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Keywords:

  • promyelocytic leukemia zinc finger (Plzf);
  • HoxD genes;
  • conserved noncoding element;
  • polydactyly

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Lx mutation in SHR.Lx rat manifests in homozygotes as hindlimb preaxial polydactyly. It was previously mapped to a chromosome 8 segment containing the Plzf gene. Plzf (promyelocytic leukemia zinc finger protein) influences limb development as a direct repressor of posterior HoxD genes. However, the Plzf coding sequence is intact in the Lx mutants. Using linkage mapping in F2 hybrids, we downsized the segment containing Lx to 155 kb and sequenced conserved noncoding elements (CNEs) inside. A 2,964-bp deletion in Plzf intron 2, never detected in control animals, is the only candidate for Lx. The deletion removes the most deeply conserved CNE in the 155-kb segment, suggesting a regulatory influence on Plzf expression. Correspondingly, using in situ hybridization and quantitative real-time polymerase chain reaction, we found a decrease of Plzf expression in Lx/Lx limb buds with concomitant anterior expansion of expression domains of its targets, Hoxd1013 genes, in the absence of ectopic Sonic hedgehog expression. Upstream regulation of Plzf in limb buds is currently unknown. We present here the first candidate Plzf cis-regulatory sequence. Developmental Dynamics 238:673–684, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

The tetrapod limb consists of three parts: the proximal stylopod, intermediate zeugopod, and distal autopod. Limb patterning is established along three axes: proximal to distal (P–D), dorsal to ventral (D–V), and anterior to posterior (A–P), each of them controlled by distinct molecular pathways (Niswander,2003).

Pentadactyly is the classic digit formula in tetrapods (Galis et al.,2001). An alteration in the number of digits (oligodactyly, polydactyly) is a result of abnormal control of autopod A–P patterning. It is a relatively frequent phenomenon, and in humans, it is considered to be the most common congenital limb anomaly (Graham and Ress,1998).

Polydactyly can be classified according to the position of the supernumerary digits as preaxial or postaxial. Preaxial polydactyly, where the supernumerary digits arise at the anterior margin of the autopod, can be further subdivided according to its dependence on ectopic expression of Sonic hedgehog (Shh). Shh expression is confined to the posterior margin of developing limb, in an organizing center called the zone of polarizing activity (ZPA; Riddle et al.,1993). Type 1 polydactyly is dependent on anterior ectopic activation of Shh similar to polydactyly induced by anterior transplantation of Shh-producing ZPA cells (Yang et al.,1997). Type 2 polydactyly is Shh-independent (Talamillo et al.,2005). In both types, the molecular pathway operating during the pathogenesis involves Hox genes (Kuijper et al.,2005; Sheth et al.,2007).

Hox genes encode transcription factors and precise activation of their transcription in time and space according to their genomic topography is required for correct definition of the body and limb plan. For limb development, two clusters—HoxA and HoxD—are essential (Tarchini and Duboule,2006). HoxD genes display asymmetric expression along the A–P axis, indicating their role in control of A–P polarity. Within the HoxD cluster, genes belonging to paralogy groups 10 to 13 (Hoxd1013) are the most important for limb development. Generally, HoxD genes are activated in two consecutive waves under different transcriptional control (Tarchini and Duboule,2006; Tarchini et al.,2006). The first phase is essential for development of stylopod and zeugopod. Hoxd1013 are expressed in posterodistal nested domains progressively restricted to successively more posterior part of the limb; hence, Hoxd13 occupies the smallest expression domain located posteriorly. The second phase is essential for autopod development. Hoxd1013 are expressed in a presumptive digit domain in a rather reverse pattern compared with the first phase; hence, Hoxd13 is expressed in the broadest domain. Under physiological situations, Hoxd1013 expression does not extend to the most anterior margin of the limb bud.

Disruption of Plzf (promyelocytic leukemia zinc finger, also known as Zbtb16 or Zfp145) function in the mouse by gene targeting leads to multiple patterning defects, including homeotic transformation of axial and limb skeleton and preaxial polydactyly (Barna et al.,2000). In addition, the males are infertile, due to deficiency of spermatogenic stem cell renewal (Buaas et al.,2004; Costoya et al.,2004). Of interest, a recent study revealed that a classic murine mutation luxoid (lu) is caused by a nonsense point mutation in Plzf (Buaas et al.,2004). Recently, loss of Plzf function was found in a clinical case. The patient had an 8 Mb deletion of paternal 11q23, including Plzf and a point mutation of Plzf on the maternal chromosome. In addition to symptoms associated with 11q23 microdeletion syndromes, the patient also presented with absence of thumbs, hypoplasia of radii and ulnae, additional vertebrae and ribs, retarded bone age, and genital hypoplasia. These traits can be ascribed to the loss of Plzf function (Fischer et al.,2008).

In addition to limb development and spermatogenesis, Plzf has been also implicated in apoptosis (Parrado et al.,2004), cell cycle regulation (Yeyati et al.,1999), tumorigenesis (Piazza et al.,2001; Felicetti et al.,2004), and hematopoiesis (Piazza et al.,2004; Labbaye et al.,2008). Its functional versatility has also been shown by identification of Plzf as a crucial member of distinct metabolic and signaling pathways controlling blood pressure (Schefe et al.,2006; Naray-Fejes-Toth et al.,2008), and angiotensin II-induced cardiac hypertrophy (Senbonmatsu et al.,2003).

Plzf acts as a transcriptional repressor, binding DNA by its C-terminal zinc finger domain containing nine Krüppel type zinc finger motifs (Li et al.,1997; Sitterlin et al.,1997), and recruiting, by its N-terminal bric-a-brac, tramtrack, brad complex/poxvirus zinc finger (BTB/POZ) domain (Bardwell and Treisman,1994; Dong et al.,1996), transcriptional corepressor complexes with histone deacetylase activity (Hong et al.,1997; David et al.,1998; He et al.,1998). Plzf also interacts through its three N-terminal zinc-finger motifs with nuclear receptors, in particular with retinoic acid receptor (RAR) alpha, blocking the RAR-RXR heterodimerization necessary for retinoic acid signaling (Martin et al.,2003). During limb development, Plzf modulates spatial expression of Hoxd1013 by transcriptional repression in the anterior part of the developing autopod. The repression mechanism includes direct binding at consensus sequences inside the HoxD cluster and recruiting repressor polycomb proteins (Barna et al.,2002). The regulation of Plzf expression itself during limb development (i.e., the factor[s] upstream of Plzf) is not known.

Mutant allele Lx in the rat was described in the 1970s in an outbred rat strain of Wistar origin (Kren,1975). The mutant rat served as a founder of an inbred strain PD/Cub (polydactylous, PD hereafter). Lx was then transferred to genetic backgrounds of BN/Cub (Brown Norway, BN hereafter) and SHR/OlaIpcv (spontaneously hypertensive rat, SHR hereafter) strains, creating thus BN.Lx and SHR.Lx.PD5 (PD5 hereafter) congenic strains, respectively. The genetic background has significant impact on both expressivity and penetrance of the mutant Lx allele: in BN.Lx it behaves as semidominant and afflicts also forelimbs. In SHR, Lx effects are strictly recessive and restricted to hindlimb autopod (Kren,1975; Kren et al.,1996). Lx is localized to rat chromosome 8 (Kren et al.,1995).

The model for our study is congenic strain PD5. PD5 homozygotes (Lx/Lx) display preaxial polydactyly of hindlimbs with 6 triphalangeal toes (plus a digit rudiment between digit I and II), accompanied by a slight alteration in the shape of tarsal bones (Fig. 1). Zeugopod and stylopod are normally formed. Forelimb is not affected. Axial skeleton is never affected. PD5xSHR heterozygotes (+/Lx) are indistinguishable from wild-type (+/+). PD5 differential segment contains 14 genes, including Plzf (Šeda et al.,2005). Because the autopod malformation resembles the phenotype observed in mouse mutant lu (Plzflu/lu) or Plzf−/−, Plzf is an obvious candidate for the Lx phenotype. However, there is an absence of zeugopod, stylopod, and axial skeleton affliction and no signs of male infertility in Lx/Lx compared with both Plzf−/− and Plzflu/lu. We showed recently that the Plzf coding sequence does not contain any sequence variant specific to rats carrying Lx (Šeda et al.,2005). These facts led us to hypothesize that Lx is a regulatory mutation in Plzf. Because the rat is not used in developmental biology as often as the mouse, we present here a side by side comparison of the limb bud development in the two species (Fig. 2). In general, the rat limb development is approximately 1–2 days delayed, proportional to its 1–2 days longer gestational period.

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Figure 1. Limb phenotype of Lx/Lx rats compared with +/Lx animals. Hind limb sample was taken at 22 days post coitum (dpc; 1 day before birth), bone is stained with Alizarin Red, cartilage with Alcian Blue. +/Lx rats show pentadactyly with diphalangeal hallux (wild-type phenotype), Lx/Lx rats possess six toes; the extra hallux (I′) is triphalangeal. There is an extra skeletal element (rudimentary digit) between the first and second toe (arrow). Tarsal skeletal elements of Lx/Lx rats are normal in number, but the anterior elements show alterations in size and shape. In particular the medial cuneiform (labeled “c”) lost its elongated shape and widened to accommodate the extra finger ray, accompanied by similar widening of the communicating navicular bone (labeled “n”). Zeugopods are comparable in Lx/Lx and +/Lx animals and do not differ from +/+ animals (data not shown; for detailed morphometric analysis of zeugopods, see Printz et al.,2003).

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Figure 2. Gross comparison of mouse (C57BL/6) and rat (SHR) early limb development. Limb buds emerge 2 days later in the rat than they do in the mouse (rat 12.5 days post coitum [dpc] ≈ mouse 10.5 dpc). At 9.5 dpc in the mouse and 11.5 dpc in the rat, we observed similar mesenchymal thickenings in the limb field in both species (data not shown). Interestingly, at the end of our observation period the difference appears closer to 3 days (rat 15.5 dpc ≈ mouse 12.5 dpc). Scale bars = 1 mm.

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In this study, we show that Plzf expression is markedly reduced in the limb bud of Lx/Lx homozygotes. Correspondingly, we found anteriorization of target posterior HoxD gene expression. Using intercross, we narrowed the chromosomal segment carrying Lx to 155 kb, containing serotonin receptor 5-HT3 subunit genes Htr3a, Htr3b (partial), and Plzf gene (partial). Surveying all conserved noncoding sequences in the region, we demonstrate that Lx homozygotes contain a 2,964-bp deletion that removes the most conserved noncoding element in the 155-kb nonrecombinant region. We conclude that this deletion is likely the cause of the mutant Lx phenotype.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Fine Mapping of Lx to a 177-kb Segment

In the PD5 congenic strain, Lx is contained in a 1.4-Mbp differential segment of PD origin. Because the Plzf coding sequence is intact in PD5, we hypothesized that the observed mutant phenotype may be a consequence of a regulatory mutation. The regulatory regions are in general less well defined compared with coding regions. In addition, they can reside anywhere in the differential segment, making it more difficult to compare comprehensively the DNA sequences. Therefore, we decided to downsize the segment carrying Lx using F2 hybrids between PD5 and BN. By analysis of 247 F2 hybrids, we found 7 recombinants inside the PD5 differential segment. Two key recombinants refined the Lx position to 177 kb (Fig. 3). This region contains 3 genes: Plzf (partial), and genes coding subunits of serotonin receptor 5-HT3Htr3a and Htr3b (partial) (Fig. 4A).

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Figure 3. Fine linkage mapping shows Lx is placed between microsatellites 21652 and 21829 (refer to Supp. Table S2 for polymerase chain reaction primers). A: Genotypes in F2 (PD5 × BN) hybrids, n = 247. The parental congenic strain PD5 (first column) carries Lx in its differential segment (of PD origin, dark), flanked by DNA of SHR origin (white). Microsatellite markers follow Mendelian rules (expected ratio PD5:heterozygotes:BN 1:2:1, D8Got72 χ2 = 4.46; P = 0.11), Lx segregation is compatible with autosomal recessive mode of inheritance (normal “+”:Lx 3:1, χ2 = 0.16; P = 0.69). Two recombinants, No. 110 and 117 are decisive for Lx placement between 21652 and 21829. B: Linkage map derived from data presented in A. Lx segment is 0.4 cM long.

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Figure 4. Nonrecombinant segment, sequencing and variation. A: Genome view of the 177 kb Lx segment (between 21652 and 21829). Annotation tracks are showed as follows: Gaps; RefSeq genes; spliced expressed sequence tags, only nonredundant included; Conservation by MULTIZ, overall conservation (“Conservation” label on the left), followed by pairwise conservation with individual genomes; x_tropicalis = Xenopus tropicalis; conservation with zebrafish (not included in MULTIZ alignment); PhastCons conserved elements, only those with LOD score > 70 shown; sequencing coverage of this study, the individual sequenced fragments numbered; variation identified in this study, the magnification does not always allow to match the genomic position exactly, exact positions are listed in Supp. Table S1; chromosome of F2 hybrid 117—SNPs in fragments 24, 3, 4, 5, 22, and 29 are heterozygous in this Lx mutant animal, excluding thus the 22-kb segment; corresponding genome features are gray. Crossing-over in F2 117 occurred between fragment 29 and 21 (dark gray on the F2 chromosome). Detailed view of the 177-kb segment with additional annotation tracks can be obtained from UCSC Genome Browser Internet page. The respective coordinates are chr8:52033239-52209946, rat genome assembly version November 2004 (version 3.4 of the Rat Genome Sequencing Consortium). B: Expanded view of the region amplified by the polymerase chain reaction in C. Black rectangle at the bottom represents the 2,964 bp deletion in Lx mutants. Deletion coordinates are chr8: 52080930-52083893. Additional tracks not shown in A depict CpG islands and repeats by RepeatMasker. C: A 2,964-bp deletion in intron 2 of Plzf is present only in Lx-carrying strains. A 3,240-bp fragment is amplified from wild-type rats, 276 bp from Lx/Lx animals. m = molecular weight marker.

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Sequencing Candidate Genes

We sequenced genomic DNA from two strains carrying Lx (PD and PD5), and two controls SHR and BN. BN/Cub is a very close relative to BN/SsnHsd, the source of the reference rat genome. Sequence of BN/Cub determined during this study is identical to the reference genome. Also, sequence of PD and PD5 is identical, as expected, because we assessed only DNA inside the differential segment of PD origin in PD5. When we found a difference between PD and any of the two controls, we expanded our analysis and compared the variant to 9 additional rat strains, including another congenic strain carrying Lx (BN.Lx), paying special attention to strains of Wistar origin (see the Experimental Procedures). Thus, we were able to exclude a role in limb development for every variant shared between Lx carrying strains and other strain(s) without limb malformation.

In addition to the Plzf coding sequence (Šeda et al.,2005), we determined the coding sequence of Htr3a and Htr3b, although their involvement in regulation of limb development is improbable—they are expressed and function mainly in the central and peripheral nervous system (Thompson and Lummis,2007). Coding sequence of Htr3a does not contain any variation. There are 3 coding SNPs (single nucleotide polymorphisms) in Htr3b, two silent (A352A, L417L) and one missense (H364R). In all three SNPs, the variant present in the Lx carrying strains is shared by other nonrelated wild-type rat strains (SD, HTG, and Wistar “BioTest”, see the Experimental Procedures section and Supp. Table S1, which is available online). L417L is a known SNP (dbSNP: rs8155004), H364R is contained in Celera BN – SD whole genome shotgun comparison (Supp. Table S1) and also in mRNA AF155044 from SD strain. None of the identified variants can thus explain the Lx phenotype.

Identification of Putative Functionally Important Noncoding Sequences

Because there is no solid evidence of presence of transcription activity other than the three above-mentioned genes in the 177-kb segment (Fig. 4A, see RefSeq and spliced expressed sequence tag tracks), and there is also no miRNA gene (data not shown), we set to find the potential regulatory elements. According to evolutionary conservation of limb development and structure in vertebrates, we presumed the regulatory sequences would be likely conserved at a similar level. Preformed conservation data for the rat genome are available at UCSC Genome Browser (URL), generated by PhastCons (Siepel et al.,2005) on sequence alignments created by MULTIZ (Blanchette et al.,2004). We selected conserved noncoding elements (CNEs) for sequencing based on two criteria: (1) PhastCons score and (2) visual inspection of the depth of conservation (in mammals, in terrestric vertebrates, in all vertebrates). We selected 31 CNEs throughout the differential segment. All elements with PhastCons logarithm of odds (LOD) score > 70 were included, as well as all those conserved at least in mammals and birds. Also, we determined the DNA sequence near the transcription start sites of the three genes (containing the promoters, possible short-range regulatory elements, and 5′ untranslated regions [UTRs]) and also 3′ UTRs. In total, including the determined coding sequences, we obtained 30 kb of high-quality sequence from each of the PD, PD5, BN, and SHR strains, corresponding to 17% of the 177-kb nonrecombinant segment (Fig. 4A, see coverage track).

Sequence Variation in the Noncoding Sequences

Beyond the 3 previously reported coding SNPs in Plzf (Šeda et al.,2005), and 3 coding SNPs in Htr3b, we identified 70 sequence variants (Fig. 4A and Supp. Table S1). There are 47 substitutions or SNPs in strict sense; 22 small indels (insertions and deletions up to 14 bp), from which 12 appear to be in a single base “run” and 5 in more complex microsatellites. In addition, during the positional cloning efforts, we identified 6 microsatellites polymorphic between the Lx-carrying and other strains (not included in the 70 variants). Last but not least, there is one substantial deletion—2,964 bp in intron 2 of Plzf (Fig. 4B,C). Of interest, only 11 of these variants were recognized previously (Supp. Table S1), all the others are novel.

Next, we examined whether the sequence variant was unique for strains carrying the Lx mutation. In the majority of cases, we found at least one strain with normal limb development that has identical sequence variant as Lx carrying strains. We excluded such variants from further analysis.

We also examined the polymorphic sites in the F2 recombinants for extra fine mapping of the recombination breakpoints. In this manner, we further reduced the nonrecombinant region to 155 kb (Fig. 4A). This criterion leads to absolute exclusion of 9 SNPs and 4 indels.

The only remaining candidate for Lx is the 2,964-bp deletion (Fig. 4B,C).

The 2,964-bp Deletion

The 2,964-bp deletion in intron 2 of Plzf was found exclusively in rats carrying the Lx mutation and was absent in all other rat strains examined (Fig. 4B,C and data not shown). The deletion removes three CNEs, two moderately conserved and one highly conserved. The most conserved CNE has PhastCons LOD score 603 (second highest in the 155-kb segment after the exon 2 of Plzf), and is conserved in all major vertebrate phyla (where genome comparison is available, Fig. 4A).

Decreased Plzf Expression in Lx/Lx Limb Buds

Plzf expression in wild-type (WT, +/+) and heterozygotes (+/Lx) was comparable both in pattern and quantity (compare Fig. 5A,D,G to Fig. 5B,E,H). In wild-type rat embryos, Plzf expression in limb buds was already established at 12.5 days post coitum (dpc; Fig. 5A). The expression spanned most of the limb bud mesenchyme. At 13.5 dpc in the forelimb bud (Fig. 5D), the expression receded from the tip of the limb bud and split, so the pattern resembled a fork in the middle of the limb bud. In the hindlimb bud, most of the mesenchyme was positive for Plzf mRNA, similar to 12.5 dpc. At 14.5 dpc (Fig. 5G), when the autopod was clearly differentiated, Plzf signal disappeared from the autopod, but remained in the more proximal portion (future zeugopod) in both fore- and hindlimb buds, being more intense in the hindlimb buds. At 15.5 dpc, the Plzf expression in both fore- and hindlimb buds was almost completely gone, with the exception of a weak staining in the proximal part of the hindlimb (data not shown). In addition to limb buds, Plzf expression was detectable in many other structures: developing central nervous system (CNS; throughout both brain and spinal cord), branchial arches, mesonephros, and the eye.

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Figure 5. Expression of Plzf throughout the limb development. Whole-mount RNA in situ hybridization of Plzf at 12.5 days post coitum (dpc) to 14.5 dpc embryos. A,D,G: wild-type (+/+) embryos. B,E,H: +/Lx embryos (displaying wild-type feet), C,F,I: Lx /Lx embryos. In all stages observed, Plzf expression in the mutant limb buds is markedly decreased in comparison to both +/Lx and +/+ limbs. We did not observe marked differences between +/Lx and +/+ limb buds. Note that, at 14.5 dpc, the future preaxial polydactyly in Lx/Lx (F) became already distinguishable by enlargement of the anterior part of the autopod. Scale bars = 1 mm.

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In Lx/Lx embryos, Plzf expression in limb buds was greatly reduced, while staining in the CNS showed pattern and intensity similar to control embryos. At 12.5 dpc (Fig. 5C) the expression was barely detectable in both limb buds, suggesting later expression onset. At 13.5 dpc (Fig. 5F) the expression was reduced, especially in the anterior mesenchyme of the hindlimb. At 14.5 dpc (Fig. 5I) in the forelimb, signal was absent in the zeugopod, and suppressed in the autopod. In the hindlimb, the autopod was negative (as it was in controls), but the zeugopod signal was greatly reduced. At 15.5 dpc, Plzf expression became undetectable in both fore- and hindlimbs (data not shown).

Confirmation of Decreased Plzf Expression by Quantitative Real-Time Polymerase Chain Reaction

We assessed the expression in limb buds also by quantitative real-time polymerase chain reaction (qPCR) with Taqman probes at 12.5 dpc and 13.5 dpc. Lx/Lx animals had decreased expression to 20–40% of the levels found in the wild-type (+/+). Heterozygotes showed significantly higher expression than mutant homozygotes, but lower than the wild-type rats (Fig. 6). We used the tail portion of the embryo, containing Plzf-expressing neural tube as the positive control. The relative expression level of Plzf was slightly higher there than in the WT hindlimb buds, and there was no significant difference among genotypes (data not shown).

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Figure 6. Plzf expression evaluation in limb buds by quantitative polymerase chain reaction (qPCR) at 12.5 and 13.5 days post coitum (dpc). Expression was assessed by a Taqman localized at the most 5′ exon junction. However, we obtained similar results with second Taqman probe at exon 5 to 6 junction. Each sample represents a pool of both fore- or hindlimbs respectively, from the given embryo. The number of unique samples is given under each column. Columns represent means ± SEM of expression level relative to wild-type (WT). P values (above columns) represent significance levels of Tukey post hoc tests modified for unequal n between the indicated groups. The statistical analysis was performed on ddCt values instead of the relative expression which is shown in the graph (see the Experimental Procedures section). Mutant limb buds at both stages have significantly lower Plzf expression compared with WT (20–40%). Heterozygotes show intermediate expression levels, consistent with the proposed cis-acting nature of the deleted Plzf intron 2 sequence.

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Plzf Target Genes Exhibit Changes of Expression Pattern

Next, to assess the functional importance of decreased Plzf expression in the Lx/Lx limb buds, we examined expression profiles of known Plzf targets. Because Plzf is a direct repressor of posterior HoxD genes (Barna et al.,2002), we focused on Hoxd10Hoxd13.

Forelimbs, which do not have any skeletal phenotype, showed no difference in HoxD expression pattern in Lx/Lx embryos compared with +/Lx at all stages examined (data not shown). There was also no change of expression in hindlimbs at 12.5 (not shown) and 13.5 dpc (Fig. 7A–D). The 13.5 dpc marks an end of the first HoxD expression phase. However, at 14.5 dpc (Fig. 7A–D), Hoxd1013 expression in the Lx/Lx hindlimb buds was extended into the anterior portion of the autopod. At the same stage, the mutant phenotype became distinguishable morphologically in the form of an enlargement of the anterior part of the autopod. Hoxd10 and Hoxd11 that are also expressed in the presumable zygopod, showed an enlarged expression domain in the anterior half of Lx/Lx zeugopod, resulting in a nearly symmetrical A–P pattern, in contrast to the marked asymmetry in control limb buds (Fig. 7A,B).

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Figure 7. Expression of Plzf target genes during hind limb development. Whole-mount RNA in situ hybridization. The top margin and bottom margin denote the anterior and posterior parts of the limb, respectively, in all panels. A–D: Whole-mount in situ hybridization of Hoxd1013 genes at 13.5 days post coitum (dpc) and 14.5 dpc in Lx/Lx and +/Lx controls. HoxD gene expression is altered in Lx/Lx embryos. A: Hoxd10 expression in hindlimbs is extended anteriorly both in autopod and zygopod at 14.5 dpc. Autopod already shows signs of accumulation of extra material, delineating the future polydactyly. B: Similar findings characterize Hoxd11 expression at 14.5, the changed expression pattern is preserved at least up to 15.5 dpc (not shown). C,D: Hoxd12 (C) expression in autopod at 14.5 dpc is “anteriorized” in a manner comparable to other posterior HoxD genes, the same is true for Hoxd13 at 14.5 (D). E: Whole-mount in situ hybridization of Shh at 13.5 dpc in Lx/Lx and +/Lx controls. No difference in Shh expression can be found between Lx/Lx and +/Lx embryos (13.5 dpc), at 14.5 dpc Shh signal is already absent (data not shown). F: Whole-mount in situ hybridization of Bmp2 at 13.5 dpc and 14.5 dpc in Lx/Lx and +/Lx controls. Bmp2 expression is anteriorized in similar way to Hoxd1013. Scale bars = 1 mm.

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We further examined the expression of Shh (Fig. 7E), because alterations of Shh function are involved in many models of preaxial polydactyly. There was no change of Shh expression in mutant limb buds in comparison to controls. Specifically, Shh expression in the mutants remained confined to the posterior ZPA with no signs of changed or ectopic expression.

Bmp2 (Fig. 7F) was expressed in the hindlimb bud mesenchyme at 13.5 dpc in a small area close to the posterior margin with no difference between mutant and control embryos. In the hindlimbs of both control and mutant embryos at 14.5 dpc, there was similar marked positivity in the presumptive third interdigital space, as well as in the clearly demarcated fourth space. The first and second interdigital spaces were not fully developed yet, and showed only moderate level of Bmp2 mRNA. In Lx/Lx embryos, there was an additional large peak of expression extending to the most anterior tip of the limb bud. Expression in the posterior autopod/zeugopod was not changed.

In summary, the expression data show reduced Plzf expression in the limb bud, especially in its anterior portion, resulting in anterior expansion of HoxD signaling.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Plzf expression pattern in wild-type rats is comparable with expression of the orthologous gene in the mouse (Avantaggiato et al.,1995; Cook et al.,1995), including the limb bud expression. In Lx/Lx mutant rats, there are substantial changes in the Plzf expression pattern in the limb buds, while the expression in other structures is comparable to the wild-type. This can explain why the Lx phenotype is restricted to limb, whereas Plzf−/− mice display affliction of the axial skeleton and defective spermatogenesis. In PD5 rats, forelimbs are not affected despite the changes in Plzf expression present in both fore- and hindlimbs. We cannot fully account for this phenomenon. There may be functional redundancy due to the fact that the forelimb bud expresses a Plzf homolog (Davies et al.,1999). In any case, restriction of the mutant phenotype to hindlimbs was observed also in Plzf−/− mice (Barna et al.,2000) and Plzf−/−Gli3−/− double null mice (Barna et al.,2005). In heterozygotes (+/Lx), in situ hybridization (ISH) data show Plzf expression comparable to +/+. However, in qPCR, the expression is lower in the heterozygotes. Presumably, the difference is due to greater sensitivity of qPCR enabling detection of slight changes in expression. Nevertheless, the qPCR result is in accord with the character of the mutation—regulatory elements in DNA normally act only in cis, predictive of intermediate expression levels in heterozygotes compared with both homozygote types.

Plzf−/− mice displayed a relatively wide spectrum of hindlimb phenotypes, afflicting all three limb segments (stylopod–autopod). In the digital arch, the following phenotypes were noted: triphallangeal hallux, with or without an additional preaxial digit, or missing hallux. Zeugopod showed separation of tibia and fibula, thickening of fibula and shortening of the whole zeugopod. Rarely, forelimbs were also affected in a corresponding manner (Barna et al.,2000). PD5 displays only limited autopod phenotype variation (Fig. 1). This may be the result of a hypomorphic nature of Lx mutation (up to 40% of WT Plzf expression level is preserved in the mutants). Such interpretation is corroborated by the fact that we observed an alteration of the second phase of HoxD expression, but not of the early first phase in Lx/Lx rats, whereas in Plzf−/− mice, both phases are affected.

In contrary to the Plzf−/− phenotype, which was analyzed on a mixed genetic background, PD5 is an inbred strain. Therefore, the phenotype of the Lx/Lx homozygotes may be stabilized at a certain severity by the uniform genetic background. Indeed, gross phenotype of other strains containing the Lx allele (Printz et al.,2003) supports this hypothesis. In BN.Lx, another congenic strain (with genetic background different from PD5, albeit also uniform), we observed 6-toed hindlimbs, shortening of the hindlimb zeugopod with distal bending and fibula thickening. The forelimbs had a well-developed thumb (in wild-type rats, thumb is rudimentary). Moreover, in BN.Lx × BN cross, hindlimb polydactyly (6 toes) is present in approximately 60% of the heterozygous offspring (Kren,1975). This does not change the fact that, in the experimental setting of this study, using SHR genetic background, Lx behaves as strictly recessive. Moreover, Lx mutation segregates in the largest rat recombinant inbred (RI) strain panel HXB and BXH (Printz et al.,2003). Each RI strain, derived by inbreeding of a F2 progeny, represents a unique, but reproducible, combination of the parental genomes (for BXH and HXB panels, parents are SHR and BN.Lx). There are 31 HXB and BXH strains including 1 subline; 19 strains and the subline are Lx/Lx homozygotes, with reproducible limb phenotypes for each strain, but a huge interstrain variation. For example, the BXH2 strain presents with forelimb polyphalangy (5 triphalangeal digits), missing thumbs in hindlimbs (4 digits), and hindlimb zeugopod affliction more pronounced than in BN.Lx. On the other pole of the variation spectrum, we can name BXH3, with limb phenotype resembling PD5. This limb phenotype spectrum is matching surprisingly well to the phenotype spectrum observed in the Plzf−/− mice.

We showed that Lx influences limb patterning independently of Shh. In contrast, the changes in posterior HoxD gene expression indicate that HoxD genes are among the effectors leading to polydactyly in Lx/Lx rats. This finding corresponds well to analysis of the Plzf−/− mice (Barna et al.,2000) that showed similar changes in expression of HoxD genes. The relationship of Shh and HoxD genes is complex and supposedly different for the first and the second phase of HoxD expression (Tarchini and Duboule,2006). It is not surprising as these two phases occur before and after Shh signaling. The first phase of HoxD expression, especially the restriction of Hoxd1013 expression in the posterior part of developing limb bud, is essential for triggering expression of Shh and maintaining correct Shh expression posteriorly in the ZPA (Zakany et al.,2004). In contrast, the second phase of HoxD (Hoxd1013) expression in developing autopod is supposed to be induced or at least modulated by Shh or by its counterplayer, Gli3 (Zuniga and Zeller,1999). As our and other's (Barna et al.,2000) works show, despite correct Shh expression in Lx/Lx rats or Plzf−/− mice, HoxD expression is anteriorized, resulting in preaxial polydactyly. We can conclude that Plzf might be a mediator of the Shh effect on HoxD genes or Plzf and Shh might be two independent factors co-defining the autopod HoxD expression pattern. The second possibility seems to reflect the reality better in the light of the findings of Barna et al. (2002). These investigators addressed a question why Plzf represses Hoxd1013 expression only in the anterior region, while its expressions extends almost throughout the limb bud. They showed that the repressive ability of Plzf is antagonized in posterior regions of the limb by signals such a retinoid acid and Shh. In accordance with these findings, it seems that a total absence of Plzf (in −/− mice) and reduced expression of Plzf in the anterior part of the limb bud (in Lx/Lx rats at 13.5 dpc, Fig. 4D) can be functionally equal and can elicit the same phenotype. Alternatively, phenotypic equivalence of Plzf−/− and Lx/Lx can be due to later onset of Plzf expression in Lx/Lx limb buds, pointing to possible importance of Plzf for the autopod at earlier stage (12.5 dpc, Fig. 4B).

The amount of programmed cell death is reduced both in Lx/Lx rats (Sedmera et al.,1998) and Plzf−/− mice (Barna et al.,2000). As Bmp2 is supposed to be a modulator or even a trigger of programmed cell death, we expected its reduced expression in Lx/Lx compared with control. Surprisingly, the only difference we observed is anteriorization of Bmp2 expression in Lx/Lx embryos, in a manner similar to posterior HoxD genes.

The involvement of Plzf in limb patterning is well documented in the mouse, thanks to analysis of Plzf−/− animals (Barna et al.,2000,2002,2005). The major novel finding of our study is the identification of a putative cis-acting Plzf regulator that may enable us to detect genes acting upstream of Plzf. Our data suggest that the regulatory element in question resides in the 2,964-bp deletion. The deleted sequence contains the most highly conserved noncoding sequence element, and the conservation is also the deepest (fish–mammals) in the region. This is in agreement with our hypothesis stating that sequence necessary for proper limb development should be conserved at least in terrestrial vertebrates, possibly even in fish (as fish fins share many common aspects with tetrapod limb development). Compared with other sequence variants (mainly SNPs) identified in the nonrecombinant region, only the deletion is exclusively associated with Lx. The only alternative explanation of Lx can be a missed sequence variant in the part of the segment that was not sequenced. However, this would be greatly surprising, as this part is not conserved.

A limb-bud-specific, long-range regulatory element driving sonic hedgehog (Shh) expression in the ZPA was described previously (Lettice et al.,2002). The Shh regulatory cis-acting element was characterized in detail (Lettice et al.,2003,2007; Lettice and Hill,2005). The importance of the Shh regulator is underscored by its point mutations responsible for human preaxial polydactyly (Lettice et al.,2007). The element identified in this study is not similar at the DNA level to the long range Shh regulator. This is not surprising, as the expression patterns of Shh (in ZPA) and Plzf (almost everywhere in the limb bud mesenchyme) are dissimilar. Another example of dissection of a regulatory region involved in limb development was provided by the mouse mutation limb deformity (ld), which is caused by disruption of a “global control region” for gremlin expression (Zuniga et al.,2004).

Our study presents the first evidence of possible molecular mechanism acting during the limb development to produce the Lx phenotype. Further analysis of the deletion would be necessary to confirm the role of the deleted CNE in limb development, e.g., by creating transgenic animals with reporter gene expression driven by the deleted element. It may be interesting to investigate whether the deleted element is a selective regulator of Plzf expression in the limb bud, given the many processes where Plzf is involved. At this stage, it seems that the regulator is not essential for the spermatogenic stem cells, as the Lx/Lx males have normal fertility. The CNS expression of Plzf is also unchanged in the Lx/Lx embryos. The deletion (2,964 bp) is too long for reliable prediction of which of the many potential transcription factor binding sites are functionally important. Moreover, the deletion contains not one, but several CNEs with varying conservation depth. To further decipher Plzf regulation through the deleted sequence, it will be necessary to perform functional studies of enhancer properties of deletion variants of the element.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Animals and Linkage Mapping

In this study, following rat strains were used: inbred strain SHR/OlaIpcv, RGD (Rat Genome Database) ID: 631848; inbred strain BN/Cub, RGD ID: 737899; congenic strain SHR.Lx.PD5, RGD ID: 1641851 (PD5 hereafter). For sequencing, we also used DNA isolated from: PD/Cub, RGD ID: 728161; BN.PD-(D8Rat39-D8Rat35)/Cub, RGD ID: 728144 (BN.Lx hereafter); CHOC/Cub, RGD ID: 737958; SD, RGD ID: 70508; SHRSP/Bbb, RGD ID: 1581618; WKY/Bbb, RGD ID: 1581635 (SHRSP and WKY DNA is kind gift of N. Hubner of MDC Berlin); HTG, RGD ID: 1302795 (kind gift of J. Kuneš, Institute of Physiology, Praha); a second colony of HTG (kind gift of L. Kazdová, IKEM Praha); outbred Wistar from Velaz Ltd., Czech Republic; outbred Wistar from BioTest Ltd., Czech Republic; WHD (Wistar hypodactylous, Liška et al., unpublished results). In addition to various Wistar rats, SHR, SHRSP, WKY, WHD, and HTG are derived from outbred Wistar rat colonies, as is PD.

All experiments were performed in agreement with the Animal Protection Law of the Czech Republic (311/1997) which is in compliance with the European Community Council recommendations for the use of laboratory animals 86/609/ECC. All experiments were approved by The Charles University Animal Care Committee.

For ISH, we used PD5 (mutant) and SHR as a wild-type control. To obtain littermates with different genotypes, SHR was crossed to PD5, and the resulting F1 hybrids were intercrossed to obtain all possible genotypes of the Lx locus in F2 littermates (Lx/Lx, +/Lx, +/+).

For linkage mapping, we generated F2 (PD5 × BN) hybrids, n = 247. We assessed their limb phenotype visually after birth. We isolated genomic DNA from the tail by phenol-chloroform extraction and ethanol precipitation, subjected it to amplification by standard PCR technique, and analyzed the products on native polyacrylamide gel electrophoresis stained with ethidium bromide. Primers for PCR correspond to established microsatellite markers in the PD5 differential segment: D8Rat42, D8Got72, D8Rat94, and D8Arb23. We also searched for additional microsatellites by Pompous (Fondon et al.,1998) or used preformatted trf (Benson,1999) data available through UCSC Genome Browser. We designed primers flanking microsatellites using Primer3 (Rozen and Skaletsky,2000), the amplicons were tested for length polymorphism. Primers for polymorphic microsatellites are listed in Supp. Table S2. We also determined, using sequencing, the genotype of F2 No. 110 in the interval between 21807 and 21829 bp; and No. 117 in the interval between 21652 bp and D8Got72 respectively, to map the crossing-over events with highest precision. While in the former case there was no reduction of the candidate region, analysis of no. 117 led to exclusion of 22 kb from the candidate region.

Search for CNEs

We inspected the nonrecombinant Lx carrying segment using UCSC genome browser. It contains preformed alignment with other vertebrate genomes, generated by MULTIZ (Blanchette et al.,2004), and the most conserved elements identified and scored by PhastCons (Siepel et al.,2005). Our (initial) inclusion criteria for sequencing were PhastCons LOD score > 70, or conservation at least among mammals and birds.

Sequencing

Tail genomic DNA was amplified by PCR with specific primers (Supp. Table S2). PCR fragments were analyzed by electrophoresis and sequenced directly using PCR primers and BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the sequencing products analyzed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems). DNA sequences were deposited in GenBank, dbSTS, and the variation in dbSNP (pending).

Whole-Mount ISH

Probes were generated from RT-PCR products (primers see Supp. Table S2). RNA was isolated from adult heart (in case of Plzf) or from whole embryos at 12.5 dpc (Hoxd1013, Bmp2, Shh). PCR products were cloned into pDrive (Qiagen, Valencia, CA) or pCR-Blunt-TOPO (Invitrogen, Carlsbad, CA). Sense/Antisense RNA was synthesized by in vitro transcription using SP6 or T7 RNA polymerase, with appropriate promoters present in the vector.

The ISH on whole-mount embryos was performed as described (Nieto et al.,1996). Briefly, embryos were fixed in 4% paraformaldehyde in phosphate buffered saline with 0.1% Triton X-100 overnight at 4°C, and then transferred into methanol. Hybridization was performed with digoxigenin labeled antisense riboprobes at 70°C over 1–2 days. Fab fragments of sheep antibody to digoxigenin conjugated to alkaline phosphatase mediated the visualization (1:5,000, Roche), n > 4 for each stage/genotype, in at least two independent replicates. The compared embryos of +/+, +/Lx, and Lx/Lx genotypes were hybridized in the same reaction, using identical hybridization mixture and equal staining time.

The qPCR

RNA was isolated from limb buds (fore- and hindlimb buds were pooled for each embryo) and tail by RNeasy Mini Kit (Hilden, Germany) according to manufacturer instructions, and reverse transcribed by SuperScript III (Invitrogen) using random hexamer primers.

Taqman probes were purchased from Applied Biosystems. Plzf assay spans exons 1-2 (assay ID Rn01418641_m1). We also used a probe/primer set amplifying exon 5–6 junction (Rn01418644_m1). GAPDH (catalog no. 4308313) was used as control. Samples were run on Applied Biosystems Real-time PCR System 7000. Each sample represented a pool of both fore- and hindlimb buds of the given embryo. Each sample was run in quadruplicate. Relative expression levels were computed using standard ddCt method, expression level in +/+ animals was set to 1. Statistical significance was determined on ddCt values using STATISTICA by analysis of variance, between group differences computed by Tukey Honest Significant Difference test modified for unequal N. The ddCt values instead of relative expression values were used because of their normal distribution.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY21859SuppTblS1.xls106KSupp. Table S1. Sequence Variants in 177-kb Region of Chromosome 8 Carrying <I>Lx</I>.
DVDY21859SuppTblS2.xls68KSupp. Table S2. Oligonucleotides Used for PCR and Sequencing.

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