Temporal Expression Pattern of Peptides in the Regenerating Caudal Fin of Teleost Fish Poecilia latipinna With Special Emphasis on krt15 and myl-1

Amongst several vertebrates, caudal fin of teleost fish has emerged as an excellent model to understand the mechanism of regeneration. By now it is well perceived that proper coordination of different regulatory signals are needed to facilitate the progression of successive stages of regeneration. Therefore, in the current study protein expression profile for caudal fin regeneration in Poecilia latipnna was brought into the focus. The pattern of protein turnover and variation in the expression level were observed across the stages of regeneration. Data from twodimensional gel electrophoresis revealed the differential expression of proteins for wound epithelium, blastema and differentiation stages when compared to resting stage. Based on the computational analysis, of the peptide expression profile and subsequent sequencing, two peptides namely Keratin type I cytoskeletal 15 and Myosin light chain-1 were studied further at transcript level. Keratin-15 was found to be up regulated at wound epithelium stage while its expression waned significantly in the blastema and differentiation stages of regeneration indicating its role in the formation of a functional wound epithelium. Western blot analysis of Keratin-15 also concurred with transcript levels of krt15. However, myl-1 transcript levels was observed to be dispensable for initial phase of regeneration whereas its elevated level in differentiation stage at both mRNA and protein level marks its noteworthy role in achieving the structural integrity of a regenerating caudal fin. This is the first observation of the involvement of Keratin-15 and Myosin light chain-1 in the regulation of regenerative process in Poecilia latipinna.


Introduction
Epimorphic regeneration involves the de-differentiation of existing tissue and recruitment of proliferative cells to form the new structure to restore the lost appendage. Such type of appendage regeneration is observed in a number of vertebrates including lizard tail, salamander limb and tail [1][2][3][4], and zebrafish caudal fin [5][6][7]. Amongst the models of vertebrate appendage regeneration, teleost caudal fin has emerged as an extensively used model to study epimorphic regeneration. Most of the caudal fin regeneration studies have been carried out in zebrafish due to its accessibility, its fast and robust regeneration and its simple architecture [8,9]. However, sailfin molly, Poecilia latipnna (Lesueur, 1821) have been served as an alternative to zebrafish, being more adaptive to the oriental laboratory conditions as well as certain experimental advantages owing to its relatively large size.
Following amputation of caudal fin in zebrafish, an epithelial layer covers the wound which gets thickened later on. Following this, mesenchymal cells migrate near the amputation plane beneath the newly formed wound epithelium, accumulate and proliferate to form the blastema and finally differentiate to replace the lost structures [5,9,[10][11][12][13][14]. Wound healing or wound epithelium formation is achieved within 12-24 hours after amputation and blastema formation is achieved in 48-60 hours post amputation. Finally, the blastemal cells differentiate and restore structures including blood vessels, bony rays, and connective tissue by 15-20 days post amputation [6,9,12,15]. This time course of caudal fin regeneration in P. latipinna differs to that of zebrafish which has been reported in this study for the first time. The aforementioned stages of epimorphic regeneration are governed by certain cellular events which are ultimately regulated at molecular level. For each process to occur in a spatio-temporal manner, a large scale of protein turnover takes place. During protein turnover, several peptides need to be synthesized newly as per the need of the tissue and many to be downregulated.
There are reports on proteomic profile in regeneration models to unravel the conserved and diverse mechanisms amongst them [16][17][18][19]. It includes the regeneration in axololtl [20,21], Xenopus laevis froglet limb-bud [22,23] and zebrafish caudal fin regeneration [24]. Herein, in P. latipinna, twodimensional gel electrophoresis was used to find out the overall expression pattern of peptides across the stages of epimorphic regeneration. This was followed by peptide sequencing of selected spots based on their differential expression pattern across the stages of regeneration.
The sequencing results yielded two peptides, keratin-15 and myosin light chain-1. As the requirement of keratin to get the structural integrity of newly formed epithelium has been reported in many vertebrates during development, adulthood and regeneration [25,26], further analysis of this peptide at transcript level was attempted. For myosin light chain-1, the gene myl-1, encodes two transcript variants expressed in fast skeletal muscle of zebrafish embryo [27] as well as it was reported in the fast twitch fibres and its knockdown has been shown to disrupt myogenesis [28]. Therefore, it was inferred that myosin light chain-1 is important during later stages of regeneration. Hence, in the current study, myl-1 transcript level was checked during the caudal fin regeneration in P. latipinna.

Animals and maintenance
Sailfin molly, Poecilia latipinna, (Lesueur, 1821), of both the sexes of the same age with an average size of 4-5cm and weigh about 5g were purchased from a commercial supplier. They were acclimatized and maintained in glass aquariums containing dechlorinated water with constant aeration, temperature range of 28-30˚C and photoperiod of 14:10 hours of light to dark cycle were maintained. The experimental protocols used in the study were carried out in accordance to the ethical guideline of Drugs and Cosmetics Rules, 1945 and was reviewed and approved by the Institutional Animal Ethics Committee (No. ZL/IAEC/  prior to the commencement of experiments.

Morphological observations and Tissue collection
One-third part of the caudal fins from all the animals was amputated in hypothermic conditions and allowed to regenerate. Fishes were observed under light microscope (Leica, USA) and achievement of each stage of regeneration was recorded at 25X magnification. Amputated fin tissues from 10 animals was collected and considered as a resting stage (R). Tissues for wound epithelium (WE) stage were collected at 24hpa. For blastema (BL) and differentiation (DF) stages, fins were collected at 60hpa and 5dpa respectively (n=10 animals per group for each experiment).

Two-dimensional gel electrophoresis
Fin tissues for all the stages, R (0hpa), WE (24hpa), BL (60hpa) and DF stage (5dpa) were collected and 10% homogenate was prepared using 2D lysis buffer containing (3-cholamidopropyl) dimethylamino-1-propane sulfonate (CHAPS), dithiothreitol (DTT) and protease inhibitor. Supernatant was collected the protein was precipitated by using cholorofrom-methanol precipitation method. The precipitated protein was solubilised in the sample buffer and the protein content was estimated by using Bradford method [29]. Absorbance was measured at 595nm with an ELISA plate reader (Metertech Σ960).
Thereafter, 40μg of each sample was subjected to firstdimensional gel electrophoresis with a 2D marker (Biorad, USA). Proteins were allowed to separate on the basis of their pI in the first-dimensional gel electrophoresis called isoelectric focusing with broad range immobilized pH gradient strip (IPG) 3-10 (Biorad, USA) followed by equilibration with DTT and Iodoacetamide (IAA). The strips were placed on to the resolving gel of SDS-PAGE and the proteins were separated further by electrophoresis. Gels were fixed with methanol:glacial acetic acid solution, stained with silver staining kit (Thermo Fisher, USA). Gel pictures were taken and analyzed using Biorad PDQuest version 8 analysis software.

Real-time PCR
All the tissues were collected in the TRIzol reagent for each stage. RNA isolation was achieved using the TRIzol method, and their purity was checked by the ratio of absorbance at 260 nm, and 280 nm and quality of RNA was ascertained with 1% agarose gel electrophoresis. 1µg DNase treated RNA was subjected to reverse transcription with cDNA synthesis kit (Applied Biosystems, USA). cDNAs were subjected to qRT-PCR analysis using select SYBR green mix (Applied Biosystems, USA) and specific primers. Primer sequences used for qRT-PCR are as follows: Forward; 5'-GGCCGTTCTTAGTTGGTGGA-3' and 18SrRNA Reverse; 5'-TCAATCTCGGGTGGCTGAAC-3'. Samples were measured in triplicate with 18SrRNA as endogenous control. The fold change values were calculated using Livak and Schmittgen method [30].

Western blot
Equal amount of proteins were loaded on to 10% SDS-PAGE and electrophoresed at 100V for 2-3 hours. Proteins were transferred onto nitrocellulose membrane with 0.45 µ pore size at 100V for 100 minutes with transfer buffer containing methanol-tris-glycine. Membrane was developed for the presence of band for Keratin-15 and MYL1 using Anti-Cytokeratin 15 antibody Rabbit IgG (1:200, EPR1614Y, Abcam) and anti-MYL1 Rabbit IgG (Sigma-aldrich, USA) and anti-β-actin mouse IgG.

Statistical analysis
The data were subjected to One Way Analysis of Variance (ANOVA) followed by Bonferroni's multiple range tests using GraphPad Prism 5.0, statistical analysis package. The values are expressed as mean ± SEM. A 'p' value of 0.05 or less was accepted as being statistically significant.

Morphology
To depict the process of caudal fin regeneration in Poecilia latipinna, gross morphological observations were made using light microscope. In Figure 1A, uncut fin can be observed along 2 with the marked amputation site. Following amputation, cut fin is seen in Figure 1B. By 24hpa, wound is covered with Apical Epithelial Cap (AEC) formed by epithelial cells which is shown in Figure 1C. Following wound epithelium stage, a bulgy structure called blastema at the end of each ray at 60hpa can be observed in Figure 1D. At 5dpa, newly formed lepidotrichia proximal to the amputation plane is visible which mark the onset of differentiation stage (Figure 1E).

Peptides profile
Computational analysis of 2D gel images of the specified stages of regeneration of teleost fish, P. latipinna caudal fin was achieved with PDQuest software and it revealed a number of spots differentially expressed. In the resting stage, a total number of 182 protein spots were present while in wound healing, blastema and regenerated stage total 196, 201 and 193 spots were recognized respectively (Table 1, Figure 2). Further, based on the analysis, two proteins namely Keratin-15 (annotated with blue triangle -pI 8.45, MW 45kDa) and Myosin light chain-1 (annotated with red circle -pI 6.68, MW 17kDa) were selected for their comprehensive study on the expression pattern in different stages of regeneration (Figure 2).

Transcript levels of krt15 and myl-1
Transcript level analysis was carried out using quantitative real time reverse transcriptase PCR. 2.34 compared to the resting stage. However, the same was found to be significantly decreasing during the BL (60hpa) and differentiation stage (5dpa) with the mean values of 0.069 and 0.0146 respectively when compared to the resting stage (Table 2, Figure 4). On the other hand, myosin light chain-1 showed reduced level during 24hpa and 60hpa but its level increased significantly in the 5dpa (Table 2, Figure 5).

Protein expression profile for Keratin-15 and myl1
To confirm our transcript level results, western blot was performed for Keratin-15 and myl-1 for all the stages for regeneration in caudal fin of P. latipinna. Protein levels of both, Keratin-15 and myl-1 did match with their mRNA expression levels. When compared to the resting stage, Keratin-15 (45kDa) was found to be increased significantly during WE stage whereas it went down during BL and DF stages (Table 3, Figure 6). Quite contrary to Keratin 15, myl-1 protein (17kDa) expression was found to be decreased during WE stage and its levels were increased noticeably during BL and DF stages.

Discussion
Vertebrate appendage regeneration has attracted a huge attention in current times. As the regenerative capability decreases with increasing complexity in tissue organization, understanding the mechanism of regeneration has become inevitable. To understand the mechanism of regeneration, a number of model organisms are available in the field of regenerative biology. Teleost fish is one such model organism which has been documented extensively for studying regeneration process. In our study, we have explored caudal fin regeneration in teleost fish, Poecilia latipinna, as a model of epimorphic regeneration to illustrate the molecular mechanism with respect to peptides.
It is previously known that regeneration involves controlled expression of peptides and transcripts of signalling molecules at each stage of epimorphosis [18,19,31]. Therefore, to identify the stage-specific expression of peptides, we performed two dimensional gel electrophoresis. When the fin tissues of the selected stages of regeneration were subjected to twodimensional gel electrophoresis, some of the spots were found to be up-regulated and expressed in a stage-wise manner. In addition, few spots exhibited down regulation, and their presence was not observed at a specific time point. Among all the spots, few remained constantly present in all the stages stating their requirement for maintaining the homeostasis of the growing tissue.
During the peptide screening, two peptides, keratin-15 and myosin light chain-1 were focused for further studies based on their differential expression level across the stages of regeneration. Keratin-15 is a type of soft alpha keratins. Keratin-15 is present in the epithelial cells and considered as epidermal stem cells marker [32]. Keratins are important constituents of intermediate filaments of the intracytoplasmic cytoskeleton of epithelial cells. Role of keratin proteins was reported during wound healing and in several healing responses [33][34][35]. It has also been observed that various keratins are expressed at high levels in regenerating tissues [36][37][38][39][40]. Imboden et al. [41] identified cytokeratin 8 as a suitable epidermal marker during zebrafish caudal fin regeneration. In the regenerating axolotl limb study, regulated expression of KRT5 was observed during blastema which was suppressed in the basal layer of the AEC. Also, KRT5 and KRT 17 were reported as useful marker gene for AEC formation in the limb [42]. Knockout study of keratin 17 in mice showed delayed wound healing [43] and its reintroduction leads to improved healing. During in-vitro study, it was suggested that keratin plays an important role in improving the speed of wound healing [44]. Moreover, keratin formulation can also improve the epidermal migration by the upregulation of keratin gene expression [45,46]. Few of the mammalian type keratin markers such as keratins 6, 16, and 17 have also been identified in the lizard [47][48][49]. Therefore, herein the expression of keratins, suggests the role of keratins for the successive formation of the epithelial layer. The possibility of keratin involvement in the formation of epithelial covering has remained high during the development of regeneration. Therefore, the study of cytoskeletal keratin 15 was undertaken further at transcript level expression during regeneration in P. latipinna. Elevated level of krt15 transcript was found at WE stage, which again confirms the role of keratin 15 in forming wound epithelium. However, expression of the same gene went down in blastema and differentiation stage. Nonetheless, western blot analysis of Keratin 15 coincided with transcript levels of krt15 and hence strengthens its requirement during WE stage in caudal fin regeneration in P. latipinna.
Other than keratin 15, our study revealed a significant change in expression of myosin light chain-1 upon peptide sequencing. myl-1 encodes two transcript variant of myosin light chain 1/3 skeletal isoform, a small polypeptide alkali light chain and functional unit of myosin, expressed in fast skeletal muscle [27]. Along with Myosin heavy chain, myosin light chain forms a functional unit for smooth muscle contractility and migration and proliferation [50].
During the proteomic study of axolotl regenerating limb it was noted that about one-third of the cytoskeletal proteins were downregulated like sarcomeric proteins of skeletal muscle such as TNNT3A, TM7, myosin light chain 3 (MYL3) and MYL5 [21]. Kelly and Buckingham [51] have reported that myl-1 expressed in mouse fast-twitch fibres. Burguie`re and his colleagues [28] had reported myl-1 as earliest expressed marker in the fasttwitch precursor cells in the zebrafish embryo, and its knockdown disrupts myogenesis. In P. latipinna, results revealed down regulation of the myl-1 gene at the wound healing and blastema stage while in the differentiation stage it was found to be increased at both transcript as well as protein level suggesting the involvement of myosin light chain 1/3 during muscle contractility for the movement of the fish, P. latipinna. Hence, the myl-1 expression during differentiation phase of regeneration points towards the active involvement myosin light chain 1/3 regulatory protein in regaining the cytoskeletal integrity.
In brief, our study bring to fore for the first time a peptide profile for caudal fin regeneration in P. latipinna. Further, based on the differential expression pattern, two proteins namely Keratin-15 and Myosin light chain-1 were sequenced and their expression pattern during different stages of regeneration was reaffirmed through both transcript as well as protein level analysis.