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Keywords:

  • limb development;
  • Sonic Hedgehog;
  • Lmbr1;
  • preaxial polydactyly;
  • Hemimelic extra-toes;
  • Shh limb-specific enhancer;
  • limb malformation

Abstract

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

In most instances of preaxial polydactyly (PPD), Sonic Hedgehog (Shh), an essential limb patterning signal, is ectopically expressed in an anterior region of the developing limb in addition to the normal posterior domain. It is thought that this anterior Shh expression leads directly to the development of the extra digits. Recent reports have identified a conserved limb-specific Shh enhancer ∼1 megabase upstream of the Shh transcription initiation site, and individual base pair changes within this region are associated with PPD. We report here that a single base pair change within this enhancer is sufficient to drive β-galactosidase expression in both anterior and posterior limb domains, similar to Shh expression in animal PPD models, whereas a wild-type construct is expressed only in the posterior limb, similar to Shh expression in normal embryos. These findings provide the first direct evidence that a single base pair change within the limb-specific Shh enhancer acts as a genetic basis for PPD. Developmental Dynamics 232:345–348, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

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

Preaxial polydactyly (PPD), or extra digit(s) on the thumb/big toe (anterior) side of the limb, is a common human limb malformation (Zguricas et al., 1999). In the developing limb, Sonic Hedgehog (Shh) is normally expressed in a region of the posterior mesoderm, called the zone of polarizing activity (ZPA), and is required for proper anterior/posterior limb patterning (reviewed in Mariani and Martin, 2003; Niswander, 2003; Tickle, 2003). Most animal models of PPD show Shh expression in both the normal posterior domain as well as in an ectopic anterior domain in the developing limb (Masuya et al., 1995, 1997; Sharpe et al., 1999). Based on experimental studies of anterior SHH misexpression, it is proposed that anterior Shh expression in the limb leads directly to the formation of extra anterior digits (Riddle et al., 1993; Lopez-Martinez et al., 1995). Of interest, in PPD, anterior Shh expression is initiated much later in limb development than posterior expression and reproducibly occurs in a restricted region of the anterior limb.

An enhancer controlling limb-specific expression of Shh was mapped recently to a region approximately 1 megabase upstream of the Shh gene, and further analysis has refined the enhancer to an approximately 800-base pair region within intron 5 of the Lmbr1 gene. This region is highly conserved across vertebrate species (Lettice et al., 2003; Sagai et al., 2004) and is syntenic with the Shh gene in all species analyzed, indicating a conserved mechanism for regulating Shh expression in vertebrate limbs. In addition, this conserved region can drive reporter gene expression specifically in the ZPA of the mouse limb, suggesting that this region acts as a long-range limb-specific enhancer (Lettice et al., 2003).

Of interest, several human families and mouse mutants affected with PPD have unique single nucleotide changes within the Lmbr1 intron 5 conserved region (Lettice et al., 2003; Sagai et al., 2004). Although it is suggested that these individual nucleotide changes cause ectopic Shh expression and thus PPD, this hypothesis has not been tested directly. Here, we report the first evidence that a single base pair change within this conserved region underlies the polydactyly phenotype.

RESULTS

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

To examine whether base pair changes within the conserved limb-specific Shh enhancer lead directly to PPD, we designed an in vivo reporter assay to test the functional significance of the base pair change from the Hemimelic extra-toes (Hx) polydactylous mouse mutant (Lettice et al., 2003; Sagai et al., 2004). We designed two reporter constructs, each containing a 1.7-kilobase fragment from Lmbr1 intron 5 cloned upstream of the hsp68 promoter and lacZ gene, that are identical except for one base pair within the conserved region corresponding to either wild-type (wt) or Hx sequence (Fig. 1A; Lettice et al., 2003; Sagai et al., 2004). Single cell mouse embryos were injected with either the wt or Hx reporter construct and were harvested and stained for β-galactosidase (β-gal) activity at embryonic days 10.5–11.5. This approach ensures that each transgenic embryo represents an independent insertion event.

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Figure 1. A single nucleotide change causes ectopic expression of a reporter gene in the developing limb. A: A portion of the Lmbr1 intron 5 conserved region used in the reporter constructs showing wild-type (wt) or Hx sequence. B–E: β-Galactosidase (β-gal) staining in transgenic mouse embryos from independent insertion lines at embryonic day (E) 11.5 (B,E), E10.75 (D), and E10.25 (C). β-Gal activity is restricted to the posterior limb in wt-injected embryos (B) and to both anterior and posterior limb regions in Hx-injected embryos (C–E). C inset: Posterior β-gal activity is already strongly detected at the onset of anterior β-gal expression. F,G: β-Gal localization in posterior and anterior limb (F) is similar to, but broader than, Shh mRNA expression in similarly staged Hx+/− limb (G; courtesy of B. Robert), likely due to perdurance of β-gal protein in Shh descendent cells (Echelard et al., 1994). H,I: Injection of the Hx construct has no effect on endogenous limb Shh expression, because Shh mRNA is detected only in the posterior limb of transgenic embryos injected with either the wt (H) or Hx (I) construct at E11.

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After injection of the wt reporter, we obtained three embryos with β-gal activity, all specifically in the posterior limb bud (Fig. 1B). This expression pattern resembles the expression pattern of Shh mRNA in an equivalently staged normal limb bud (Echelard et al., 1993). After injection of the Hx construct, we obtained six embryos with β-gal activity, five of which showed robust and reproducible staining in both posterior and anterior regions of the limb bud, similar to Shh expression in the Hx mouse limb (Blanc et al., 2002; Fig. 1F,G). We note that each of the β-gal staining domains observed is larger than the corresponding Shh mRNA expression domain in either wt or Hx embryos. This increase may be due to the perdurance of β-gal activity (Echelard et al., 1994) in marked Shh descendent cells (Fig. 1F; Harfe et al., 2004). However, we point out that the posterior β-gal expression domain is broader in embryos injected with the Hx construct than in those injected with the wt construct (Fig. 1B,D), correlating with the observations that the posterior Shh mRNA expression domain becomes enlarged distally and extends anteriorly beneath the apical ectodermal ridge in Hx mutant embryos (Blanc et al., 2002). This finding supports the hypothesis that the Hx mutation relieves normal repression in the limb bud and allows the observed expansion of the posterior ZPA domain. Importantly, we also found that posterior β-gal activity is already very strong when anterior β-gal is first detectable, reproducing the timing of Shh expression in models of PPD (Masuya et al., 1995, 1997; Sharpe et al., 1999). Thus, the Hx construct β-gal expression pattern recapitulates the spatiotemporal distribution of Shh mRNA in PPD limbs.

DISCUSSION

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

The results described here have led us to further consider the mechanisms that normally act to confine Shh expression to the ZPA, as well as those that lead to ectopic anterior Shh expression in polydactylous mutants. Recent data have suggested that the juxtaposition of 5′ Hoxd gene expression in the posterior of the limb, in combination with other factors such as dHAND and GLI3, plays a major role very early in limb development in establishing the posterior Shh expression domain (te Welscher et al., 2002; Zákány et al., 2004). These transcription factors likely bind to regions within the limb-specific Shh enhancer to permit early posterior Shh expression in the limb. In addition, Hoxd12 and dHand, genes that are known to be important for early posterior Shh expression, cause ectopic Shh expression and PPD when misexpressed in the anterior mesoderm of the developing limb (Knezevic et al., 1997; Charite et al., 2000; Fernandez-Teran et al., 2000). Taken together, these data suggest that the same conditions are likely necessary for both early posterior and late anterior Shh expression.

Our data, and that of others, further suggest that the conserved limb-specific Shh enhancer contains both positive and negative regulatory elements that are necessary for proper asymmetric expression of Shh in the limb. We hypothesize that transcriptional repressors, which would bind to the negative regulatory elements within the conserved region, normally prohibit anterior Shh expression in regions outside of the ZPA. However, if these repressor binding sites are mutated, the repression is relieved, leading to expansion of the normal posterior Shh domain and the ectopic anterior Shh expression domain. To date, six individual base pair changes linked to human and animal PPD have been isolated and are widely distributed throughout the 800-base pair limb-specific Shh enhancer (Lettice et al., 2003; Sagai et al., 2004). Furthermore, our data demonstrate that, in at least one PPD mutant, Hx, a single nucleotide change within the conserved limb-specific Shh enhancer leads to altered Shh expression in both the anterior and posterior limb domains, suggesting that the overall conformation of the enhancer is important for both positive and negative regulation. Clearly, the next step in determining the mechanism for long-range limb-specific cis-regulation of Shh both in the posterior and anterior of the developing limb is to determine the trans-acting factors, such as Hox genes and other transcription factors, that contribute to the asymmetric localization of Shh and proper anterior/posterior patterning of the adult limb structure. Our results offer the first direct experimental evidence that a single base pair change in the long-range limb-specific Shh enhancer can lead to ectopic anterior Shh expression, in turn serving as a genetic basis for PPD.

EXPERIMENTAL PROCEDURES

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

Generation of Mouse Wild-Type and Hx 1.7-kb Fragment Reporter Constructs

Mouse genomic DNA from Hx heterozygous mice was purchased from Jackson Laboratories (Bar Harbor, ME). DNA was diluted to 200 ng/μl for use in polymerase chain reaction (PCR). Primers were designed so that a 1,676-base pair fragment containing the conserved region (as reported in Lettice et al., 2003) could be amplified. Primers used were as follows: 5′ XmaI Hx primer, 5′-TCCCCCCGGGCTTGCTTTTGTTGTAGGGATTTTAC-3′ (the XmaI site is underlined); 3′ Hx primer, 5′-CCTCACACACACGCATGGTAC-3′. PCR was performed using High Fidelity Polymerase Mix (Roche). The PCR band was gel purified and cloned into pGEM-T Easy (Promega) for sequencing. Once error-free clones were obtained either containing wt sequence or the Hx single nucleotide difference, the clones were digested with XmaI and HindIII (using an internal HindIII site on the 3′ end of the fragment). The insert band was gel purified and cloned into the XmaI and HindIII sites of the hsp68/lacZ expression construct (kindly provided by Ralph DiLeone). Positive clones were then resequenced and used in subsequent experiments.

Preparation of DNA for In Vivo Mouse Transient Transgenic Experiments

Approximately 50 μg of either wt or Hx mouse reporter construct DNA was linearized with NotI and electrophoresed at 20 volts overnight on an 0.8% agarose gel, prepared without ethidium bromide using SeaKem agarose. A small slice from either side of the gel was removed and stained with ethidium bromide, and the portion containing DNA was removed from the slices. These pieces were then realigned with the remaining unstained portion of the gel and used as templates to cut out the fragment containing the DNA. The DNA was then electroeluted with ammonium acetate, ethanol precipitated, and resuspended in a small volume of TE, pH 7.4. The resuspended DNA was then further purified using an elutip-d column, and the final amount was resuspended in TLE buffer (10 mM Tris, pH 7.5; 0.1 mM ethylenediaminetetraacetic acid). This highly purified DNA was then used in injection experiments at a concentration of ∼1–2 ng/μl.

Transgenic Embryo Production

The wt or Hx DNA was injected into the pronuclei of one-cell mouse embryos derived from C57BL/6 females. The injected embryos were transferred into oviducts of pseudopregnant ICR females, and embryos were harvested at different stages of limb development.

LacZ Staining of Embryos

Embryos were harvested into ice-cold phosphate buffered saline (PBS) and fixed for 1 hr at 4°C (fixative: 2% formaldehyde, 0.2% glutaraldehyde, 0.2% NP-40 in PBS), washed three times in PBS, and incubated in stain at 37°C overnight (stain: 5 mM KFeCN, 5 mM KFeCN, 0.2% NP-40, 2 mM MgCl2, 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside in PBS). Embryos were then rinsed in PBS, refixed for 20 min in 4% paraformaldehyde, and rinsed and stored in PBS. For some embryos, the left forelimb and hindlimb were harvested after first fixation step and whole-mount in situ hybridization was performed according to standard protocol on harvested limbs.

Acknowledgements

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

We thank Kathy Helmuth at the University of Wisconsin Transgenic Animal Facility for performing transgenic mouse injections; Ralph DiLeone for providing the hsplacZ construct; Benoît Robert for the unpublished image in Figure 1G; and Xin Sun, Sean Carroll, Richard Clark, Matthew Harris, and members of the Fallon laboratory for helpful comments on the manuscript. J.F.F. was funded by grants from the National Institutes of Child Health and Development and the University of Wisconsin Medical and Graduate Schools.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Blanc I, Bach A, Robert B. 2002. Unusual pattern of Sonic hedgehog expression in the polydactylous mouse mutant Hemimelic extra-toes. Int J Dev Biol 46: 969974.
  • Charite J, McFadden DG, Olson EN. 2000. The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development. Development 127: 24612470.
  • Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA, McMahon AP. 1993. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation on CNS polarity. Cell 75: 14171430.
  • Echelard Y, Vassileva G, McMahon AP. 1994. Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development 120: 22132224.
  • Fernandez-Teran M, Piedra ME, Kathiriya IS, Srivastava D, Rodriguez-Rey JC, Ros MA. 2000. Role of dHAND in the anterior-posterior polarization of the limb bud: implications for the Sonic hedgehog pathway. Development 127: 21332142.
  • Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin C. 2004. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118: 517528.
  • Knezevic V, De Santo R, Schughart K, Huffstadt U, Chiang C, Mahon KA, Mackem S. 1997. Hoxd-12 differentially affects preaxial and postaxial chondrogenic branches in the limb and regulates Sonic hedgehog in a positive feedback loop. Development 124: 45234536.
  • Lettice LA, Heaney SJ, Purdie LA, de Beer P, Oostra BA, Goode D, Elgar G, Hill RE, de Graaff E. 2003. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 12: 17251735.
  • Lopez-Martinez A, Chang DT, Chiang C, Porter JA, Ros MA, Simandl BK, Beachy PA, Fallon JF. 1995. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage. Curr Biol 5: 791796.
  • Mariani FV, Martin GR. 2003. Deciphering skeletal patterning: clues from the limb. Nature 423: 319325.
  • Masuya H, Sagai T, Wakana S, Moriwaki K, Shiroishi T. 1995. A duplicated zone of polarizing activity in polydactylous mouse mutants. Genes Dev 9: 16451653.
  • Masuya H, Sagai T, Moriwaki K, Shiroishi T. 1997. Multigenic control of the localization of the zone of polarizing activity in limb morphogenesis in the mouse. Dev Biol 182: 4251.
  • Niswander L. 2003. Pattern formation: old models out on a limb. Nat Rev Genet 4: 133143.
  • Riddle RD, Johnson RL, Laufer E, Tabin C. 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75: 14011416.
  • Sagai T, Masuya H, Tamura M, Shimizu K, Yada Y, Wakana S, Gondo Y, Noda T, Shiroishi T. 2004. Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome 15: 2334.
  • Sharpe J, Lettice L, Hecksher-Sorensen J, Fox M, Hill R, Krumlauf R. 1999. Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch. Curr Biol 9: 97100.
  • te Welscher P, Fernandez-Teran M, Ros MA, Zeller R. 2002. Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling. Genes Dev 16: 421426.
  • Tickle C. 2003. Patterning systems—from one end of the limb to the other. Dev Cell 4: 449458.
  • Zákány J, Kmita M, Duboule D. 2004. A dual role for Hox genes in limb anterior-posterior asymmetry. Science 304: 16691672.
  • Zguricas J, Heus H, Morales-Peralta E, Breedveld G, Kuyt B, Mumcu E, Bakker W, Akarsu N, Kay S, Hovius S, Heredero-Baute L, Oostra B, Heutink P. 1999. Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36. J Med Genet 36: 3240.