Hox C6 expression in blastemas from the tail and different levels of the limb. The upper section of the Figure shows Southern blot analyses of the product of amplification of Hox C6 E1-E2, Hox C6 Ex-E2, NvHBox-4, and GPX; only one of the triplicate assays is shown. The lower part of the Figure shows the linear regression of the PCR-amplification in each tissue (mean of three replicates). The relative abundance of each band was measured with an image analyzer (BIOIMAGE-VISAGE 110S from Millipore Corp., Ann Harbor, MI). Abbreviations: C, control samples that were loaded in identical amount on all blots hybridized with the same gene; numbers, rounds of amplification; BT, blastemal tail (reaction started with 1 mg of poly(A)+ RNA); BHLP, blastemal hindlimb proximal (reaction started with 1 mg of poly(A)+ RNA); BHLD, blastemal hindlimb distal (reaction started with 1 mg of poly(A)+ RNA); BHLD(3X), blastemal hindlimb distal (reaction started with 3 mg of poly(A)+ RNA); log O.D., log of absorbance for each band.

assay was precise enough to reveal a threefold difference between two samples. The PCR-amplification of Hox C6 E1-E2 cDNA showed major differences of expression between different tissues. The level of transcript was almost undetectable in the tail and slightly higher in BHLP compared with BHLD; the difference was lower than threefold because BHLD (3X) is higher than BHLP. The PCR-amplification of Hox C6 Ex-E2 cDNA showed similar amounts of transcript in BHLP and tail tissue and lower amounts in BHLD. The difference is evaluated to be threefold because the amounts of transcript are similar in BHLP and BHLD (3X). We already reported a threefold difference of expression between proximal and distal regeneration blastemas by analyzing the intensity of Hox C6 signal on Northern blot (Savard et al., 1988).

and E2 on Figure 1A). One contained the entire intron and part of E1 as well. One of these libraries was also screened with an Ex probe, and a single clone was isolated. Analysis showed that it contained DNA sequences from position 207 to 481 (Figure 2B) and an additional 500 bp of intronic sequences. We concluded that the extra 500 bp were intronic DNA after we PCRamplified an identical DNA fragment in both the tail cDNA clone and genomic DNA (data not shown). We examined the l02 genomic phage (Figure 1A) for the presence of Ex sequences but found none in the 9 kb of DNA upstream of E1, which may explain why we were unable to PCR-amplify genomic DNA with Ex and E2 primers: the distance separating these exons was too great for the assay. Spliced Hox C6 RNA in newt limb and tail

Hox C6 cloning in tail cDNA libraries We screened 3 × 106 plaques from two independent newt tail cDNA libraries (unamplified) with an E2 probe. Three positive clones were isolated, which on analysis were found to contain E2 sequences and various lengths of intron (DNA region between E1

In view of a possible difference in the RNA processing of the limb and tail, we used PCR amplification to analyze the Hox C6 RNA in each tissue. We primed the reverse transcription of the poly(A)+ RNA with a random hexamer and then used a mixture of oligonucleotides specific to E1 and

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controls in which the RT enzyme was omitted demonstrated that the poly(A)+ RNA preparations were not contaminated with genomic DNA (data not shown). Hox C6 expression in the adult frog

Figure 5. Southern blot analysis of PCR-amplified Hox C6 E1-E2 cDNA. The products of amplification were separated on a gel and blotted for subsequent hybridization with a probe of the intron separating E1 and E2 (In) and a probe of the second exon (E2). The oligonucleotide specific to In (5'-TCTACGATCATTTAAGGAGAG-3') revealed a 1.568 kb band corresponding to the unspliced Hox C6 RNA. The oligonucleotide specific to E2 (redundant oligonucleotide recognizing the amino acid sequence DRQVKIWFQNRRKEK) revealed the unspliced Hox C6 band (1.568 kb) and an additional 0.434 kb band that corresponds to the spliced Hox C6 E1-E2. Abbreviations: M, DNA marker phiX174 + HaeIII; FL, forelimb; T, tail; HLs, hindlimb skin; HLm, hindlimb muscle; Un, amplified newt genomic DNA; Bam, amplified newt genomic DNA + BamHI.

E2 for PCR amplification. By knowing the structure of the gene (Figure 1A), we can predict that the detection of a 1568 bp band represents the unspliced RNA, whereas a 434 bp band represents the spliced RNA. Two different probes were used to detect the amplified DNA, one specific to the intron that can detect only the unspliced product, the other specific to E2 and able to detect the two forms of the product (Figure 5). Unspliced and spliced PCR products were found in all tissues. However, the proportion of unspliced RNA was high in the tail sample and low in forelimb and hindlimb samples. We amplified genomic DNA with the same primers to control the specificity of the assay; the undigested product comigrated with the unspliced cDNA, and a BamHI restriction digest produced DNA fragments of expected molecular masses (Figure 5). Additional

To determine whether sustained expression of Hox C6 in adult appendages is a phenomenon particular to the newt, we looked for its expression in the adult Xenopus laevis (Figure 6A). A Northern blot of poly(A)+ RNAs from adult Xenopus embryos was hybridized serially with XlHBox-1 (Hox C6) and two normalizing cDNAs (actin types 5 and 8). The Hox C6 probe revealed a single rare 1.8 kb transcript in the embryo, but no detectable signal was found in the adult forelimb or hindlimb samples. Hybridization of the same blot with type 5 cytoskeletal actin shows that the loading of RNA in each lane is comparable. Finally, hybridization with type 8 cytoskeletal actin led to the conclusion that no significant contamination of the muscle fractions by skin tissue had occurred. RNase protection analysis of Xenopus RNA preparations from adult Xenopus limbs and Xenopus embryos showed that Hox C6 RNA was present in all these samples (Figure 6B). This result contrasts with the results of Northern blot analysis, which detected Hox C6 expression only in embryonic RNA (Figure 6A). The integrity of the RNA samples used in the protection assay was analyzed by Northern blot with the normalizing actin (type 5 cytoskeletal actin) and 18 S (ribosomal RNA) probes (Figure 6B). These analyses suggest that similar regulative pathways control the expression of Hox C6 in the adult newt tail and the adult Xenopus limbs.

Discussion Differential expression of Hox C6 in the newt limb and tail We isolated two Hox C6 transcripts that share the same DNA-binding domain and differ in their 5' extremities. Molecular analysis showed that these transcripts encode an alternative splice of coding exon 1. Examples of such differential splicing occur in the Xenopus and human Hox C6 homologs (Cho et al., 1988; Simeone et al., 1988). In both cases, the gene expresses two types of transcript: a short one (1.8 kb) similar to the E1-E2 transcript we previously reported (Savard et al., 1988) and a long one (2.2 kb) that possesses an extra exon 1 located about 10 kb upstream. This exon differs from the newt Ex in that it does not make use of the same splicing event to join the homeobox-containing exon (E2), and it does not carry a long ORF in phase with the homeodomain. Our analysis of Hox C6 expression in adult newt tissue indicates that at least two mechanisms are operating. In the liver, no expression of Hox C6 RNA was detected. In the limb and tail, Hox C6 expresses two transcripts that differ in their exon 1 sequences

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885

Figure 6. Analysis of Hox C6 expression in Xenopus tissue. A, Northern blot hybridization of adult X. laevis limb and embryonic poly(A)+ RNA samples hybridized serially with probes of E1 and actin (type 8 and type 5 cytoskeleton actin). Abbreviations: HLs, adult hindlimb skin; HLm, adult hindlimb muscle; FLs, adult forelimb skin; FLm, adult forelimb muscle; E, embryos of stage 35 to 55. B, RNase protection analysis of Hox C6 expression in Xenopus tissue. An antisense riboprobe from the Xenopus Hox C6 E1 was hybridized with various RNA fractions of adult and embryonic Xenopus tissues; the mixture was subsequently digested with RNase and loaded on a denaturing gel. The protected fragment is 214 bp long. Abbreviations: FL, adult forelimb; HL, adult hindlimb; E, embryo stage 35 to 55. RNA fractions: total, 25 mg; poly(A)−, 25 mg; poly(A)+, 5 mg. The probes actin and ribosomal 18 S were used on Northern blot hybridization to verify RNA integrity.

(Ex-E2 and E1-E2). The Ex-E2 form is present in similar amounts in both tissues, whereas significantly higher levels of the E1-E2 form are present in the limbs. The differential pattern of expression of these transcripts suggests they may have distinct roles. The exon shared by the limb and tail harbors the DNA binding domain; it is therefore possible that this portion of the Hox C6 protein is necessary to bind specific genomic sequences, whereas the portion coded by the first exon is involved in regulating genetic events implicated in the determination of limb and tail. In Xenopus, expression of the two forms of Hox C6 protein shows precise regulation along the anteroposterior axis of the embryo, suggesting they also may have distinct developmental roles (Oliver et al., 1988a,b). Previous analyses of the human Hox C locus have shown that the chromosomal region covering at least four genes (Hox C5, C6, C8, and C9) of the cluster functions as a single transcription unit of about 45 kb (Simeone et al., 1988; Oudejans et al., 1990). Several polyadenylation signals would be used, giving rise to a number of polyadenylated RNAs, which in turn are spliced to generate mature mRNAs. The present analysis shows that this situation is probably similar in the newt and that the Hox C transcription unit remains active in the appendages of adult amphibians. Our results demonstrated that some posttranscriptional mechanism may limit the amount of

mature Hox C6 E1-E2 transcript in the tail and that one level of regulation might be splicing. It is also possible that message instability contributed to the failure to detect a mature E1-E2 transcript in the tail, and it should be noted that both the newt and Xenopus 3'-untranslated regions contain three AUUUA destabilizing sequences (Shaw & Kamen, 1986; for a review, see Jackson & Standart, 1990).

Expression of Hox C6 along the proximal-distal axis of the limb Another interesting characteristic of Hox C6 expression is the graded accumulation of both transcripts along the proximal-distal axis of the limb. It is known that there is graded expression of many homeobox genes in the developing limb bud, a phenomenon that might provide a molecular address for the spatial identity of cells, information necessary to the formation of different limb structures (Izpisu´a-Belmonte et al., 1991). A gradient of expression of Hox C6 along the proximal-distal axis of the developing limb has been reported for Xenopus (Oliver et al., 1988a,b, 1989), chicken (Oliver et al., 1990), and mouse (Oliver et al., 1988a,b, 1989). The graded expression of Hox C6 in the regeneration blastema of the adult newt limb suggests that a similar system of positional

JMB—MS 594 886 specification occurs during regeneration. It will be important to determine, by using cytochemical assays, whether the cellular content of Hox C6 products varies continuously along the limb or there is differential representation of distinct cell types in the blastema at different levels. It is noteworthy that in situ hybridization would not discriminate between tissues expressing primary transcripts and those expressing both primary and mature transcripts. The newt Ex is included in at least two types of transcript (1.8 and 1.5 kb), the longest harboring the Hox C6 homeodomain. We have not yet characterized the shortest. In view of what is known about the differential splicing of the human Hox C6 gene (Simeone et al., 1988), it would not be surprising to find that Ex is present in the messenger RNAs for other homeobox genes of the same cluster.

Differential expression of Hox C6 in anurans and urodeles We reported that limbs from adult Xenopus do not express Hox C6; we did this demonstration by comparing 5 mg of embryonic poly(A)+ RNA with 25 mg of adult forelimb poly(A)+ RNA on Northern blot (Savard et al., 1988). In this work, Northern blot analysis revealed no Hox C6 accumulation in the adult Xenopus forelimb and hindlimb tissues, but expression was readily detectable by RNase protection. This finding demonstrates that the Hox C6 transcriptional unit is still active in the limbs of the adult Xenopus and suggests that posttranscriptional mechanisms might limit its accumulation. It would be interesting to compare the time course of loss of the mature Hox C6 transcript with the progressive loss of regeneration ability in premetamorphosed anurans (Dent, 1962; Kurabuchi, 1990). At least six other homeobox genes are expressed in the adult newt limb (Beauchemin & Savard, 1992, 1993; Simon & Tabin, 1993; Beauchemin et al., 1994). The sustained expression of these genes in the adult urodele limb is an interesting possibility for explaining an underlying predisposition for regeneration. Because the principal function of homeobox genes during development is one of positional determination, it is conceivable that their sustained expression in adult tissue represents the maintenance of a positional memory that is passed on to blastemal cells during the process of dedifferentiation. Whether Hox expression in the adult urodele is related to some morphogenetic properties of the regeneration fields remains an open question. This mechanism is not universal in that newt Hox D10 and D11 are not detectable in any adult tissue, but they appear in the blastema mesenchyme after amputation (Brown & Brockes, 1991; Simon & Tabin, 1993). Nonetheless, it will be important to understand the significance of the mechanism whereby the newt Hox C6 remains on in the adult limb, but its homologous version in Xenopus does not.

Regulation of Hox C6 in Regeneration

Materials and Methods Animals Adult N. viridescens were purchased from C. D. Sullivan Co. Inc. (Nashville, TN). Anaesthesia and surgical procedures were as reported (Savard et al., 1988). The bud of blastema tissue was cut off at midbud stage (according to the classification of Iten & Bryant, 1973) between days 13 and 16 post-amputation. The newts generally grew another midbud stage blastema in fewer than 12 days; this second blastema, along with subsequent ones, were harvested. Blastemas and other tissues were routinely stored in liquid nitrogen before RNA extraction. cDNA cloning and sequencing We used two different newt-tail cDNA libraries, previously described by Brown & Brockes (1991) and Beauchemin & Savard (1992). Plaques (3 × 106 ) were blotted onto nitrocellulose membranes and screened with E2 or Ex DNA probes. Positive clones were purified and subcloned for subsequent analysis. Nucleotide sequence determinations were done by the dideoxy termination method (Pharmacia Ltd), and sequence analysis was performed using the sequence analysis software package of the genetics computer group from the University of Wisconsin (Devereux et al., 1984). Probes and hybridization The newt E1 DNA probe corresponds to the N71 cDNA (Savard et al., 1988); the DNA fragment was also used to produce sense and antisense riboprobes. The newt E2 DNA probe corresponds to the PvuII-Xhol DNA fragment included in the 3'-untranslated region of the p158 cDNA clone (Savard et al., 1988). NOR is a 700 bp cDNA that was fortuitously cloned (Savard et al., 1988); it is the newt version of the DNAbpA gene (Sakura et al., 1988), and it detects a single abundant transcript that is ubiquitously expressed except in the brain, where it is dramatically reduced, and in the skin, where it is moderately reduced (Beauchemin & Savard, 1992). The newt Ex probe is a cDNA fragment that covers the sequence from nt 1 to nt 522 (Figure 2B). The Xenopus E1 probe is an EcoRI-BamHI DNA fragment (214 bp) subcloned from the genomic clone AC1 (Carrasco et al., 1984). The Xenopus type 5 cytoskeletal probe is a 210 bp cDNA fragment from the 3'-untranslated region of X1-424 (Mohun et al., 1984). The Xenopus type 8 cytoskeletal actin probe is a 1300 bp cDNA fragment from the translated region of XlcA1 (Mohun et al., 1984). The ribosomal 18 S is a cDNA that was fortuitously cloned from a newt cDNA library. For Northern blot analysis, RNA was electrophoresed on 1% (w/v) agarose gel containing 0.66 M formaldehyde (Maniatis et al., 1982). The RNA was transferred to a Gene-screen filter and hybridized according to the method suggested by the supplier (New England Nuclear). After hybridization, the blots were washed 3 × 20 minutes in 0.1 SSC/1% (w/v) SDS at 60°C and exposed for 7 to 14 days at −70°C, except for NOR, actin and 18 S probes, which were exposed for only 4 to 24 hours (SSC is 0.15 M Nacl, 0.015 M sodium citrate, pH 7.0). RNase protection Synthetic antisense RNA probes (0.5 × 106 cfs/min with specific activity of 00.5 × 108 cts/min per mg) and RNA

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Regulation of Hox C6 in Regeneration samples (25 mg with both total and poly(A)− RNA and 5 mg with ploy(A)+ RNA) were incubated overnight at 45°C in 30 ml of hybridization buffer (80% (v/v) formamide, 0.04 M NaPipes (pH 6.7), 0.4 M NaCl, 1 mM EDTA). Then the mixture was incubated for 30 minutes at 30°C in 300 ml of RNase buffer (0.01 M Tris-HCl (pH 7.5), 0.3 M NaCl, 5 mM EDTA) containing 6.2 mg/ml RNase A (Boehringer) and 420 U/ml RNase T1 (Boehringer). SDS (0.1% final concentration) and proteinase K (166 mg/ml final concentration) were added and incubated for 30 minutes at 37°C. Then tRNA carrier (10 mg) was added in the poly(A)+ samples. The mixtures were phenol chloroform extracted and ethanol precipitated. The pellet was resuspended with denaturing sequencing loading buffer and loaded onto a denaturing 6% (w/v) polyacrylamide gel. After migration, the gel was dried onto 3 MM paper and exposed to X-ray film (XAR-5, Kodak) in the presence of intensifying screens, generally for seven to ten days at −70°C.

PCR amplification Poly(A)+ RNA samples (1 mg) were reverse transcribed for two hours at 37°C using 200 units of Moloney reverse transcriptase (RT) (BRL) in RT buffer containing 1.0 mM dNTPs, 50 units RNasin (Pharmacia), 10 mM DTT, and 0.5 pmol of oligo(dT)-primers. The mixture was filtrated onto a Centricon 100 spuncolumn (Amicon Division). Three samples (5 ml) of each cDNA were then incubated with 2.5 units of Taq DNA polymerase (Pharmacia) in 50 ml of PCR reaction buffer (67 mM Tris (pH 8.8), 6.7 mM MgCl2 , 170 mg/ml BSA, 17 mM (NH4 )2 SO4 , 1.5 mM dNTPs, and 10% (v/v) dimethyl sulfoxide containing 150 ng of the appropriate primer (E1 downstream; Ex downstream; E2 upstream). The reactions were run with a programmable thermal controller (PTC-100, MJ Research Inc.) under the following conditions: a hot start (94°C for five minutes, ice for two minutes; Taq addition, 52°C for two minutes and 72°C for two minutes) and various numbers of regular cycles (94°C for 35 seconds, 52°C for 45 seconds, 72°C for two minutes with an extension cycle of five seconds). During the amplification procedure, 10 ml samples were taken after various numbers of cycle and loaded on agarose gel that were subsequently blotted and hybridized with specific oligonucleotides (chosen in the amplified region). The blots were exposed on X-ray films at room temperature for various lengths of time. The signals revealed on X-ray films were processed with an image analyzer to measure their intensity (BIOIMAGEVISAGE 110S from Millipore Corp., Ann Harbor MI). We PCR-amplified several genes from each cDNA reaction. The following controls were done to ensure that the assay was quantitative. (1) One mg of total RNA from mouse liver was added to all the samples before cDNA synthesis; three aliquots of each reaction were primed with oligonucleotides specific to the mouse glutathion peroxydase gene (GPX) and PCR-amplified; the GPX signal was expected to be similar in all samples if cDNA synthesis proceeded normally. (2) cDNA synthesis of distal hindlimb blastema (BHLD) was done with both 1 mg and 3 mg of newt RNA; therefore, a quantitative difference was expected for each newt gene tested by PCR amplification (NvHBox-4 and Hox C6). (3) The number of PCR cycles run with each gene was chosen carefully to ensure that the rate of amplification was in the exponential phase. (4) Each cDNA sample was PCR-amplified in triplicate. (5) Each agarose gel was loaded with a control sample (BT) to normalize for differences occurring in the procedure of blotting, hybridization, and X-ray exposure. (6) All blots

from the same PCR amplification were hybridized in the same bag and exposed to X-ray films in the same cassette. (7) Finally, the absorbance of each band was represented on a semi-log scale to draw linear curves from which we statistically analyzed the linear regression between every tissue. The PCR-RACE cloning of Ex was performed with 1.0 pmol of oligonucleotide specific to E2 (E2-upstream-1) to prime the cDNA synthesis of limb and tail poly(A)+ RNA. The single-stranded cDNAs were filtrated on a Centricon 100 spun-column and poly(A) tailed with 15 units of terminal deoxynucleotidyl transferase (BRL) for 15 minutes at 37°C in 200 ml of DNA-tailing buffer (0.1 M potassium cacodylate (pH 7.2), 2 mM CoCl2 , 0.2 mM DTT). The first series (30 rounds) of PCR amplification was done with 10 ml of the previous reaction and 150 ng of both oligonucleotides Ro and E2-upstream-1 in 50 ml of PCR reaction buffer containing 2.5 units Taq DNA polymerase. The second series (30 rounds) of PCR amplification was done with 1 ml of the first amplification reaction and 150 ng of both oligonucleotides Ri and E2-upstream-2. The final amplification products were cloned in the PCR-1000 vector (TA-cloning kit, Invitrogen Corporation). List of oligonucleotides with the orientation of priming: Hox C6 Ex downstream, 5'-CTCTCGCCCGCCCCATA-3'; Hox C6 E2-upstream-1, 5'-AATGCTATATTGTCGTGG-3'; Hox C6 E2-upstream-2, 5'-ATCCATATTCATTGTGCCAGT-3'; Hox C6 E1 downstream, 5'-AGTGTCCAGGAGCAGAAG-3'; NvHBox-4 downstream, 5'-ATGTGGATGTGAGTGTGT-3'; NvHBox-4 upstream, 5'-CTCTCTTTGTGCGGTTTA-3'; GPX downstream, 5'-GAGGCACCACGGTCCGGGAC-3'; GPX upstream, 5'-AAGCGGCGGCTGTACCTGCG-3'; Ro upstream, 5'-GACTCGAGATATCATCGA(T)17-3'; Ri downstream, 5'-GACTCGAGATATCATCGA-3'.

Acknowledgements We thank Dr Jeremy Brockes and Phillip Gates for stimulating discussions. We are indebted to Dr Tom Moss for providing the Xenopus embryos. We are also grateful to Ms Diane Castilaw for her help in the translation of the manuscript. P.S. holds a scholarship from the ‘‘Fonds de la recherche en sante´ du Que´bec’’. This work was funded by the Medical Research Council of Canada (MT-10664).

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Edited by J. Karn (Received 21 March 1994; accepted in revised form 4 April 1995)