Expression of specific corneous beta proteins in the developing digits of the Japanese gecko (Gekko japonicus) reveals their role in the growth of adhesive setae

Comparative Biochemistry and Physiology, Part B 240 (2020) 110370

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Expression of specific corneous beta proteins in the developing digits of the Japanese gecko (Gekko japonicus) reveals their role in the growth of adhesive setae ⁎

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Feifei Wang, Mingyue Chen, Fengna Cai, Peng Li , Jie Yan , Kaiya Zhou Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Gekko japonicus Setae Corneous beta proteins mRNA expression In situ hybridization

Geckos possess strong adhesion ability, even can climb on smooth surface. Previous studies have shown that the setae of geckos play a crucial role in their ability to climb on vertical walls. But the biological molecular mechanism of their adhesion ability remains unclear. In the present study, the expression patterns of corneous beta proteins (CBPs) genes related to claws, scales, and feathers development (named as ge-gprp-9, ge-gprp-10, ge-gprp11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 respectively) in the developing pad lamellae of different embryonic stages (stage 34, stage 36, stage 39, and stage 42) of the Japanese gecko Gekko japonicus were detected using fluorescence quantitative PCR approach. The results showed that there were significant upregulated expression of CBPs mRNA at embryonic stage 39 with the embryonic continuous maturation and the highest expression level was detected at embryonic stage 39 or stage 42. Moreover, the expression levels of four CBPs genes ge-gprp-9, ge-gprp-10, ge-gprp-11, and ge-gprp-12 in the embryonic and adult development of gecko were detected by fluorescence in situ hybridization technique. The results from in situ hybridization detection revealed that the positive signals of these CBPs genes expression were the same in the developing pad lamellae of G. japonicus. The positive signals of eight CBPs genes were mainly found in the setae tissue, oberhautchen, and β layer, which suggests these CBPs genes are involved in the growth of setae.

1. Introduction Reptiles are ectothermic amniotes whose skin is cornfield and well adapted to land conditions. During the evolution of vertebrates from aquatic to terrestrial life, the ability to survive and reproduce on land was acquired through cornification of the epidermis to prevent mechanical damage, ultraviolet radiation and water loss, and amniotic egg reproduction. Recent study has definitely indicated that the differences between keratinization and cornification, cornification indicates the addition of specialized proteins to intermediate filaments (IF)-keratins and keratinization indicates accumulation of IF-keratins (Alibardi, 2016). In reptiles, the scales contain different components of the stratum corneum, often called intermediate filaments proteins (IF-keratins, i.e. EX-α-keratins) or corneous beta proteins (CBPs, i.e. EX-βkeratins) (Review see Alibardi, 2016; Holthaus et al., 2019). Generally, the genes encoding CBPs are located on the section of the genes encoding epidermal differentiation complex (EDC). Some, previously thought to be β-keratins, are now thought to be CBPs, because some sequences have different properties. The CBPs are completely different

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type/family of proteins forming filaments of 3–4 nm in diameter, and their genes, composition, filaments formation, localization etc. are completely different from IF-keratins (Alibardi, 2016; Holthaus et al., 2019). These IF-keratins are made of 8–10 nm keratin filaments or aggregation cytokeratin. They are differ in physicochemical, biochemical and cytological properties (Toni et al., 2007). In reptiles, especially geckos, the unique digit pads, also known as pad lamellae. A pad lamella is a transformed and modified scale that bears setae on its surface. The presence of pad lamellae allows the gecko to operate freely on vertical surfaces with varying degrees of smoothness (Gamble et al., 2012). Study on the climbing ability of gecko indicated that the adhesion ability of the gecko digit pad is due to the van der Waals between setae and object surface (Autumn et al., 2002). Setae exist in digits as well as tail of some geckos (Bauer, 1998; Alibardi and Bonfitto, 2019). The system of setae is multi-grade structure, with more than ten pad lamellae extended from the bottom of each digit. Each tip of pad lamellae stretches thousands of setae. Moreover, the terminal of each setae gives rise to numerous branching that terminal with small/ nanoscale endings termed spatulae, the sites of adhesion (Alibardi,

Corresponding authors at: College of Life Sciences, Nanjing Normal University, #1 Wenyuan Road, Nanjing 210023, Jiangsu, China. E-mail addresses: [email protected] (P. Li), [email protected] (J. Yan).

https://doi.org/10.1016/j.cbpb.2019.110370 Received 21 July 2019; Received in revised form 5 October 2019; Accepted 14 October 2019 Available online 24 October 2019 1096-4959/ © 2019 Elsevier Inc. All rights reserved.

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2009; Hallahan et al., 2009; Zhao et al., 2008). The formation of setae in specific climbing pad lamellae is a specialized process of differentiation of the oberhautchen layer of the gecko epidermis that derives from the general shedding cycle of lizards that includes two epidermal generations with setae, outer and inner (Alibardi, 2009). Before molting, the two generation of setae are present, the outer setae and the forming inner generation of setae underneath (Alibardi, 2009; Russell and Eslinger, 2017). The complex structure consists of six layers, from outside inward including oberhautchen, β layer, mesos region layer, α layer, lacunar layer, and clear layer (Alibardi and Toni, 2005). The β layer contains proteins that increase the strength of the epidermis. These proteins protect the scales from mechanical damage (Holthaus and Alibardi, 2018). The α layer is surrounded by more flexible proteins which are surrounded by lipid materials, forming a stretchable layer that reduces water loss. The adherent setae on the pad lamellae of reptiles, formed by the interaction between oberhautchen layer and clear layer of the epidermis (Alibardi and Toni, 2005). Different types of setae have different branching patterns, which presumably depend on the structure of the cytoskeleton formed by transparent cells in the clear layer. The latest comparative genomics analysis showed that the expansion of the CBPs family plays an important role in the adhesion ability of the G. japonicus. The results came from the comparative analyses of the CBPs family of the three reptile species, G. japonicus, Anolis carolinensis and Alligator sinensis (Liu et al., 2015). Data on the geckos (G. gecko, Tarentola mauritanica, Hemidactylus turcicus) other than G. japonicas indicate that most CBPs belong to the Ge-CPRPs (gecko cysteineproline-rich proteins) (Dalla Valle et al., 2009; Dalla Valle et al., 2007; Dalla Valle et al., 2005; Hallahan et al., 2009). We also noted that previous studies revealed cysteine is really the specific amino acid for setae CBPs while glycine and glycine-cysteine-rich proteins are more generally found in normal scales and in the claw (Hallahan et al., 2009; Dalla Valle et al., 2009; Alibardi and Toni, 2009; Alibardi, 2014). In order to reveal which members in the genetic family are associated with the formation of setae, eight representative CBPs genes namely scale keratin-like (LOC107118250, ge-gprp-9), claw keratin-like (LOC107118347, ge-gprp-10; LOC107118267, ge-gprp-11), beta-keratinrelated protein-like (LOC107118369, ge-gprp-12), claw keratin-like (LOC107121854, ge-gprp-13), keratin-associated protein5-3-like (LOC107112857, ge-gprp-14), beta-keratin-related protein-like (LOC107118276, ge-gprp-15), and feather keratin B-4-like (LOC107114932, ge-gprp-16) were selected in the present study. The reason for choosing these eight genes was due to the higher expression levels among CBPs genes which we found in the digits in previous experiments (transcriptome sequencing and quantitative experiments). For easier readability, the eight genes were tentatively named after gegprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp15, and ge-gprp-16. Fluorescence quantitative PCR (qPCR) and in situ hybridization techniques were used to detect the expression patterns of these CBPs genes in the setae of the toes at four different embryonic stages of G. japonicus. The present study was made for characterizing these proteins with the following purpose to improve the understanding of the molecular/chemical-physical mechanism of adhesion.

Table 1 Based on illumina high-throughput sequencing, 22 corneous beta proteins genes of Gekko japonicus were obtained. Subject id

Subject description

XP_015276176.1 XP_015276040.1 XP_015276061.1 XP_015276151.1 XP_015272025.1 XP_015280351.1 XP_015272026.1 XP_015275986.1 XP_015275976.1 XP_015276022.1 XP_015275958.1 XP_015270656.1 XP_015264924.1 XP_015276054.1 XP_015275998.1 XP_015276069.1 XP_015276157.1 XP_015269542.1 XP_015284961.1

Beta-keratin-related protein-like [G. japonicus](ge-gprp-12) Scale keratin-like [G. japonicus](ge-gprp-9) Claw keratin-like [G. japonicus](ge-gprp-11) Claw keratin-like isoform X1 [G. japonicus](ge-gprp-10) Feather keratin [G. japonicus] Claw keratin-like [G. japonicus](ge-gprp-13) Feather keratin B-4-like [G. japonicus](ge-gprp-16) Claw keratin-like [G. japonicus] Claw keratin-like isoform X2 [G. japonicus] Feather keratin 4-like [G. japonicus] Keratin-associated protein 10-7-like [G. japonicus] Keratinocyte-associated protein 3 [G. japonicus] Keratinocyte differentiation factor 1 [G. japonicus] Keratin-associated protein 4-7-like [G. japonicus] Keratin-associated protein 5-1-like [G. japonicus] Beta-keratin-related protein-like [G. japonicus](ge-gprp-15) Beta-keratin-related protein-like isoform X2 [G. japonicus] Keratin-associated protein 5-3-like [G. japonicus](ge-gprp-14) Keratinocyte-associated transmembrane protein 2 [G. japonicus] Keratin-associated protein 9-1-like [G. japonicus] Scale keratin-like [G. japonicus]

XP_015276130.1 XP_015270495.1

was set on a regular time, usually at night (Rivera-Perez et al., 2010). In order to ensure the integrity of all eggs and better collection, pregnant female geckos were kept separately in small feeding boxes. Dry vermiculite and water according to the mass ratio of 1:1 were configured to the eggs hatching material. We put the eggs into a hatch matrix of the box and placed them in the incubator of 28 °C. According to the embryonic development stages of G. japonicas (Zhao et al., 2017), stage 34 (13 days ~ 19 days), stage 36 (22 days ~ 28 days), stage 39 (33 days ~ 40 days), and stage 42 (46 days ~ 56 days) were selected for subsequent experiments. And the embryonic development characteristics of the four stages were collected and observed. 2.2. Transcriptome sequencing Illumina high-throughput sequencing technology was applied in various fields of biology and reap lots of achievement. Transcriptome sequencing was important significance for screening key genes. In the present study, G. japonicus, a common species in Nanjing, was used as experimental material. Illumina high-throughput sequencing technology was used to carry out deep sequencing of genes expressed in the skin (setae part) of the digit of G. japonicus and 22 CBPs sequences (Table 1) were obtained and further analyzed. 2.3. Sequence analysis In this study, eight CBPs genes were named according to the characteristic of amino acid content. Bioinformatics analysis was performed on the obtained ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15 and ge-gprp-16 genes, and the full-length cDNA of these genes (nucleotide and amino acid sequences shown in Supplementary material 1) was verified by BLAST from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih. gov). The alignment of genes in the conserved central region were excluded using MEGA version 6 (Tamura et al., 2013). To investigate the physical and chemical properties of CBPs, some physical and chemical properties of these CBPs were studied using online software protparam (http://web.expasy.org/protparam/). The secondary structure of CBPs was performed by PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/). The phylogram of CBPs genes from G. japonicus, Anolis carolinensis and Alligator mississippiensis was computed by the programs MEGA 6.0 (Tamura et al., 2013) and NJPlot (Vidal and Hedges, 2005) to verify the

2. Materials and methods 2.1. Animal culture and sample collection In the present work, the procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Normal University [SOXR (Jiangsu) 2015–028]. We collected females which ready to lay eggs between May and July of 2018 from various localities in Nanjing (32°03′N, 118°45′E), eastern China. Then transported them to our laboratory at Nanjing Normal University and raised them in 500 mm × 300 mm × 250 mm (length × width × height) plastic cages. The temperature of the incubation room was kept at 24–28 °C. Feeding 2

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1 min, 95 °C for 15 s. Two biological replications and three technical duplications were designed (see data in Supplementary material 3), which were all operated on ice box. Statistical tests were carried out using SPSS software 13.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed using one-way analysis of variance (ANOVA) and Turkey's post-hoc Duncan's multiple range tests (Li et al., 2018a, 2018b). Statistical significance was considered when p values were < 0.05 (Lozito and Tuan, 2016).

relationship between CBPs genes and other homologous. For phylogenetic analysis (see data in Supplementary material 2), regions of ambiguous alignment were excluded and the alignment of genes in conserved central region was used (Hallahan et al., 2009; Ng et al., 2014). 2.4. Preparation of total RNA and quantitative real-time PCR The epidermis of the pad lamella in the embryos at stage 34, stage 36, stage 39, and stage 42 of G. japonicus were quickly transferred to a 1.5-ml centrifuge tube. Total RNA was extracted from pad lamellae using TransZol™ Up Plus RNA Kit (TransGen, Beijing, China) according to the product manual (Dalla Valle et al., 2011). The concentration and purity of total RNA were determined by a NanoDrop ND2000C spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For all RNA samples, A260/A280 and A260/A230 ratios were in the range 2.0–2.1 and 1.9–2.0 respectively. The first strand cDNA was synthesized by using PrimeScript® RT Master Mix (Perfect Real Time) kit (TaKaRa, Dalian, China), reaction system as references. Quantitative real-time PCR (qRT-PCR) was carried out in Applied Biosystems StepOnePlus™ Real-Time PCR System (Thermo, USA). Genespecific primers were designed by Primer Premier 5 (Premier, Canada) according to the sequences of the eight genes (ge-gprp-9, ge-gprp-10, gegprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16) and these primers were also shown in Table 2. The expression levels were normalized to the β-actin gene. According to the β-actin gene sequence (GenBank ID: AF199487) in the cDNA library of the Anolis carolinensis, the reference gene primers were designed as described above. The gene-specific primers were also shown in Table 2. It is estimated that the amplification fragment length is about 200 bp, and the reaction system and reaction conditions are as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and annealing at 60 °C for 1 min, with a final at 95 °C for 15 s, 60 °C for

2.5. Preparation of probes During embryonic development, in situ hybridization detection of mRNA can provide the spatiotemporal expression of genes (Dalla Valle et al., 2010). According to the sequences of ge-gprp-9, ge-gprp-10, gegprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 genes, the gene-specific primers (Table 2) of the nearly 3′-terminal were designed by Primer Premier 5 software (Lalitha, 2000). The amplified products were recovered and cloned into the T-easy vector then sequenced after subcloning. The antisense probe was synthesized according to DIG RNA Labeling Kit (Sp6/T7) of Roche (Basel, Switzerland). 2.6. In situ hybridization procedure We used posterior pad lamellae of sacrificed animals and four embryos at different development stages respectively. The tissue was infiltrated in the fixative (formulated with DEPC-treated water) immediately for 2–12 h after it was cleaned. After fixation, the tissue was dehydrated with gradient alcohol and then dipped in wax and embedded. Then these paraffin-embedded tissue were sliced for slides by spreading machine. Slices were obtained using the oven sheet at 62 °C for 2 h. After 2 h roasting, paraffin sections dewaxed paraffin sections dewaxed to water. According to the fixed time of tissue, the slices were boiled in the retrieval solution (Cacl2/Tris) for 10 min and cooled naturally. According to the characteristics of different tissues, protease K (20 μg/ml) was added for 37 °C digestion for 20 min. The use of proteinase K is advantageous because it can degrade RNase potentially present in the tissue. Rinse three times with PBS buffer solution after washing with pure water. Sections with 4 μm thick were stained with 6 ng/μl probe (Skieresz-Szewczyk et al., 2017; Volpi et al., 2018).

Table 2 Primers used in this study. Primer

Sequence

Purpose

β-actin-F β-actin-R q-ge-gprp-9-F q-ge-gprp-9-R q-ge-gprp-10-F q-ge-gprp-10-R q-ge-gprp-11-F q-ge-gprp-11-R q-ge-gprp-12-F q-ge-gprp-12-R q-ge-gprp-13-F q-ge-gprp-13-R q-ge-gprp-14-F q-ge-gprp-14-R q-ge-gprp-15-F q-ge-gprp-15-R q-ge-gprp-16-F q-ge-gprp-16-R ge-gprp-9-F ge-gprp-9-R ge-gprp-10-F ge-gprp-10-R ge-gprp-11-F ge-gprp-11-R ge-gprp-12-F ge-gprp-12-R ge-gprp-13-F ge-gprp-13-R ge-gprp-14-F ge-gprp-14-R ge-gprp-15-F ge-gprp-15-R ge-gprp-16-F ge-gprp-16-R

5′-TTGTGAGGATGCTGGATGAGA-3′ 5′-CCATCTCCTGCTCGAAGTCC-3′ 5′-GTCGTGGCAGCATCTGTTGA-3′ 5′-GAAAGCAATGGACACCCCCA-3′ 5′-TTCTGTGGGAGGCAACACTC-3′ 5′-GTACCCCTCCTCAGGCCATA-3′ 5′-GATCGGGTTCTCGTTTTGGC-3′ 5′-GAACAGATACTGGCACGACG-3′ 5′-GTGAACTGAAGAACTTTTGTGTGA-3′ 5′-AGGGAAATGAAGCAGCCAAGAAA-3′ 5′-CTTGCGGTGTTGGATCATGC-3′ 5′-GGATGACGACTTCAGACCCC-3′ 5′-GGGGGTTACCCAAAACACAG-3′ 5′-AGCATCCTCCACTTTGCTGG-3′ 5′-ATCCCAGGACCCATCCTCTC-3′ 5′-CCTCCATAATGGCTGCCGA-3′ 5′-ACCAGATCCCTCCATVGGAA-3′ 5′-ATAGCCGCAGGTGCTGTATC-3′ 5′-ATGTCTGCCGACTGCGG-3′ 5′-CAGTAGAGCCCTTCTTCCAAGG-3′ 5′-GTCCACAGTTTGCTGTCCCA-3′ 5′-CAGTAGAGCCCTTCTTCCAAGC-3′ 5′-GGCTGGCTACTGTGGTCC-3′ 5′-GTTTCCAAGGATCCCACCCT-3′ 5′-CCACTGTGGTCCCGAGTTCA-3′ 5′-AGGCCATAACCAGAGCCG-3′ 5′-TGCGGTGTTGGATCATGCTA-3′ 5′-GGTCCACAAGGATCACAGGC-3′ 5′-AACAACAACAGAGAGGAGGGA-3′ 5′-GCTTCTGGGGAAGGACTTTCA-3′ 5′-TCACCAGACGCTTCTTCCTTC-3′ 5′-AGCCTCCATAATGGCTGCCG-3′ 5′-GTGTCCTCAAGCAGTCTCGG-3′ 5′-ACCTCACGCATGACAGTTCA-3′

qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone Gene clone

3. Results 3.1. Characterization of eight CBPs genes The online server ProtParam and PSIPRED were used to analyze the physicochemical characteristics/parameters of eight gecko glycineproline-rich proteins (GE-GPRP-9, GE-GPRP-10, GE-GPRP-11, GEGPRP-12, GE-GPRP-13, GE-GPRP-14, GE-GPRP-15, and GE-GPRP-16). The results showed that the molecular weight of CBPs were 8–18 kDa and the theoretical isoelectric points were approximately 8.5 (Supplementary Figs. S1–S2). Each of the eight CBPs contained at least two or four β-strands sequences (Supplementary Fig. S1). The amino acids contents showed that glycine, leucine and proline are the three amino acids with the higher percentage in the composition of these proteins. The relationship between G. japonicus CBPs genes and other homologous genes was shown in Fig. 1. Phylogenetic profiling suggested that the CBPs genes originated by duplication of primordial gene. The partial amino acid sequence of CBPs known as core-box is conserved. However, homologous core-box does not exist in GE-GPRP14. We suspect GE-GPRP-14 does not have the typical core-box characteristics of CBPs family (Supplementary Fig. S1) or we only got a partial sequence of GE-GPRP-14 from the N-terminal that does not include the core-box region. In terms of genetic relationship, G. japonicus is more closely related to A. carolinensis. Both G. japonicus and A. carolinensis have adhesion and climbing ability due to their similar CBPs 3

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Fig. 1. Comparative presentation of 20 genes (obtained using MEGA version 6) of three species (Gekko japonicus, Anolis carolinensis and Alligator mississippiensis). Phylogram of G. japonicus, A. carolinensis and A. mississippiensis corneous beta proteins was built by using the programs MEGA and NJPlot. G. japonicus in green background, A. carolinensis in blue background, A. mississippiensis in red background. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

family are associated with the formation of setae, eight representative CBPs genes were selected in the present study. According to the nomenclature adopted in previous studies for these proteins (Dalla Valle et al., 2009; Dalla Valle et al., 2007; Dalla Valle et al., 2005; Hallahan et al., 2009), the eight CBPs genes namely scale keratin-like (LOC107118250, ge-gprp-9), claw keratin-like (LOC107118347, ge-gprp10; LOC107118267, ge-gprp-11), beta-keratin-related protein-like (LOC107118369, ge-gprp-12), claw keratin-like (LOC107121854, gegprp-13), keratin-associated protein5-3-like (LOC107112857, ge-gprp14), beta-keratin-related protein-like (LOC107118276, ge-gprp-15), and feather keratin B-4-like (LOC107114932, ge-gprp-16) were selected to investigate their roles in the growth of setae in the G. japonicus. These Ge-GPRPs are rich in glycine and proline. Genome and transcriptome analyses identified a number of CBPs (formerly beta-keratins) in the developing, adhesive setae of Gecko japonicus.

structure. The ge-gprp-14 is most distant from the other 19 genes, because of the lack of core-box. Core-boxes are highly conserved regions of 20 amino acids comprised within the 34 amino acid region forming β-strands (Dalla Valle et al., 2007; Dalla Valle et al., 2009; Alibardi, 2013, 2016). The CBPs detected in the present study contained two or four β-strands sequences (Supplementary Fig. S1). In addition, a seven amino acids region, generally consisting of a CINQIPP-motif, indicated as the specific-box for lizards-geckos (Hallahan et al., 2009), is also highly conserved within of G. japonicus and A. carolinensis. Therefore, we verified that this region is a distinguishing symbol for lepidosaurians vs. archosaurian. Other regions are less conserved (Alibardi, 2014). 3.2. Transcriptome sequencing It is important to analyze the gene expression pattern of the skin (setae) of digit. Illumina high-throughput sequencing technology was used to screen for key genes that related to the skin (setae) development of digit of G. japonicas. All RNA-seq raw data were deposited in the National Center for Biotechnology Information BioSample database (accession number: SAMN12786172). Relevant bioinformatics analysis was carried out on the sequencing results, and a total of 22 CBPs sequences were obtained from the measured data and further analyses were carried out (Table 1). To investigate which members in the genetic

3.3. Developmental stage division and main distinguishing features of Gekko japonicus From oviposition to hatching, the processes of embryonic development were observed and recorded in this study (Fig. 2). In the period of embryo stage 34, the separation between digits of both forelimbs and hindlimbs was visible and digits began to condense. Digits were surrounded by interdigital webbing structures. Dorsal and ventral surfaces 4

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Fig. 2. Developmental staging series for the Gekko japonicus (stage 34, stage 36, stage 39, stage 42, Bar = 1 mm). A~C. stage 34. D~F. stage 36. G~I. stage 39. J~L. stage 42. f. Forelimb; h. Hindlimb; c. pad lamellae. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of all digits were transparent with fleshy structure. At embryonic stage 34, there was no obvious tendency of bulge on the ventral surface of digits or forming setae appeared. At embryonic stage 36, the skeletal system developed gradually and began to form claws. The interdigital webbing structure between degeneratation and outline of digits was initially formed. The tip of digits was slightly expanded compared to the root of digits. The transparency of digits was reduced totally. When embryo developed to stage 39, the pattern on the ventral surface of all digits was visible and the pad lamellae began to form. The scales on the dorsal surface of digits become clearly visible due to the appearance of pigmentation (Guerra-Fuentes et al., 2014; Roscito and Rodrigues, 2012). At embryonic stage 39, the distension in the tip of digits was more obvious than that at stage 36. Although there was no seta on the pad lamellae, we speculated that the material related to the formation of setae began to accumulate during this period and forming digital scales aside the very beginning of pad lamellae. Our intention is to investigate which stage is critical for setae formation. Embryos at stage 42 were collected and observed. The results revealed that the inner setae have formed at embryonic stage 42.

3.4. Expression level of eight CBPs genes in four different developmental stages Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 genes in the G. japonicus during four embryonic stages (34, 36, 39 and 42). A. carolinensis βactin was selected as an internal reference gene (GenBank accession number: AF199487.1). The primers were showed in Table 2. Results revealed that the eight CBPs genes were expressed extensively from embryo stage 34 to stage 42 (Fig. 3). The expression levels of these CBPs genes were greatly increased at embryo stage 39. The expression patterns of the eight CBPs genes at different developmental stages of G. japonicus can help to predict their function. Compared with embryonic stage 34, the expression levels of the eight CBPs genes decreased or increased slightly at stage 36. The morphological characteristics of digits showed marked differentiation but no stratum corneum formed in embryonic stage 36. We speculate that the expression levels probably in response to corneous morphological changes of embryonic development of G. japonicus. Such morphological changes were consistent with the fluorescence quantification results. At embryonic stage 39, the expression levels of the eight CBPs genes are greatly increased. The inner hair cells under the skin of the pad lamellae 5

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Fig. 3. Relative expression of ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 in different stages during embryonic development of Gekko japonicus. The stage 34 was used as calibrator for each stage. Bars represent mean ± SD (n = 3). Different letters indicates statistically significant differences (P < 0.05).

may be accumulated in the long filaments of the CBPs and the apical part of setae passes upward at stage 39. In the period from embryonic stage 39 to stage 42, the expression levels of the eight CBPs genes showed two different variation trends: compared with that at embryonic stage 39, the expression levels of ge-gprp-9, ge-gprp-10, ge-gprp11, ge-gprp-12, ge-gprp-15, and ge-gprp-16 genes at stage 42 were decreased significantly. On the contrary, the expression levels of ge-gprp13 and ge-gprp-14 increased significantly at stage 42. We collected

embryos at stage 42 that were incubated for 55 days and cracked their shells in advance and observed its morphological characteristics. Premature infants of G. japonicus could climb as the adults. The phenotypic characteristics of G. japonicus infant at embryonic stage 42 was probably the most similar to that of adult stage. At embryonic stage 42, digits development tends to be finished, therefore the expression levels of ge-gprp-13 and ge-gprp-14 increased. It suggest that different CBPs genes are assigned with different regulatory functions at stage 42 and 6

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Fig. 4. In situ hybridization of pad lamellae visualized by colorimetric reaction (bluish violet). In situ hybridization in four different developmental stages. Control showing absence of cRNA in the pad lamellae (F). A. embryonic stage 34; B. embryonic stage 36; C. embryonic stage 39; D. embryonic stage 42; E. embryonic stage 42 larger version. Bar: 25 μm; l: pad lamellae; nc: newly differentiated cell layer; fsg: first setae generation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the differential expression of these CBPs genes, we also sliced digits at embryonic stage 36 by in situ hybridization and microscan. The results were the same, there was no positive signal or no morphogenesis of setae, as that at embryonic stage 34 (Fig. 4A–B). However, these CBPs genes were strongly expressed at embryonic stage 39 and stage 42 (Supplementary Figs. S3–S13). Such phenomena match the results of these CBPs genes mRNA expression from fluorescence quantification detection. In situ hybridization detection revealed that all of ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 genes in the longitudinal section at embryonic stage 39 and stage 42 showed obvious positive signals in digits and there was no significant difference in the expression sites (Fig. 4C–D). At embryonic stage 39, gene positive signals mainly detected in epidermal, but the differentiation of cell layer beneath the epidermis did not show up. Beginning of pad lamellae formation but not yet of the setae at stage 39, no obvious signal is seen in the forming oberhautchen but mainly a nuclear labeling. The differentiation of subepidermal cell layer was more obvious at stage 42. At this stage, the first setae generation (FSG, forming underneath the embryonic epidermis) formed and the positive signal was concentrated on the FSG and nucleus. The oberhautchen

different CBPs genes may have different expression patterns. The analyses of statistical significance indicated all the eight CBPs genes, gegprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp15, and ge-gprp-16 showed significant differences at embryonic stage 39 with other developmental stages. 3.5. In situ hybridization in four different developmental stages and adult stage using specific probes of the eight CBPs genes The cRNA probes of ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, gegprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 produced blank or weak labeling in the longitudinal section pad lamellae of G. japonicus at embryonic stage 34 and only showed the pattern similarity to the negative control (Figs. 4 and 5, Supplementary Figs. S3–S11). In negative controls, omitting the probes, no signal was seen. At embryonic stage 34 (Fig. 4A), the stained part of setae was not seen from the overall shape of slices. According to the morphological characteristics of digit at embryonic stage 34, stage 36 and stage 39 (Fig. 2), we found that no obvious setae were observed at these stages, while the claw forms from stage 36 and the pad lamellae forms from stage 39. To further examine 7

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Fig. 5. Results of in situ hybridization detection of ge-gprp-16 gene expression at embryonic stage 34, 36, 39 and 42 of Gekko japonicus, a: green fluorescent signal; b: DAPI counterstained nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Results of in situ hybridization and fluorescence detection visualized by colorimetric reaction and of genes in the adult digits. A~B. In situ hybridization in adult stages. C~D. Control showing absence of cRNA in the pad lamellae. Bar: 25 μm; l: pad lamellae; cl: clear layer; fsg: first setae generation; ob: oberhautchen layer; e: epidermis; fβ: forming beta layer; be: differentiating cells of the beta layer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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the composition and the situ hybridization provided images. Therefore, subsequent studies on setae can start from the functional level of different amino acids. The localization and function of different amino acids at different developmental stages were further studied in order to identify the amino acids that play an important roles in gecko adhesion. Our results suggest that these CBPs genes play an important role in the embryonic development of G. japonicus. Due to setae originated by modification of scales and required the participation of relevant proteins, the expression of ge-gprp-10, ge-gprp-11, and ge-gprp-15 were as strong as others and showed no significant difference at stage 42. However, the expression of ge-gprp-13 at stage 42 was similar to that at stage 39, and the expression of ge-gprp-9, ge-gprp-12, ge-gprp-14, and gegprp-16 showed significant differences at stage 42 with that at other developmental stages and the first setae generation have formed. These results provided more evidences that the ge-gprp-9, ge-gprp-12, ge-gprp14 and ge-gprp-16 were involved in the growth of first setae generation. The first setae generation and cell layer had appeared at embryonic stage 42. Positive signals mainly existed in β-layer suggests that the growth of setae closely related to β-layer. During the differentiation of β layer, the β cells located above the basal layer form a CBPs filament bundle and gradually move upward (Alibardi et al., 2007). The spine process elongation and external keratin materials encapsulating the bundle of CBPs filaments due to the interaction of the β-layer cells with the oberhautchen cells and finally forming setae (Alibardi, 2009). During the growth of setae, clear cells in the clear layer and cytoskeleton proteins with unknown components form a thin film around the setae. Clear cells in the clear layer can produce multiple elongations that can infiltrate into the spinous process of oberhautchen and these clear cells also play an active role in forming setae (Alibardi et al., 2011). Setae branching may derives from clear layer. Previous studies suggested that setae tips of different shapes are related to these cytoskeleton proteins in the clear layer (Alibardi, 2009, 2018; Alibardi et al., 2011). During setae growth, various cytoskeletal proteins of unknown composition produced in clear cells form a resistant but dynamic pellicle around the plasticity setae (Alibardi, 2009). The formation of terminal spatulae with different morphology is probably determined by the specific deposition of these cytoskeletal proteins around the growing tips of setae (Dalla Valle et al., 2007; Alibardi, 2009, 2018), but further researches are needed on this point. The present study has emphasized (possibly for the first time) on spatiotemporal expression patterns and the specific functions of CBPs genes in G. japonicus. Previous study on G. japonicas have retrieved 72 CBPs, previously indicated as β-keratins (Liu et al., 2015). In this work, we selected eight CBPs genes and try to identify regulatory gene(s) that are involved in the adhesion ability of G. japonicus. These CBPs genes belong to a large homologous gene family. Specific members of this family are specifically expressed during the development of different appendages (Strasser et al., 2014). Therefore, it is very important to explore the specifically expressed patterns of CBPs genes during setae formation of G. japonicus (Bhattacharjee et al., 2016). Although our study did not cover all of CBPs genes in G. japonicus, these CBPs genes that we selected have certain representative in functional significance. It has been confirmed that the eight CBPs genes have certain functions during setae growth by experiments. The expression levels varied significantly at different stages and the differences were the greatest before or after the formation of pad lamellae. These genes encode important structural proteins for the formation of setae. In subsequent experiments, we could downregulate these CBPs genes (ge-gprp-9, gegprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16) or the others in the pad lamellae for identifying specific functions of CBPs genes (Vitulo et al., 2017). The further studies on the localization and possible roles of other CBPs genes in epidermis of G. japonicus will allow a more complete understanding of their process of cornification. In the pad lamellae of digits of some geckos, setae can grow over 100 μm in length and their apical part begins to branch and form

clear layer interface showing the bottle-like shape characteristics of oberhautchen cells from which setae are produced and interface with the clear layer cytoplasm (Fig. 4E). Therefore, we believe that embryonic development of G. japonicus mainly carried out the accumulation of material at the stage 39 and stage 42 before formation of setae and differentiation of various cell layers. The results for the other genes were presented in Supplementary Figs. S3–S13. The two in-situ hybridization methods complement each other in detecting gene expression: the CISH method allows a better histological detection of the differentiation layers while the FISH technique better evidence the signals. A small amount of nuclear staining in this image is normal, which is often associated with a longer fixation time. The morphological characteristics of setae were most clearly expressed in the slices of adult stage (Fig. 6, Supplementary Figs. S8, S10, S12, S13). Due to the limitation of technical factors or samples was in renewal stage, outer epidermal generation had fallen off but the inner setae forming were shown (Fig. 6A). Moreover, obvious external setae were present and obvious positive signals could be seen on setae by fluorescence in situ hybridization detection (Fig. 6B). Positive signals also existed in β-layer and oberhautchen layer. 4. Discussion This study focused on expression patterns of CBPs genes in the setae of G. japonicus which can provide support for crawling on vertical surfaces. This characteristic had been studied extensively for many years, and numerous breakthroughs had been made in physics and bionics (Endoh et al., 2018; Guo et al., 2012; Li et al., 2018a, 2018b). In recent years, valuable researches had been done on genetics and valuable works on amino level. Glycine-rich proteins are found mainly in setae and their terminal spatula (Hallahan et al., 2009; Alibardi, 2009). β- and α-corneous layers of lizard epidermis are composed of proteins that are rich in different glycine and cysteine-glycine, the former for mechanical resistance and water-solubility of β layer, and the latter for resistance and elasticity of α and β-corneous layers (Alibardi, 2009; Alibardi and Toni, 2005; Maderson et al., 1998). According to the analysis results in the Supplementary Figs. S1–S2, we can verified that most of the CBPs here detected possess a net basic/positive charge. Studies on amino acids of G. japonicus setae indicated that seta was mainly composed of CBPs that rich in glycine, proline, valine, serine and leucine (Dalla Valle et al., 2007; Hallahan et al., 2009). Among above amino acids, glycine may be related to the materials accumulation of β layers and may have a significant influence on the flexibility and stickiness of gecko setae. Glycine is highly hydrophilic and can stabilize the protoplast colloid and metabolic processes in tissues, so it can reduced the freezing point and prevented cell dehydration. Among the eight CBPs proteins, all the N-terminal regions are rich in cysteine and the central regions are rich in proline. The N-terminal regions are not conservative and the central regions rich in proline are very conservative (Dalla Valle et al., 2007). Proline is mainly involved in the formation of setae in the gecko stratum corneum (oberhautchen) of gecko pad lamellae. Valine can promote the growth of organisms, repair tissues and provide the energy. The concentration of proline and valine in the core-box region of CBPs contributes to the formation of β folding. Therefore valine and proline are important for the stabilization of CBPs. These amino acids may contribute to the formation of β-strand regions (Alibardi et al., 2007). Morever, cysteine is related to the formation of oriented CBPs bundles in setae (Alibardi, 2009). Cysteine is a sulfurcontaining amino acid that is involved in the production of disulfide. In addition, cysteine (Cys) is mainly related to the formation of the apex bifurcation of setae. However, the content of cysteine (Cys) in the eight CBPs mentioned above are not high, which suggest that the CBPs are mainly involved in the accumulation process of substances in the layer of G. japonicus setae. The low cysteine content found here may indicate the extracted mRNAs were collected not only from the pad lamellae but also from the developing claws from stage 36 onward, explaining both 9

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Acknowledgement

multiple ends, known as spatulae (Alibardi, 2009). In contrast to G. japonicus, the setae of A. carolinensis do not form branches at setae tips, and A. mississippiensis do not form setae. These CBPs sequences retrieved from G. japonicus, A. carolinensis and A. mississippiensis included in the alignments were showed in Fig. 1. Two phylogenetic trees were inferred: one from A. mississippiensis sequences only, and another from G. japonicus and A. carolinensis sequences described above. As mentioned earlier, core-boxes are highly conserved within CBPs of G. japonicus, A. carolinensis and A. mississippiensis (Alibardi et al., 2007; Alibardi and Toni, 2009; Fraser and Parry, 2011). Moreover, specificbox CINQUIP for Lizard-geckos, is also highly conserved within CBPs of G. japonicus and A. carolinensis, but the lizard pre-core box is different between lizards (lepidosaurians) and crocodilians (archosaurian) and is a specific tag for G. japonicus and A. carolinensis in comparison to A. mississippiensis. Understanding the chemical composition and conformation of proteins in setae will help to understand the adhesion mechanism (Green et al., 2017). Recently, study on the Tokay gecko revealed the importance of protein composition for the adhesion mechanism (Alibardi, 2018). The fine structure of setae reminds us that the structure observed in the mature branchlets of feathers is largely determined by the accumulation of feather corneous beta protein (Alibardi, 2009; Hackett et al., 2008). Whether these proteins evolved from a common ancestral protein that found in stem reptiles remains unknown (Sawyer et al., 2003; Widelitz et al., 2007). Our study can provide new ideas from the genetic level. Besides the study on gecko adhesive devices is possible to develop new adhesion devices by simulating the microstructure of setae (Alibardi, 2009). There are many efforts on setae, which are worth exploring (Peattie et al., 2007). The present pioneer study requires the analysis of other embryonic stages for future and planned analyses in order to obtain more data on the proteins involved in setae formation and mechanism of adhesion.

The authors are extremely grateful to the two anonymous reviewers for great helpful comments on the manuscript. For financial support, the authors appreciate the National Natural Science Foundation of China (Grants 31672269 and 31000949 to JY and 31000954 to PL), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJA330001), Program of Natural Science Research of Jiangsu Higher Education Institutions of China (Grant No. 17KJD240001 to PL), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP, PPZY2015B117). References Alibardi, L., 2009. Cell biology of adhesive setae in gecko lizards. Zool. (Jena) 112 (6), 403–424. https://doi.org/10.1016/j.zool.2009.03.005. Alibardi, L., 2013. 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5. Conclusion In the present study, we characterized (possibly for the first time) the differential expression patterns of eight CBPs genes in four developmental stages (stages 34, 36, 39, 42) of G. japonicus. As these CBPs genes (ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp14, ge-gprp-15, and ge-gprp-16) are strongly expressed at stage 39 and stage 42, we postulate that they may be involved in the growth of the pad lamellae and play a regulatory role in the accumulation of stratum corneum material. Our results suggest that these CBPs genes play an important role in the embryonic development of G. japonicus. Moreover, in situ hybridization is used to investigate the expressed locations of these CBPs genes. The cRNA probes of the eight CBPs genes produced strong labeling in β-layer and setae of G. japonicus at stage 39, stage 42, and adult stage. These results are consistent with the mRNA expression from fluorescence quantitative PCR detection, which further confirms that eight CBPs genes are involved in the growth of setae. This study improved our knowledge of the biological functions of these CBPs genes in G. japonicus. Finally, we find that the first setae generation was grown at stage 42. Overall, our results narrowed the time range when setae begin to appear, especially from embryonic stage 39 to stage 42. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2019.110370. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. The authors declare that they have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company. 10

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