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Keywords:

  • gecko lizards;
  • differentiating epidermis;
  • β-keratins comparation;
  • mRNAs;
  • sequencing

Abstract

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

The β-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of β-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3′- and 5′-rapid amplification of cDNA ends analyses five specific gecko β-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine–rich proteins of 16.8–18 kDa containing 169–191 amino acids, especially 17.8–23% glycine, 8.4–14.8% proline, 14.2–18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (β-pleated fold) likely responsible for the formation of β-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher β-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and β-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian β-keratins. Developmental Dynamics 236:374–388, 2007. © 2006 Wiley-Liss, Inc.


INTRODUCTION

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

The epidermis of scales of lizards comprises cyclically alternating epidermal generations (shedding cycle; Maderson et al.,1998). A hard type of keratin, β-keratin, is produced in some epidermal layers (oberhautchen and in its spinulae, and in the β-layer), but its synthesis is halted in the following layers (mesos, α-, lacunar, and clear layer) where only softer α-keratin remains. Keratins are synthesized during the renewal phase of the shedding cycle of lizard epidermis, which terminates with the molt of the external (outer) epidermal generation. After shedding, the epidermis enters in a resting phase in which proliferation is reduced or interrupted until the following renewal phase.

The β-keratins in both reptilian and avian skin are made of β-pleated sheets (strands) aggregated into a densely packed lattice and show a typical X-ray and ultrastructural pattern of 3- to 4-nm electron pale filaments (Baden and Maderson,1970; Fraser et al.,1972; Gregg and Rogers,1986; Fraser and Parry,1996). Generally, β-keratins have a smaller molecular weight (10–22 kDa) than α-(soft)-keratins (40–68 kDa; Wyld and Brush,1979,1983; Sawyer et al.,2000; Alibardi and Toni,2006a,b). The number of β-keratins in reptilian epidermis, their amino acid sequences, and spatial conformation are only known for a lizard claw (Inglis et al.,1987), and for a scale keratin (Dalla Valle et al.,2005) to date. The latter study showed the expression of β-keratin mRNAs in both oberhautchen and β-cells of lizard epidermis. To understand the molecular nature of β-keratins and their genomic organization, more species of reptiles are currently under analysis. The goal of these studies is to address the process of epidermal differentiation in the epidermis of the first amniotes, the reptiles, as the base to understand the process of cornification of amniotes (Alibardi,2003).

Among reptilian species, particular interesting is the case of geckoes possessing modified scales in their digits, termed climbing lamellae, that allow these lizards to crawl on vertical or upside-down surfaces (Maderson,1970; Hiller,1972; Alibardi,1998; Figs. 1, 2). The structure and mechanism of adhesion of climbing setae in geckoes are presently under intense study due to the possible applications to create new types of dry adhesives. The adhesion is provided by Van der Waal's forces and capillary interactions between the substrate and millions of microscopic setae of 0.5–2.0 μm by 30–120 μm in length that are present on the climbing lamellae of digital pads (Autumn et al.,2000,2002; Huber et al.,2005). The setae correspond to extremely elongated spinulae of the oberhautchen layer of the epidermis of geckoes (Fig. 2 compare B1 with C1). The length of setae increases toward the tip of the pad lamellae (Fig. 2C2), and in some species of geckoes can reach over 100 μm (Maderson,1970). Because spinulae and setae derive from elongation of oberhautchen cells, they should contain the same proteins, with the possible exception of the most apical, adhesive portion of the setae, termed spatula.

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Figure 1. A,B: Images of the two studied species, Tarentola mauritanica with regenerating tail (A) and Hemidactylus turcicus (B). C–F: The aspects of regenerating tail during progressive stages of tail regeneration in T. mauritanica are shown C: Blastema at 12 days. D: Elongated cone (3 weeks) with initial scaling near the stump. E: Lengthening regenerating tail (approximately 5 weeks old) with diffuse scaling. F: Scaled regenerated tail at approximately 9 weeks postamputation. Scale bars = 5 mm in A,B,D–F; 2.5 mm in C. cn, normal tail; cr, regenerated tail.

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Figure 2. A–C: Schematic representation of normal scales (A,B) and pad lamellae (A–C) of gecko digits. B: In normal scales, two epidermal generations (outer and inner) alternate, producing short spinulae from the oberhautchen layer (B1 is a detail of the square in B). C: In the specialized scales forming the pad lamellae (square in C is magnified in C1), the spinulae produced in the inner oberhautchen cells elongates into setae, until they reach the final length of setae present in the outer generation (C1). The apex of each setae terminates with a spatular shape, the site of adhesion with the substrate. The longest setae are located toward the tip of scale (C2). BA, basal (germinal) layer; CL, clear layer; DL, differentiating epidermal layers; HI, hinge region; IEG, inner epidermal generation; IG, inner epidermal generation; IS, inner setae; ISP, inner setae; Oα, outer α-layer; Oβ, outer β-layer; OEG, outer epidermal generation; OG, outer epidermal generation; OPS, outer spinulae; OS, outer setae; SPT, spatular ending of setae.

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Setae elongate by the addition of a special resistant type of β-keratins at their base (Alibardi,1997,1998,1999,2003; Alibardi and Toni,2005). These keratins constitute a large part of the material of setae and form long bundles oriented along the main axes of setae. Previous studies on gecko epidermal proteins have indicated that β-keratins with a molecular weight of 10–22 kDa are present and that they resemble those of normal scales (Thorpe and Giddings,1981; Alibardi and Toni,2005; Rizzo et al.,2006).

Specific information on the amino acid composition and sequence of β-keratin during differentiation of gecko epidermis are missing. To address this problem, in the present study, we have isolated and sequenced, by reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA ends (RT-PCR/RACE) analysis, scale keratin mRNAs from normal and regenerating epidermis of two species of geckoes. The presence of the mRNAs in the scales of geckoes has been studied by in situ hybridization to determine the sites of expression of the proteins. The study also aims to determine whether gecko β-keratins are present in both general scales and specialized climbing pad lamellae, and to compare these proteins with those previously sequenced in lizards to detect possible affinities. The study is part of a broad genomic and proteomic program of our laboratory aiming to understand the evolution of β-keratins in reptiles and birds.

RESULTS

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

Histology of Scales and Epidermal Regeneration

The two species of geckoes used in the present study (Tarentola mauritanica, Tm, Fig. 1A; and Hemidactylus turcicus, Ht, Fig. 1B) possess a great climber capability, and their movements on walls are extremely fast at summer temperatures (25–35°C). These geckoes also have a very efficient process of autotomy and release their tail readily, which is rapidly regenerated. In our experiments, the tail was largely regenerated within 6–7 weeks after loss, although variability was very broad among individuals (Fig. 1C–F). Like in lizards (Dalla Valle et al.,2005), after tail loss a regenerative blastema covered by a smooth and dark epidermis was formed within 10 days (Fig. 1C). The blastema elongated in the following 7–10 days and produces a coniform tail covered by a smooth, dark epidermis (Fig. 1D). The epidermis formed scales starting from the proximal region attacked to the tail stump. At 3–5 weeks postamputation, most of the elongated tail was scaled (Fig. 1E). At 2 months postamputation, the long regenerated tail was evenly scaled but lacked the typical spines present in normal tuberculate scales (Fig. 1F).

Most of gecko scales (normal or fully regenerated) were covered by thin corneous layers, that consisted in an external β-layer merged to an oberhautchen layer with microornamentation. The latter are only visible at high magnification or using the electron microscope (data not shown). Beneath the β-layer, an inner dark α-layer was present (Figs. 2, 3). In the regenerating tail epidermis at 2–3 weeks postamputation, the number of layers of spinosus cells increased to 6–10 in comparison to the 2–4 cell layers present in normal epidermis in postshedding stages (Fig. 3A,B). Keratinocytes of the regenerating epidermis became fusiform and flat before forming a variably thick α-keratin corneous layer (the wound epidermis). At 4–5 weeks postamputation, neogenic scales were forming from epidermal pegs that grew into the dermis (data not shown). In the central part of epidermal pegs, localized in proximal regions of the regenerating tail near the original tail, a differentiating oberhautchen and β-layers were seen beneath the wound epidermis (Fig. 3C,D). The oberhautchen formed a dense line that was made of short spinulae, along which shedding later occurred. After the formation of a compact β-layer beneath the oberhautchen, the sloughing of the external wound epidermis at 3–5 weeks allowed the regenerated (neogenic) scales to become visible (see Fig. 1D–F).

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Figure 3. A–F: Histology of normal (A) and regenerating (B–D) scales and of pad lamellae (E–F). A: A fully regenerated scale (similar to a normal scale), which has differentiated a new β-layer (arrow) and is completing the formation of the α-layer (arrowhead). B: Regenerating stratified but undifferentiated epidermis with superficial wound epidermis forming a thin corneous layer (arrow) at 2 weeks postamputation. C: Detail of the central part of a regenerating peg at 3 weeks of regeneration, showing the differentiation of the spinulae (arrows) of the oberhautchen layer in contact with the clear layer. Underneath, fusiform cells of the β-layer are differentiating. D: Regenerating scales with caudal orientation (top figure) become evident underneath the wound epidermis of the proximal areas of regenerated tail at 3 weeks postamputation. The arrows indicate the formation of the β-keratinized layer beneath the wound epidermis. E: A longitudinal section of a pad lamella in the renewal phase, showing in detail the differentiating cells of the oberhautchen layer. Long setae (still growing) produced from hypertrophic oberhautchen cells are directed toward cells of the clear layer. F: Detail of overlapped pad lamellae with mature, long setae seen in longitudinal section. Scale bars = 10 μm. d, dermis; in inner scale surface; o, outer scale surface; re, regenerating epidermis; s, setae; T, scale tip; w, wound epidermis.

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In digital pads, both in renewal phase or in postshedding condition (resting), scales were modified into more expanded lamellae in which long setae (over 20 μm) were present (Fig. 3E,F). The growing setae of pad lamellae derived from hypertrophic differentiating oberhautchen cells of higher dimension than those of normal scales (compare Fig. 1C with 1E). Therefore, the narrow and dense line made by oberhautchen spinulae of normal or regenerating scales corresponds to a wide band of setae in renewing pad lamellae. After shedding of the external epidermis, the long and mature setae remain on the outer scale surface.

Immunocytochemistry Using β-Keratin Antibodies

In both species, only the oberhautchen layer, which is fused with the β-layer of regenerated and normal scales, were immunostained with antibodies produced against avian or reptilian keratins. In particular, the β-layer appeared immunoreactive for a specific antibody produced against a lizard β-keratin of 15–16 kDa (Fig. 4A; see Alibardi and Toni,2006a,b). Also, setae of pad lamellae were labeled using β-keratin–specific antibodies, such as the universal or the β-1 antibody (Fig. 4B; Sawyer et al.,2000). The ultrastructural study confirmed the immunolabeling in the oberhautchen layer merged with the β-layer, including in the elongating setae of renewal epidermis, or in the mature setae merged with the β-layer of postshedding (mature normal or regenerating) scales (Fig. 4C).

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Figure 4. Immunocytochemistry of gecko epidermis (H. turcicus). A: A normal limb scale with immunofluorescent β-layer using the lizard β-keratin antibody. B: A digit pad lamella scale showing the immunoreactive (lizard β-keratin antibody) oberhaucthen and setae. C: Detail of a forming setae of inner generation, in which the filaments are intensely immunolabeled (arrows) with the β-1 antibody. D: Ultrastructural detail of β-1 immunogold-labeled oberhautchen merged with the β-layer, with the base of outer setae. cl, cytoplasm of clear cells surrounding the seta; clear d, dermis; h, hinge region; ob, oberhautchen-β-layer; s, setae; t, scale tip. The asterisk indicates a tangentially sectioned area of a setae. Scale bars = 10 μm in A,B, 250 nm in C,D.

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Cloning of the Gecko Tm and Ht β-Keratin cDNAs

To clone gecko Tm keratins, we used, for the 5′-RACE analysis, a primer (Ker10, Table 1), which was selected from the nucleotide sequence previously determined for this protein in the lizard P. sicula (Dalla Valle et al.,2005). We obtained a fragment that was used, after direct sequencing, to design two more antisense and sense primers specific for the gecko Tm keratin sequence (gec1F and gec1R, gec2F and gec2R, see Table 1). These primers were used in two rounds of amplification with 5′- and 3′-RACE cDNAs to obtain fragments of approximately 400 and 300 bp, respectively, that were cloned and sequenced. Sequencing of three to four clones showed that the fragments were composed by a well-conserved central coding region with 5′- or 3′-untranslated terminal regions (UTR) less conserved in each clone. Because it was impossible to correctly overlap the different fragments, we chose from the conserved region between the different 5′- and 3′-clones a pair of primers (5-gecTm and 3-gecTm) capable of amplifying the entire coding region. After cloning and sequencing the obtained fragment, we found a sequence compatible with previously sequenced 5′- and 3′-UTR of the complete gecko Tm β-keratin cDNA (Ge-gprp-1). This sequence was deposited in EMBL/Gene Bank with accession number (AC) AM162665. Another complete sequence was amplified using the antisense primer 3-gecTm with the 5′-RACE cDNAs as a template, and the 5′-RACE outer and inner primers (Table 1). This sequence, named Ge-gprp-2, was deposited with AC AM263204. The last sequence (Ge-gprp-3), obtained with the 5-gecTm and 3-gecTm primers and covering only the coding region, was deposited with AC AM263205. The results obtained with the cloning of 5′- and 3′-RACE fragments have, however, indicated the presence of more then three different transcripts coding for β-keratins reported above.

Table 1. Primers Used in RT-PCR and 5′- and 3′-RACE Analysesa
PrimerSequencesPositionbAccession no.
  • a

    RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends.

  • b

    Nucleotide position in the deposited sequence.

Ker-105′-CAACGGCACATGGAGTGTTG-3′+ 355 [RIGHTWARDS ARROW] + 336AJ890445
Uni-ker45′-CAGGTGGGATCTGGTTGAT-3′+ 372 [RIGHTWARDS ARROW] + 354AM162665
gec1F5′-ACCATCCCTGGACCCATCCTC-3′+ 340 [RIGHTWARDS ARROW] + 360AM162665
gec1R5′-GAGGATGGGTCCAGGGATGGT-3′+ 360 [RIGHTWARDS ARROW] + 340AM162665
gec2F5′-ACCTGCAGAAGTCATGCTCCA-3′+ 300 [RIGHTWARDS ARROW] + 320AM162665
gec2R5′-TGGAGCATGACTTCTGCAGGT-3′+ 320 [RIGHTWARDS ARROW] + 300AM162665
5-gecTM5′-AGACTCATCTCCACCACACAG-3′− 23 [RIGHTWARDS ARROW] − 3AM162665
3-gecTM5′-AAGTCCATGAGAATTTAGCA-3′+ 554 [RIGHTWARDS ARROW] + 535AM162665
Gec-4F5′-TCCTTCCTGCATCAACCAGA-3′+ 270 [RIGHTWARDS ARROW] + 289AM263205
gec-3R5′-CATGGAGTGTTGCCTCCCAC-3′+ 371 [RIGHTWARDS ARROW] + 352AM263206
5-gecHt5′-TCATCTCTCTCGTTCTCTTGC-3′− 64 [RIGHTWARDS ARROW] − 44AM258989
3-gecHt5′-CAGTCCAGTCCATTATGTCAG-3′+ 631 [RIGHTWARDS ARROW] + 611AM258989
gecTm-intr5′-AGCGTCATCTCTCTCACT-3′− 68 [RIGHTWARDS ARROW] − 51AM162665
gecHt-intr5′-GCTTCATCTCTCTCGTTC-3′− 67 [RIGHTWARDS ARROW] − 50AM258989

The deduced amino acid sequence derived from gecko Tm keratin cDNA Ge-gprp-1 is based on an open reading frame (ORF) of 540 bp, which starts from a putative initiation methionine 68-bp downstream from the 5′-end and continues to a stop codon TAA (Fig. 5). The 3′-UTR is 258 b long. The ORF encodes a putative protein of 179 amino acids with a calculated molecular mass of 16,8 kDa. The ATG in all the transcripts has a nucleotide context that corresponds to the proposed consensus sequence for the initiation of translation (Kozak,1986). A canonical AATAAA poly-A signal falls within the expected range of 10–25 b upstream of the poly-A site. Ge-gprp-2 encodes for a putative protein of 179 amino acids, identical (at the amino acid level) to Ge-gprp-1 as the nucleotidic differences were localized in the 5′-UTR. Ge-gprp-3 encodes for a putative protein of 177 amino acids and presents 30 differences in the nucleotidic sequence, leading to 16 differences at the amino acid level respect to Ge-gprp-1 and 2; moreover the sequence is 2 amino acids shorter than the previous two.

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Figure 5. Nucleotide sequences of gecko Tm β-keratins and the deduced amino acid sequences. One-letter symbols of encoded amino acids are shown below the DNA sequence. The numbers refer to the nucleotide and amino acid positions at the end of each line. Small lines indicate nucleotides or amino acid lacking. The in-frame translation start codon as well as the stop codon and the putative polyadenylation signal are boxed. The oligonucleotide primers used to amplify the complete coding region are double-underlined. The sequences are available at the EMBL/GenBank/DDBJ database under accession numbers AM162665, AM263204, and AM263205 for Ge-gprp-1, 2, and 3, respectively.

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As regard gecko Ht, the sequencing of four clones containing the fragment amplified with the cDNAs from 3′-RACE analysis, and performed in two rounds of PCR with the sense primers gec1F and gec2F, shows the presence of at least four different transcripts with low identity in the 3′-UTR. Two different transcripts were instead found after cloning the fragment obtained by 5′-RACE analysis with an antisense primer specific for gecko Ht (gec3R). A set of primers (5-gecHt and 3-gecHt) selected to amplify the complete coding region of gecko Ht was used to clone the β-keratin sequence of gecko Ht. From three overlapping fragments, we obtained the complete gecko Ht β-keratin cDNA (Ge-gprp-4) that was deposited with AC AM258989. Another sequence, complete in the coding region but not in the 5′-UTR, was found using the sense primer 5-gecHt with the 3′-RACE cDNAs and the 3′-RACE outer and inner primers. This sequence, named Ge-gprp-5, was deposited with AC AM263206.

The deduced amino acid sequence derived from gecko Ht keratin cDNA Ge-gprp-4 is based on an ORF of 576 bp, which starts from a putative initiation methionine 79-bp downstream from the 5′-end and continues to a stop codon TAA (Fig. 6). The 3′-UTR is 262 b long. The ORF encodes a putative protein of 191 amino acids with a calculated molecular mass of 18 kDa. Also for gecko Ht β-keratin transcript, the ATG has a nucleotide context that corresponds to the proposed consensus sequence for the initiation of translation and a canonical AATAAA poly-A signal is present in the expected position. Ge-gprp-5 encodes a putative protein of 169 amino acids with a calculated molecular mass of 16 kDa. The comparison of the two proteins presented in Figure 6, shows that they have only 67% of identity at the amino acid level. Table 2 reports the percentage in amino acids for the five deduced proteins.

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Figure 6. Nucleotide sequences of gecko Ht keratins and the deduced amino acid sequences. One-letter symbols of encoded amino acids are shown below the DNA sequence. The numbers refer to the nucleotide and amino acid positions at the end of each line. Small lines indicate nucleotides or amino acid lacking. The in-frame translation start codon as well as the stop codon and the putative polyadenylation signal are boxed. The oligonucleotide primers used to amplify the complete coding region are double-underlined. The sequences are available at the EMBL/GenBank/DDBJ database under accession numbers AM258989 and AM263206 for Ge-gprp-4 and 5, respectively.

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Table 2. Amino Acid Composition, Molecular Weight, and pI of β-Keratins
 Ge-gprp-1 Ge-gprp-2Ge-gprp-3Ge-gprp-4Ge-gprp-5
  1. aaa, amino acid; MW, molecular weight; pI, isoelectric point.

aa179177191169
MW16.816.818.016.2
Theoretical pI8.868.759.499.34
 %%%%
Ala (A)168.9147.9199.9158.9
Arg (R)42.252.884.284.7
Asn (N)42.242.331.642.4
Asp (D)00.000.000.000.0
Cys (C)42.252.852.674.1
Gln (Q)31.731.731.621.2
Glu (E)21.131.721.021.2
Gly (G)3720.73318.64423.03017.8
His (H)00.021.110.500.0
Ile (I)126.795.194.7116.5
Leu (L)168.9179.6168.4116.5
Lys (K)10.610.600.000.0
Met (M)21.110.621.021.2
Phe (F)52.852.884.274.1
Pro (P)1910.61910.7168.42514.8
Ser (S)3117.33218.13116.22414.2
Thr (T)63.463.452.663.6
Trp (W)00.000.000.000.0
Tyr (Y)42.231.752.610.6
Val (V)137.3158.5147.3148.3
Asp + Glu2 3 2 2
Arg + Lys5 6 8 8

Northern Blotting Analysis and Expression Analysis

The expression of gecko Tm keratins was analyzed also by Northern blotting by using two samples of total RNAs extracted from normal tail epidermis, regenerating tail epidermis, and epidermis of the digital pads of gecko Tm (Fig. 7). One band slightly lower then 1 kb, consistent with the cloned transcripts length, is detected with both normal and regenerating tail epidermis. Although less RNA has been loaded for normal tail, the signal is clearly more intense with the RNA extracted from regenerating tail. Moreover, no signal was observed with the RNA extracted from gecko digital pads.

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Figure 7. A representative Northern blot analysis of total RNAs extracted from gecko Tm epidermis with a gecko Tm digoxigenin (DIG) -labeled antisense cRNA β-keratin probe. Arrows and numbers indicate the positions of RNA standards. In the lower panel, methylene blue-stained 28S and 18S ribosomal RNAs of each mRNA sample are shown. A: Normal tail epidermis. B: Regenerating tail epidermis. C: Epidermis of digital pads.

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Northern hybridization analysis failed to give a positive signal with total RNA extracted from normal epidermis and digital pads of gecko Ht (data not shown). Unfortunately, an adequate amount of RNA from regenerating epidermis was not available in this species.

To confirm the higher expression of β-keratins in the regenerating tail epidermis, an RT-PCR analysis with the set of primers gec4F-gec3R, common to β-keratins of both geckoes, was performed at 20-24-28 and 32 PCR cycles, starting with similar cDNA quantities. The normalization of cDNAs was performed on the basis of results obtained by means of PCR amplification with a set of primers for 18S rRNA (data not shown). Although the sample number is very low, in both cases, the expression level was higher with RNA extracted from regenerating tail epidermis compared with normal epidermis (Fig. 8).

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Figure 8. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of β-keratin expression performed at different PCR cycles (24-28-32) with cDNA of regenerating tail epidermis (R) and normal tail epidermis (N) of gecko Tm and Ht. Sample D corresponds to RT-PCR analysis performed at 40 PCR cycles with epidermis of the digital pads. C−, negative control; MW (molecular weight), DNA size marker.

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Finally, an RT-PCR analysis was carried out using RNA extracted from epidermis of gecko Tm and Ht digital pads. A positive signal was obtained only with the gecko Ht samples shown in Figure 8.

Southern Blotting Analysis and Genomic DNA Amplifications

To estimate the possibility that β-keratins belong to a multigene family, and the number of β-keratin gene copies, we used digestion of genomic DNA with restriction enzymes followed by Southern blotting analysis. The enzymes used, SalI, EcoRI, and XbaI, have no restriction site within the known gene sequences for geckoes β-keratins and, thus, should result in several bands representing a minimum of gene copies.

Using genomic DNA of gecko Tm, Southern blotting analysis produced two to at least six (but probably more) bands ranging from 0.6 to more then 12 kb, indicating the existence of at least six gene copies without restriction site (Fig. 9). Only two bands, one of each corresponding to the uncut genomic DNA, was obtained with the sample digested with SalI. The higher signal intensity with respect to the XbaI and EcoRI restricted samples could be explained by the occurrence, in the same fragments, of more then one copy of the gene without restriction sites between them. Another explanation for this result is the occurrence of DNA fragments of approximately the same length, migrating at a similar position in the gel.

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Figure 9. Southern blot analysis of genomic DNA of geckoes Tm and Ht. The restriction enzymes used are indicated on the top of the lanes. The sizes of the fragments hybridizing with the probes are estimated from the 1-kb DNA ladder (Invitrogen).

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Three fragments, one more intense then the other two, were found for gecko Ht genomic DNA digested with XbaI and EcoRI, whereas only one band, corresponding to uncut genomic DNA, was found with SalI (Fig. 9). These results are consistent with the presence of multicopy gene in the genome of gecko Ht, as previously indicated for gecko Tm.

To analyze the presence or absence of introns inside the coding region of β-keratin, we amplified genomic DNA and cDNA of both species of gecko with the pair of primers “5-gecHt - 3-gecHt” and “5-gecTm - 3-gecTm.” The intron analysis inside the 5′-UTRs was instead performed with a common antisense primer, Uni-ker4, selected on a conserved region of gecko and lizard β-keratin cDNAs and two specific sense primers covering the beginning of the 5′-UTR (gecTm-intr and gec-Ht-intr). In both cases, the fragment size of the amplification products from both genomic DNA and cDNA was the same (data not shown), indicating that introns are absent in the ORF of these genes as well as in the 5′-UTRs. This finding was further confirmed by sequencing of genomic DNA amplifications. Moreover, using the primers 5-gecTm - 3-gecTm and 5-gecHt - 3-gecHT for gecko Tm and Ht (named A and B in the explicative draft of Fig. 10, panel B), with genomic DNA and 3 min of extension instead of 20 sec, a band corresponding to the β-keratin coding region (between 600 and 700 pb) together with a higher fragment of more then 3 kb could be amplified in both gecko species (lines “1” in Fig. 10, panel A).

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Figure 10. Panel A: Polymerase chain reaction (PCR) analysis of the possible tandem genomic organization and orientation of the β-keratin genes. C−, negative control; 1, genomic DNA amplification with the A and B primers; 2, nested PCR with the C and D primers using the DNA extracted from the higher amplified products in the line 1. Panel B: Explicative draft of possible gene tandem organization and orientation within the gecko genome (see text for details).

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After gel extraction, the higher fragments were demonstrated to contain sequences corresponding to β-keratin (lines “2” in Fig. 10A). This finding was demonstrated by a nested PCR performed with internal primers (5-gecTm-gec1R for gecko Tm and gec4F-gec3R for gecko Ht, named C and D in Fig. 10B). This result suggests the possibility that β-keratin genes are clustered with the same orientation.

In Situ Hybridization

Single-strand cDNA or cRNA digoxigenin (DIG) -labeled probes, directed against the sequence of both Tm or Ht β-keratins coding regions (see above), were used for studying the expression of the isolated mRNAs in the normal and regenerating epidermis of gecko scales. The advantage of using regenerating scales, as previously explained (Dalla Valle et al.,2005), is due to the induction of the differentiative process in the epidermis in which β-cells form a β-layer (Fig. 3D). The hybridization product has been detected by anti-DIG alkaline phosphatase antibody (Fig. 11A–C, page 4). This staining better identifies the single layers of the epidermis of neogenic scales and the cells expressing the specific mRNA. The cRNA and, less intensely, the cDNA antisense probe (not shown), exclusively label differentiating cells of the oberhautchen and the β-layer of neogenic scales of Tm (Fig. 11A–D). No labeling was seen in the undifferentiated wound or in normal epidermis or in the dermis. The negative control (omitting the probe, data not shown) and the sense cRNA probe did not or weakly labeled β-cells of neogenic scales (Fig. 11D).

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Figure 11. In situ hybridization using a cRNA probe and alkaline phosphatase detection in Tm. A: Low-magnification view of three regenerating scales, showing labeled cells forming the axial oberhautchen and β-layer (arrows). Dashes indicate the outline of forming scales. B: Detail of the labeled cells of the oberhautchen and initial β-layer cells (arrow) located in the central, axial part of the regenerating scale (dashes underlie the boundary of the epidermal peg). C: High-magnification detail of reactive cells of the oberhautchen cells (arrows) in contact with nonreactive cells of the clear layer, and the reactive differentiating β-cells (arrows) underneath (arrowheads). D: The sense control section of regenerating scales (underlined by dashes), showing absence of reactivity in the forming oberhautchen and β layers (arrows). aR, antisense probe; ba, basal layer. cl, clear layer; de, dermis; dashes underlie the basal layer of scales. H, hinge region; la, lacunar cells; sR, sense probe (control). Scale bars = 20 μm in A, 10 μm in B,D, 5 μm in C.

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The labeling remained localized within the thinning β-layer toward the scale tip. Below the β-layer, the labeling rapidly disappeared in suprabasal cells. The corneous layer, and granules of the clear layer sometimes were nonspecifically stained, as confirmed by the negative and sense controls.

DISCUSSION

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

The present study on the regenerating tail of geckoes confirmed that neogenic scales are formed in a proximodistal gradient as in other lizards and represents an optimal model for cloning and characterization of β-keratin (Alibardi,2003; Dalla Valle et al.,2005). The random collection of normal, postshedding epidermis (including the epidermis of pad lamellae) often does not yield enough specific mRNA for analysis. The study shows that gecko β-keratins present common but also specific features in comparison to those of lizards (Fig. 12). Gecko sequences resemble that of the lizard P. sicula (Dalla Valle et al.,2005) but are very different from that of a hard keratin protein isolated from the claw of the varanus lizard (Inglis et al.,1987). Gecko β-keratins are a little larger (1–2 kDa) than β-keratins of P. sicula, and this finding may be a specialization of tuberculate scales of geckoes.

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Figure 12. Comparative analysis of deduced lizard (P. sicula), geckoes (T. mauritanica and H. turcicus), and avian β-keratins (F-ker, S-ker, C-ker) using the MCOFFEE program. Double-underline indicates the initial and central regions of high identity. Asterisks indicate identity; colons indicate strong similarity; dots indicate weak similarity. Li-gprp-1, AJ890445; Li-gprp-2, AM259048; Li-gprp-3, AM259049; Li-gprp-4, AM259050; Li-gprp-5, AM259051; Ge-gprp-1, AM162665; Ge-gprp-2, AM263204; Ge-gprp-3, AM263205; Ge-gprp-4, AM258989; Ge-gprp-5, AM263206; S-ker, P04459; F-ker, P02450; C-ker,P25692.

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Northern blotting analysis gives a positive signal only in gecko Tm and showed a higher β-keratin expression in regenerating tail epidermis. This difference very likely depends on the absence of differentiating β-cells in the randomly sampled epidermis from Ht, so that the specific mRNAs were too low in number for detection with Northern analysis. This finding also explains why, in setae epidermis, the RT-PCR analysis revealed the presence of the transcripts only in Ht (β-cells were probably differentiating) but not in Tm (β-cells were probably absent). The higher expression of RNA during regeneration was confirmed in both geckoes by RT-PCR analysis and is related to the presence of numerous differentiating oberhautchen and β-cells.

The study indicates that the sequenced β-keratins are present to both normal (body) scales and in the modified pad lamellae but does not clarify whether scales and pad lamellae also possess other, specific β-keratins. Ongoing proteomic studies, however, have detected similar protein patterns in both normal scales and pad lamellae (Toni and Alibardi, unpublished observations). As a result of the above indications, in the following discussion, we consider the cloned β-keratins to be present in oberhautchen cells of both generalized scales (with spinulae) and pad lamellae (with setae).

The differentiation of oberhautchen and β-cells in regenerating scales and pad lamellae follow the same cytological stages observed in developing scales and in normal scales during the renewal phase of the shedding cycle (Maderson et al.,1998). The elongation of oberhautchen spinulae into setae is apparently supported and moulded by the action of the fibrous cytoskeleton present in the cytoplasm of clear cells that surrounds setae (Alibardi,1997,1998,1999). It is unknown whether and how clear cells of climbing pad lamellae induce this exceptional elongation of oberhautchen spinulae. The in situ hybridizations study has clearly shown the presence of transcripts for the glycine-proline-serine–rich proteins in oberhautchen cells that produce spinulae, and in β-cells of gecko epidermis. Therefore, setae also contain basic glycine-proline–rich proteins that associate into a three-dimensional conformation suitable to polymerize into linear, long parallel cables in spinulae and setae. The presence of the transcripts for the cloned glycine-proline-serine–rich proteins has been confirmed for the setae of Ht by RT-PCR, although in situ hybridization was not available for setae.

The isolation and sequencing of three epidermal cDNAs from gecko Tm and two cDNAs from gecko Ht, the protein structures of the deduced proteins, and their sites of expression have confirmed that the transcripts encode at least five different β-keratins (Figs. 5, 6, 12). However, the results obtained with 5′- and 3′-RACE analyses suggest the presence of a higher number of transcripts in both animals. The putative encoded proteins have high identity in gecko Tm (90% between Ge-gprp-1/2 and Ge-gprp-3), whereas the two isoforms found in gecko Ht show lower identity (64%), also due to the different lengths between them (Figs. 5, 6, 12). Whereas in the two species of gecko the identity is 56%, between lizard (Dalla Valle et al.,2005; Alibardi et al.,2006) and gecko Tm the identity decrease to 45%, and to 36% between lizard and gecko Ht.

Despite the differences, all reptilian proteins contain a conserved central region rich in proline, consisting of a stretch of 35–40 amino acids that presents some homology with avian feather, scale, and claw keratins (Fig. 12). In particular, the sequence underlined in Figure 12, indicates as core-box, shows a 42–53% homology with avian keratins, and seems implicated in the formation of the framework of β-keratin filaments (Gregg and Rogers,1986; Brush,1993; Fraser and Parry,1996).

The deduced proteins have a lower molecular weight (16.8 and 18 kDa) and different amino acid composition (Table 2) from cytokeratins (Fuchs and Marchuk,1983; Steinert and Freedberg,1991; Fuchs and Weber,1994; Coulombe and Omary,2002). The prediction of the isoelectric point for these proteins using the ProtParam tool (http://www.expasy.ch/tools/protparam.html) indicates that they are basic proteins (8.9–9.4), confirming proteomic data indicating that at least some β-keratins of reptilian epidermis are basic (Alibardi and Toni,2006a,b). The deduced proteins contain 179–177 (Tm) and 169–191 (Ht) amino acids, are glycine-rich (18–23%), serine-rich (16–18%), and proline-rich (8–15%; Table 2). Cysteine, tyrosine, and histidine are very low in these proteins (0–4%). The glycine-proline-serine–rich proteins of gecko scales, together those of scales of P. sicula and of the claw of V. gouldii represent the first 11 reptilian “β-keratins” of which the primary sequence is known. These studies suggest that lizards and geckoes have the ability to synthesize glycine-proline–rich proteins that are used to form the hard corneus layers of their scales.

Southern blotting analysis has revealed that multiple copies of the gene are present in the genome of both geckoes, as it was previously suggested by cloning and sequencing of multiple transcripts encoding for β-keratins in neogenic scales of lizards. The study has also shown that lizard (including gecko) β-keratins genes are devoid of introns inside the coding regions. The PCR analyses performed with elongated PCR extension has suggested that these genes are clustered in the genome with the same orientation (Fig. 10). This disposition is also present for different β -keratins in the avian genome (Gregg and Rogers,1986).

Despite that β-keratins are phylogenetically more recent than α-keratins of intermediate filaments, their evolution in reptilian epidermis remains unknown (Alibardi et al.,2006). The homology of the central region of gecko (and P. sicula) proteins with scale–feather keratins is interesting, as also the latter protein is polymerized into long cables in elongating barb and barbule cells during feather development (Gregg and Rogers,1986; Brush,1993; Fraser and Parry,1996). Therefore, the presence of a central, conserved region in reptilian and avian keratins suggests that these proteins had a common, ancestral protein in stem-reptilian progenitors of modern reptiles and birds. This was previously indicated by immunological results (Alibardi and Toni,2006a,b). The knowledge of the primary sequences of more and more reptilian epidermal proteins may help to reveal the molecular evolution of these small and resistant proteins tailored for making hard epidermal derivatives such as scales, claws, beaks, and feathers.

EXPERIMENTAL PROCEDURES

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

Tail loss was obtained by grabbing the tail to induce its release by autotomy in adults of two species of gecko lizards: the Mediterranean gecko Tarentola mauritanica (n = 13) and the small pink gecko Hemidactylus turcicus (n = 6). Tail regeneration occurred at temperature ranging between 25 and 35°C. During tail regeneration, the epidermis regenerated neogenic scales, which were formed from the proximal regions near the tail stump and progressed toward the apex of the regenerating tail (Figs. 1C–F, 3C,D). The regenerating tail was collected by induced autotomy in the proximal portion of the tail, and the skin with the subcutis and some muscle/lipid tissue was isolated and immediately fixed. Two digits, including the climbing pad lamellae, were clipped in each animal, and immediately collected and fixed. From the same animals, tail regeneration was induced two times within a period of 2 months and half in sequence (first regeneration and second regeneration, which occurred with similar sequences in the indicated 2.5-month period). This procedure allowed doubling the available sampling, and more tissues available for the study and mRNAs extraction were obtained. Among the geckoes studied, six specimens of H. turcicus and eight specimens of T. mauritanica were used for sampling small biopsies (1–3 mm) of the skin for the microscopic study, and the remaining tissues were used for RNA and DNA extraction from both normal and regenerating skin.

Immunocytochemistry

Skin fragments of 2–5 mm in length were fixed at room temperature for 5–6 hr in 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Tissues were dehydrated and embedded in the hydrophilic resin Bioacryl (Scala et al.,1992). Sections of 2–4 μm or 40–90 nm thickness were collected, respectively, for light and ultrastructural cytochemistry, as previously reported (Alibardi and Toni,2005). For histological study, sections were stained in 0.5% toluidine blue. For immunocytochemistry, a rat polyclonal antiserum, produced against a lizard β-keratin of 15–16 kDa was used (Alibardi and Toni,2006b) at the dilution of 1:40–50 in 0.05 Tris buffer, pH 7.6. The antibody–antigen complex was detected by a goat anti-rat conjugated with fluorescein isothiocyanate diluted 1:50 in 0.05 M Tris buffer. In controls, the primary antibody was omitted or incubated with nonimmune serum.

Other tissues were sectioned with an ultramicrotome for collecting thin sections on Nickel grids for the ultrastructural study. The sections were incubated in Tris buffer with 2% bovine serum albumin with the rat antiserum against lizard β keratin (dilution 1:50) or with the β-1 antiserum (dilution 1:200), a rabbit antiserum against a chicken scale keratin of 14–16 kDa (Sawyer et al.,2000). After rinsing the sections on the grids in buffer, the secondary antibody against rat or rabbit (IgG conjugated with 10 nm gold) was applied for 1 hr, the sections were rinsed in buffer again and stained for 6 min in 2% aqueous uranyl acetate. Grids were observed under a CM-100 Philips transmission electron microscope.

Tissue Preparation and Nucleic Acids Extraction

Samples of gecko skin were collected from all the specimens. At the time of tissue collection, the tails were amputated by autotomy. Tissues were immediately frozen in liquid nitrogen and stored at −80°C until analyzed. Total RNA was extracted using the commercial product Trizol (Invitrogen, Milan, Italy) according to the manufacturer's instructions. The RNA samples were kept at −80°C until use. Genomic DNA was extracted, from the remaining tail tissue after skin collection, with the Genomic DNA Purification Kit (Fermentas, M-Medical, Milano), according to the manufacturer's instructions.

Cloning of Gecko Keratin cDNA

The gecko Tm and Ht keratin cDNAs were cloned using a two-step strategy based on the use of 5′- and 3′-RACE analyses that were performed with the FirstChoice RLM-RACE kit (Ambion, Milan, Italy), following the manufacturer's instructions. The cloning progression and the primers used are described in the Results section.

For the 3′-RACE, the cDNA was synthesized by incubating, at 42°C for 1 hr, 1 μg of gecko Tm and Ht skin total RNA extracted from regenerating epidermis in 20 μl of the first-strand buffer, which was supplemented with M-MLV Reverse Transcriptase, RNase inhibitor, 0.5 mM of dNTPs, 2 μl of the 3′-RACE adapter. A total of 1 μl of the first-strand mixture was added to 50 μl of the PCR buffer containing 200 μM of dNTPs, 0.2 μM of the 3′-RACE Outer Primer, 2.5 U of Biotherm Taq DNA polymerase (Società Italiana Chimici, Rome, Italy), and a sense primer. The amplification procedure consisted of 2 min at 95°C followed by a touch-down PCR reaction with annealing temperatures decreasing from 68–62°C to 56–50°C over 16 cycles and the final 24 cycles maintained at 56°C. The extension phase of the last cycle was prolonged by 10 min. If necessary, diluted products were subjected to second rounds of amplification using the 3′-RACE inner primer and another specific 5′-sense primers.

For 5′-RACE, 10 μg of total RNA was treated with calf intestinal phosphatase to remove 5′-phosphates from truncated mRNA or non-mRNA, leaving a 5′-OH. The resulting dephosphorylated RNA was processed with tobacco acid pyrophosphatase to remove the 5′-cap from full-length mRNAs. This treatment leaves a 5′-phosphate required for ligation. A 5′-RACE adapter was ligated to the 5′-end of the mRNA using T4 RNA ligase. Subsequently, first-strand cDNA synthesis was made by reverse transcribing the ligated mRNA with random decamers. Keratin transcripts were then PCR amplified using the 5′-RACE outer primer and a specific antisense primer. The cDNA obtained was further amplified by a second PCR using a second gene-specific antisense primer and the 5′-RACE inner primer.

The resultant amplicons from 3′- and 5′-RACE analyses were purified from the sliced gel bands and the gel-purified fragments were ligated into a pGEM-T vector using a pGEM-T Vector System I, according to the supplier's recommendations (Promega, Milan, Italy). Plasmids from positive colonies were purified, and four or more clones were sequenced. To define more precisely the nucleotide and amino acid sequences of gecko keratins, cDNAs produced by reverse transcription with random hexamers and the ThermoScript RT-PCR System Kit (Invitrogen) were amplified with primers flanking the ORF (see the Results section). PCR products were cloned, and two or more independent clones were sequenced. The same cDNA was also used to perform expression analysis by PCR.

cRNA and cDNA Probes Synthesis

To prepare sense and antisense cRNA probes, recombinant plasmids containing the whole coding region were linearized by restriction cleavage and used as a template. The cRNA transcripts were DIG-labeled by in vitro transcription using a DIG RNA Labeling kit (Roche Diagnostics, Milan, Italy) and T7 and SP6 polymerase. Using the same plasmids, double-strand and single-strand cDNA probe were performed by normal and asymmetric PCR with DIG-labeled dNTPs (Roche).

Northern Blotting Analysis

Total RNAs from gecko Tm and gecko Ht epidermis were electrophoresed through 1.1% formaldehyde-denaturing gel, blotted onto a nylon membrane positively charged (Roche, Milan, Italy), and baked at 80°C for 2 hr. The High Range RNA Ladder (M-Medical) was used as a size standard. The 28S and 18S rRNAs were visualized by methylene blue staining to check RNA loading and integrity. The membranes were hybridized overnight at 68°C with DIG-labeled cRNA antisense probe encompassing all the coding region of gecko Tm and Ht keratin mRNA in 5× standard saline citrate (SSC) containing 50% formamide, 0.02% sodium dodecyl sulfate, 0.1% lauroylsarcosine, 1% blocking reagent, and 100 μg/ml of transfer RNA. After incubation with an anti-DIG antibody, the signals were detected using the CPD-Star DIG Luminescent reagent, according to the manufacturer's instructions. The signals were revealed by exposing the membranes to an X-ray film for different intervals of time (30–120 min). The analysis was performed two times.

Genomic Southern Blotting Analysis

Genomic DNA samples (12 μg) were individually digested with restriction endonucleases for 3 hr (EcoRI, XbaI) and for 18 hr (SalI) at 37°C. All enzymes were purchased from Promega (Milan, Italy). The digested samples were fractionated on gels of 1% agarose in TBE buffer and blotted onto a nylon membrane positively charged (Roche) using 20× SSC (3 M NaCl, 300 mM sodium citrate, pH 7.0) as transfer buffer. Membranes were hybridized with a DIG-labeled DNA probe corresponding to the whole coding region of gecko Tm and Ht β-keratins. DIG-labeled bands were visualized as described above.

Nucleotide Sequencing

Sequencing was performed on double-stranded DNA using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit (Applied Biosystems, Monza, Italy). Electrophoresis of sequencing reactions was completed on the ABI PRISM model 377, version 2.1.1 automated sequencer. The homology searches were carried out using the Basic Blast program version 2.0 at http://www.ncbi.nlm.nih.gov/BLAST/, whereas the alignment was performed using the ClustalW program at http://www2.ebi.ac.uk/clustalw/. The poly (A+) signal was found with the “HCpolya:Hamming Clustering poly-A prediction in Eukaryotic Genes” (http://l25.itba.mi.cnr.it/∼webgene/wwwHC_polya.html). Amino acids alignment was done using the MCOFFEE program (http://www.igs.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi).

In Situ Hybridization

From two Tm and four Ht geckoes with regenerating tail showing scale neogenesis (3–5 weeks), the tail was collected and fixed in 4% formaldehyde as above. Tissues were dehydrated in ethanol at increasing concentration, infiltrated in xylene, and embedded in wax.

Tissues of 6–7 μm thickness were collected using a rotary microtome, dewaxed with xylene, and hybridized using the antisense cDNA probes or antisense cRNA probes. As controls, sense cDNA or cRNA probes were used, as well as negative controls (omitting the probes), following a hybridization protocol previously indicated (Dalla Valle et al.,2005). The hybridizing medium contained a mix of 50% formamide, 4× SSC, 0.1% Tween-20, 50 μg/ml tRNA, 100 g/ml 50 mg/ml, 10 mM EDTA, 50 μm/ml heparin, 0.5% Blocking reagent (Roche 1096176).

For hybridization with cDNA probes, an overnight incubation at 40–42 °C in the hybridization medium containing 1–2 ng/μl of DIG-labeled probe was carried out. For hybridization with cRNA probes, an overnight incubation at 60°C in the hybridization buffer containing 1–2 ng/μl of DIG-labeled probe was carried out. Hybridization was followed by washed at progressively decreasing concentrations of SSC (2× SSC, 0.5 SSC, 0.2 SSC, 0.1 SSC, increasing stringency) until the phosphate-saline-Tween-buffer (PBT buffer). Sections were incubated for 2 hr at room temperature with anti-DIF alkaline phosphatase-conjugated antibody (Roche, Mannheim, Germany) diluted 1: 500 in PBT buffer. Detection was done with PBT buffer containing 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4 chloro-3-indolyl phosphate (BCIP) as substrates, as indicated by the manufacturer (Roche, Mannheim, Germany).

Acknowledgements

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

The study was financed by 60% Grants from the Universities of Padova and Bologna, and by self-support (L.A.).

REFERENCES

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