Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2021.12235 MMR-0-0-12235 Articles Identification of differentially expressed proteins involved in fetal scarless wound healing using a rat model of cleft lip YanYu 1 2 3 LiuHong 2 YiJiarong 3 ChenZizi 3 ChenJia 3 ZhangJianfei 4 GaoKewa 3 5 HeSiqi 3 5 WangAijun 5 JinPing 1 HuFeng 6 ZhouJianda 3 Department of Endocrinology, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China Department of Plastic and Reconstructive Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China Department of Burns and Plastic Surgery, The Second Hospital of South China University, Hengyang, Hunan 421000, P.R. China Department of Surgery, Surgical Bioengineering Laboratory, University of California Davis, Sacramento, CA 95817, USA Department of Dermatology, Hunan People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan 410000, P.R. China Correspondence to: Dr Jianda Zhou, Department of Plastic and Reconstructive Surgery, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, P.R. China, E-mail: zhoujianda@csu.edu.cn Dr Feng Hu, Department of Dermatology, Hunan People's Hospital, The First Affiliated Hospital of Hunan Normal University, 61 Western Jiefang Road, Changsha, Hunan 410000, P.R. China, E-mail: doctorzhoujianda@163.com 08 2021 22 06 2021 24 2 596 06052020 07122020 Copyright: © Yan et al. 2021 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge-shaped cleft lip-like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription-quantitative (RT-q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper-left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper-left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro-inflammatory factors, including TNF-α, were also different between two groups. Label-free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT-qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

 cleft lip scarless healing label-free quantification proteomics parallel reaction monitoring National Science and Basic Resources Survey Special Foundation, China 2017FY101204 Technology Innovation Guide Program of Hunan Province, China 2017SK50124 Science and Technology Major Project of Hunan Province, China 2019SK1010 Science and Technology Major Project of Hunan Province, China 2019SK1015 The present study was supported by a grant from the National Science and Basic Resources Survey Special Foundation, China (grant no. 2017FY101204), the Technology Innovation Guide Program of Hunan Province, China (grant no. 2017SK50124), the Science and Technology Major Project of Hunan Province, China (grant no. 2019SK1010) and the Science and Technology Major Project of Hunan Province, China (grant no. 2019SK1015). Introduction

The cleft lip is a very common congenital oral and maxillofacial malformation, often accompanied by cleft palate and alveolar cleft. Although surgical repair techniques are continuously being improved, numerous patients still experience inevitable secondary scar formation after surgery. In recent years, with the development of prenatal diagnosis and treatment technology (1), intrauterine surgery has made it possible to correct developmental deformities, such as a cleft lip.

The concept of scarless healing was first proposed by Burrington (2) in 1971. It was later observed that fetal skin wounds that occur during early pregnancy can heal quickly and restore intact skin barrier functions. In contrast, fetal skin damage that occurs in the third trimester of pregnancy can result in the formation of scar tissue similar to that of an adult (3). Therefore, the different manifestations of scarless healing of mammalian fetal wounds are related to the gestational age of the fetus (4). Dang et al (5) and Longaker et al (6) demonstrated that this transition period from scarless healing to scar formation occurred between day 16.5 of gestational age (GA) and GA18.5 in rats and mice, which have a gestation period of ~21.5 days. Lorenz et al (7) and Cass et al (8) suggested that when 1–2 mm incisions are inflicted on fetal rats, the transition period of scarless healing to healing with scar formation was still between 16.5 (GA16.5) and 18.5 days (GA18.5).

This phenotypic difference in fetal wounds has inspired further examination of the specific underlying mechanisms. Initially, it was hypothesized that the reason for early scar repair was that the fetus developed in amniotic fluid, which is rich in growth factors and extracellular matrix (ECM) components (9,10). Previous studies typically utilized large animal models to study the presence of scars following repair (11,12). However, only a few studies have reported the use of a fetal rat cleft lip wound model to establish the effectiveness of surgical repair at different gestational ages. Moreover, due to the short gestation period of rats, the experimental cycle can be shortened, and the experiment can therefore be repeated.

Given the importance of this process, the present study, screened out several specific markers of early fetal scarless repair. The present study aimed to gain insight into the occurrence and mechanisms of scarless repair, and to identify new clinical targets for the prevention and treatment of scars.

 Materials and methods <sec> <title>Animals

A total of 36 SPF-grade adult Sprague-Dawley (SD) rats (female; mean weight, 250 g; age, 12 weeks) were obtained from the Third Xiangya Hospital of Central South University Animal Experiment Center (Hunan, China) and divided into two groups that received surgery once their fetuses reached GA16.5 or GA18.5, respectively (n=18 each). The following housing conditions were implemented: A temperature between 25±2°C, relative humidity of 55±15%, ventilation rate of 10–20 times per hour, time-controlled artificial lighting (12-h day-night cycle) and ad libitum access to food and water. The experiments were supervised throughout and were performed in accordance with animal experimentation ethics.

 Preliminary study on different repair modes applicable to fetal rats with artificial cleft lip wounds

Fetal rats located away from the uterine horn were selected to prevent subsequent abortion, as described previously (13). In the current study, rats were anesthetized with 30 mg/kg pentobarbital sodium intraperitoneally before surgery. A wedge-shaped cleft-like defect was created on the upper-left lip of the fetal rats. The upper-right lip did not receive any treatment and was used as a control condition. The fetal rats were then returned to the uterus. Fetal rats from the GA16.5 and GA18.5 groups were then removed three days post-surgery as previously described (4) (i.e., at GA19.5 or GA21.5, respectively). All fetuses and rats were euthanized using carbon dioxide (30% volume displaced/min). Death was confirmed using cervical dislocation. A total of three fetal rats were obtained from each pregnant rat, for a total of 54 fetal rats from both GA16.5 and GA18.5 groups, and the survival rate was calculated. Tissue samples from the surgical site on the upper-left lip and asymmetrical sections from the upper-right lip were collected from the fetal rats for histological examination, including hematoxylin and eosin (H&E) staining, Masson's Trichrome staining and type-I collagen immunohistochemical staining as previously described (14–16).

The upper-left lip tissue samples from the GA16.5 group were defined as group 1, whereas the upper-right lip tissue samples from the GA16.5 group were defined as group 2. In addition, the upper-left lip tissue samples from the GA18.5 group were defined as group 3, whereas the upper-right lip tissue samples from the GA18.5 group were defined as group 4. Each subgroup included 27 samples. Protein expression was compared between group 1 and 2, group 3 and 4, as well as group 3 and 1. Label-free quantification PRM was performed as previously described (17) and was used to detect the differentially expressed proteins among the different groups. MaxQuant 1.5.6 (https://www.maxquant.org) and Perseus 1.4 (https://www.maxquant.org/perseus/) were used to analyze the results of label-free quantification PRM: Volcano plots were generated for differentially expressed proteins: Y-axis, -log10(P-value); x-axis: log2(ratio). The points distributed outside the two vertical borders and above the horizontal border represented the proteins with significant differences; proteins with at least a 1.5-fold change in expression and P<0.05 were considered significant. Subsequently, bioinformatics analysis, including GO and KEGG pathway analysis, was performed to identify differentially expressed proteins (18).

 Experimental verification of tissue repair proteins in fetal rats with artificial cleft lip wounds

The mRNA levels of the differentially expressed molecules were assessed using reverse transcription-quantitative (RT-q) PCR, as previously described (19). Differentially expressed levels of proteins were detected by label-free quantification PRM as previously described (17).

 Statistical analysis

GraphPad Prism 8.0 (GraphPad Software, Inc.) and SPSS 22.0 (IBM Corp.) were used to perform calculations and carry out statistical analysis. Student's t-test was used to compare differences between two groups. The experimental data from each group were analyzed for congruence of variance before the t-test were applied. The FDR values were within 0.01 in the comparisons. Mixed ANOVA followed by Sidak's post hoc test was used to analyze the differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Gross observation

All fetal rats were observed before delivery. The nasolabial cleft was first observed before surgery and images were captured to facilitate the observation of changes in the fetal rats from the GA16.5 and GA18.5 groups. We observed the same area again 72 h post-surgery to identify differences. The cuneiform tissue of the upper-left lip was removed by microsurgery to create a cleft lip wound. The changes in the fetal rats were observed macroscopically. In the GA16.5 group, the upper-left cleft lip wound completely healed 72 h after surgery (i.e., GA19.5) and the continuity of the upper lip tissue was restored. Only a slight depression was observed in the surgical area. The upper-left lip tissue was nearly symmetrical with that of the right side. However, in the GA18.5 group, the cleft lip wound was not completely healed 72 h after surgery (i.e., GA21.5); a clear scar was observed in the surgical area, and the upper lip was asymmetrical on both sides due to wound contracture (Fig. 1).

 Histological analysis

In the GA16.5 group 72 h after surgery, the tissue of the upper-left lip wound demonstrated complete regeneration when observed under the microscope (Figs. 2–4). The results of H&E staining demonstrated complete epithelialization of the upper-left lip, and the structure of new follicles was detected under the epidermis. Compared with the normal skin of the upper-right lip, a slight depression in the cleft part of the upper-left lip and thickening of the skin was noted, whereas inflammatory cell infiltration and neovascularization were not apparent (Fig. 2). Masson's Trichrome staining revealed collagen fibers under the epidermis, demonstrating a fine reticular and emerging follicular structure (Fig. 3). Immunohistochemical analysis indicated no obvious difference in the amount of type-I collagen in the upper-left cleft lip area and the rest of the upper lip (Fig. 4).

In the GA18.5 group, the position of the wound was easily identified by a distinct scar on the upper-left lip. H&E staining demonstrated that partial epithelialization occurred in the upper-left cleft lip area. Compared with the normal skin of the upper-right lip, the upper-left lip displayed a clear scar, new capillary formation around the wound and increased fibroblast proliferation and ECM volume, whereas structural components of hair follicles were not observed under the epidermis (Fig. 5). Masson's Trichrome staining demonstrated the absence of new follicular structure and the presence of dense collagen fibers under the epidermis (Fig. 6). Immunohistochemical analysis in the upper-left cleft lip wound demonstrated an increase in type-I collagen expression and fiber density, as well as a more compact structure and absence of adnexal skin (Fig. 7), compared with normal upper lip tissue.

Immunohistochemical analysis of cell proliferation markers was also carried out. Compared with GA16.5 fetal rats, the expression of Ki67 and CD31 slightly increased in the GA18.5 group following surgery. By contrast, the expression of CK10 decreased in the GA18.5 group, compared with the GA16.5 group (Fig. 8).

 RT-qPCR analysis of inflammatory factors

The relative mRNA expression levels of the pro-inflammatory factors TNF-α, IL-10 and TGF-β were evaluated in the two groups of fetal rats. The mRNA levels of TNF-α and IL-10 were significantly higher in GA18.5 rats, compared with GA16.5 rats. Furthermore, the mRNA expression levels of TGF-β were significantly reduced in the GA18.5 group (Fig. 9).

 Protein identification and differential protein screening

Compared with group 1, 57 differentially expressed proteins were identified in group 2, of which 37 were upregulated and 20 were downregulated. A comparison of groups 3 and 4 revealed 312 differentially expressed proteins, of which 171 were upregulated and 141 were downregulated. Lastly, compared with group 1,289 differentially expressed proteins were identified in group 3, of which 151 were upregulated and 138 were downregulated. Only 50 differentially expressed proteins and their multiple variations were upregulated or downregulated between all groups (Tables I–IV). The distribution of the differentially expressed proteins among the selected samples is presented as volcano plots (Figs. 10–12).

 Bioinformatics analysis

Gene ontology (GO) enrichment analysis was performed on the differentially expressed proteins, and their properties were generally described as biological process (BP), molecular function (MF) or cellular component (CC). The first 10 GO enrichment results from each group are displayed in Fig. 13. The results demonstrated that 73, 542 and 376 differentially expressed proteins were significantly enriched between groups 1 and 2, 3 and 4 and 3 and 1, respectively. The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified the possible pathway related to the differentially expressed proteins between groups (Fig. 14).

In addition, the interaction network of the differentially expressed proteins that regulate wound repair were analyzed. Examples of the interaction networks of the differentially expressed proteins involved in wound repair are as follows: i) Smad4, Tgf1i1, Ptpn6 and Hmox1 in group 1 and 2; ii) S100a9, Fgg, Anxa1, Fgb, Plg and S100a8 in group 3 and 4; and iii) CD36, S100a9, S100a8, Cd9Fgg, Anxa1, Fgb, Plg and S100a8 in group 3 and 1 (Fig. 15).

 RT-qPCR analysis of possible target protein in cleft lip repair

RNA was extracted from tissue samples with TRIzol® reagent and the quality was checked using gel electrophoresis. Relative mRNA levels were analyzed using RT-qPCR (Fig. 16). The relative mRNA expression levels of Smad4 were significantly higher in group 2, compared with group 1 (P<0.05). Moreover, the relative mRNA expression levels of Fabp5 were significantly lower in groups 4 and 1, compared with group 3 (P<0.05). Additionally, the relative mRNA expression levels of S100a4 were significantly lower in group 4, compared with group 3 (P<0.05). S100a8 and S100a9 were significantly higher in group 3, compared with in groups 1 and 4 (P<0.05).

 Immunofluorescence results

Immunofluorescence staining of Smad4, Fabp5, S100a4, S100a8 and S100a9 was performed on samples from both the GA16.5 and GA18.5 groups 72 h post-surgery. The expression levels of all five proteins increased in GA18.5 compared to GA16.5, and the differences were statistically significant (P<0.05; Fig. 17).

 PRM analysis of differential protein expression

The differences in multiple variations of Smad4 expression were compared between groups 1 and 2. The panel reaction monitoring calculated this difference as 0.557, indicating downregulation in group 1 compared with in group 2 (P=0.043) (Table II). In contrast, no statistically significant differences were observed between groups 3 and 4. The difference in multiple variations of Fabp5 between groups 3 and 4 was calculated as 2.91, indicating upregulation in group 3 compared with in group 4 (P=0.024) (Table III). Additionally, the expression levels of Fabp5 were upregulated (P=0.01) in group 3 compared with in group 1; however, the difference between the variations present in groups 1 and 2 was not statistically significant. The difference in the multiple variations of S100a4 and S100a8 between groups 3 and 4 was calculated as 2.897 and 92.828, respectively, indicating an upregulation of the expression levels of both proteins in group 3 (P=0.001 and P=0.002, respectively) (Table III). Furthermore, the difference in the multiple variations of S100a8 between groups 3 and 1 was 25.933, which indicates upregulation in group 3 (P=0.002) (Table IV). However, the differences were not statistically significant between groups 1 and 2. The difference in the multiple variations of S100a9 was 30.191 and 19.538 between groups 3 and 4 and groups 3 and 1, respectively, suggesting upregulation in group 3 (P=0.0004 and P=0.001, respectively) (Tables III and IV). In contrast, the difference in the multiple variations of S100a9 between groups 1 and 2 was not statistically significant (Tables II–IV).

 Discussion

In recent decades, various animal models of congenital cleft lip have been successfully established through surgical induction (4,20). It has been suggested that intrauterine cleft lip repair can effectively improve this defect and reduce the impact of scars on normal facial development after birth. Thus, it also provides a new way for the effective repair of congenital cleft lips. In the present study, pregnant SD rats were used to establish a fetal rat model of cleft lip wound at two time points, GA16.5 and GA18.5. The different pregnancy models were induced by using two different repair methods of a cleft lip wound of the fetus (21,22). The exact gestational age is particularly important for the results of the repair of cleft lip in the fetal rats. Thus, the use of a rat model provides an added advantage in that the exact time of conception can be replicated, thereby minimizing differences between groups.

The present findings confirmed the hypothesis that fetal rat defects can be regenerated during early pregnancy without scar formation (23). It was also demonstrated that fetal rat defects could not be completely regenerated in late pregnancy and resulted in scarring (24). Furthermore, the expression of pro-inflammatory factors was different between the two groups. However, these observations were only made at one time point (72 h) after constructing cleft lip wounds in fetal rats. Future studies are needed to examine samples collected at different time points following surgery. Another shortcoming of this study entails the lack of comparison between the cleft lip wound repairs of fetal rats at different ages, such as the fetus in the early stages of pregnancy, or in newborn and/or adult rats. Label-free quantitative proteomics were used to examine proteins that play important roles in the postoperative repair process of fetal cleft lip. Protein expression was examined in four groups of samples. In addition, bioinformatics analysis was conducted to identify potential biological markers, providing a theoretical reference and methodological basis for the examination of relevant mechanisms underlying fetal intrauterine scar repair. However, further studies are required to determine whether any one protein or several proteins, plays a key role in wound healing.

Smad4 belongs to the family of Smad proteins and is a common mediator in the signal transduction processes of the TGF-β family (25). TGF-β expression can lead to fibroblast proliferation and ECM deposition (26,27). The present findings indicated that the mRNA and protein expression levels of Smad4 were downregulated in the scar-free repair group.

Furthermore, the mRNA and protein expression levels of Fabp5 were upregulated in the scar formation group. Therefore, it may be hypothesized that Fabp5 could be involved in the fibrosis of the fetal cleft lip wound, which may be mediated by the TGF-β signaling pathway (28–30).

S100a4 is a member of the S100 calcium-binding protein family, and its expression is associated with various non-neoplastic diseases, such as chronic obstructive pulmonary disease and cardiac hypertrophy (31–35). The present study demonstrated that the mRNA and protein expression levels of S100a4 were upregulated in the scar formation group, which may be associated with scar repair of fetal rat cleft lip wounds.

S100a8 is also a member of the S100 calcium-binding protein family (36–42). mRNA and protein expression levels of S100a8 were significantly upregulated in the scar repair group in the present study, indicating a potential role for S100a8 in the process of fetal cleft lip wound healing.

Current reports frequently associate S100a9, a member of the calcium-binding protein family S100, with infectious diseases, immune diseases and tumors, such as non-small cell lung adenocarcinoma (43–45). mRNA and protein expression levels of S100a9 were significantly upregulated in the scar formation group. Therefore, we speculated that S100a9 may play an important role in the process of fetal wound healing. However, whether the reduced expression levels of Fabp5, S100a4, S100a8 and S100a9 in the third trimester of pregnancy would reduce or worsen scar formation remains unclear. Further functional testing and regulatory studies are required to confirm the role of these five differentially expressed proteins in fetal wound repair.

The cleft lip is a very common congenital condition that often leaves life-long scarring. The present study identified five differentially expressed proteins, namely Smad4, Fabp5, S100a4, S100a8 and S100a9, that may be potential biomarkers of the scarless repair process in fetal rat cleft lip wounds. These findings may facilitate the discovery of new clinical targets for the prevention and treatment of scars. However, the role of these proteins in fetal wound repair and potential underlying mechanisms require further examination.

 Acknowledgements

Not applicable.

 Funding

The present study was supported by a grant from the National Science and Basic Resources Survey Special Foundation, China (grant no. 2017FY101204), the Technology Innovation Guide Program of Hunan Province, China (grant no. 2017SK50124), the Science and Technology Major Project of Hunan Province, China (grant no. 2019SK1010) and the Science and Technology Major Project of Hunan Province, China (grant no. 2019SK1015).

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

YY, FH and JZho conceived and designed research. YY and FH performed animal experiments and staining. YY, HL, PJ, FH, JY, KG, SH and JZha performed PCR and label-free quantification PRM. YY, FH, JC, JY, ZC, AW and JZha analyzed data. YY and FH prepared figures. YY drafted the manuscript. FH and JZho edited and revised the manuscript. YY, FH, HL AW, PJ and JZho approved the final version of the manuscript. FH and JZho confirmed the authenticity of all of the raw data. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

This study followed the regulations stipulated by the People's Republic of China regarding the Management of Experimental Animals and was approved by The Animal Experiment Management and Medical Ethics Sub-committee of The Third Xiangya Hospital of Central South University, Hunan, China (approval no. 2014-S168).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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Macroscopic appearance of the cleft lip in the GA16.5 and GA18.5 groups. Arrows indicate the surgical site on the upper-left cleft lip of the fetus. (A-D) Macroscopic appearance of the upper-left lip of a fetus from the GA16.5 group (A) prior to surgery, (B) during surgery, (C) immediately after surgery and (D) 72 h after surgery. (E-H) Macroscopic appearance of the upper-left lip of a fetus from the GA18.5 group (E) prior to surgery, (F) during surgery, (G) immediately after surgery and (H) 72 h after surgery. GA, gestational age.

H&E staining of upper lip tissue 72 h following model establishment in the GA16.5 group. (A) H&E staining of the upper lip tissue. The slight depression in the upper-left lip identifies the location where the cleft lip-like defect was created. The site of the defect is completely epithelialized. Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) New follicular structures can be observed under the epidermis of the upper-left lip. Magnification, ×400. GA, gestational age; H&E, hematoxylin and eosin.

Masson's Trichrome staining 72 h following model establishment in the GA16.5 group. (A) Masson's Trichrome staining of the upper lip tissue. The slight depression in the upper-left lip identifies the location where the cleft lip-like defect was created. Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) The fine network of collagen fibers (stained blue) and new hair follicle structures are visible under the epidermis of the upper-left lip. Magnification, ×400. GA, gestational age.

IHC staining 72 h following model establishment in the GA16.5 group. (A) IHC staining of the upper lip tissue. The slight depression in the upper-left lip identifies the location where the cleft lip-like defect was created. Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) The expression of type-I collagen in the upper-left lip is almost indistinguishable from the upper-right lip. Magnification, ×400. GA, gestational age; IHC, immunohistochemistry.

H&E staining of upper lip tissue 72 h following model establishment in the GA18.5 group. (A) H&E staining of the upper lip tissue. A clear depression in the upper-left lip identifies the location where the cleft lip-like defect was created. The site of the defect is not completely healed. Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) No new hair follicle structures were observed under the epidermis. New capillaries, an increased number of fibroblasts and an increased volume of extracellular matrix can be observed around the wound. Magnification, ×400. GA, gestational age.

Masson's Trichrome staining 72 h after model establishment in the GA18.5 group. (A) Masson's Trichrome staining of the upper lip tissue. A clear depression in the upper-left lip identifies the location where the cleft lip-like defect was created Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) The collagen fibers (stained blue) are visible and no new hair follicle structures are observed under the epidermis of the upper-left lip. Magnification, ×400. GA, gestational age.

IHC staining 72 h after model establishment in the GA18.5 group. (A) IHC staining of the upper lip tissue. A clear depression in the upper-left lip identifies the location where the cleft lip-like defect was created Magnification, ×40. (B) Normal subepidermal structure of the upper-right lip. Magnification, ×400. (C) The expression of type-I collagen in the upper-left lip is higher compared with the upper-right lip. Magnification, ×400. GA, gestational age; IHC, immunohistochemistry.

Immunohistochemical analysis 72 h after model establishment in the GA16.5 and GA18.5 groups. (A and B) Ki67 staining of upper lip tissue from (A) the GA16.5 group and (B) the GA18.5 group. (C and D) CD31staining of upper lip tissue from (C) the GA16.5 group and (D) the GA18.5 group. (E and F) CK10 staining of upper lip tissue from (E) the GA16.5 group and (F) the GA18.5 group. (G) IOD values for Ki67, CD31 and CK10 staining in both groups. *P<0.05; ***P<0.001; ****P<0.0001. GA, gestational age; IOD, integral optical density.

Relative mRNA expression levels of TNF-α, IL-10 and TGF-β. ***P<0.001; ****P<0.0001. GA, gestational age.

Volcano plot of the differentially expressed proteins in group 1 and group 2. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats. GA, gestational age.

Volcano plot of the differentially expressed proteins in group 3 and group 4. Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

Volcano plot of the differentially expressed proteins in group 3 and group 1. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

GO enrichment analysis of the differentially expressed proteins. GO enrichment analysis of the differentially expressed proteins in (A) group 1 and group 2, (B) in group 3 and group 4 and (C) in group 3 and group 1. GA, gestational age; GO, Gene Ontology; p.adjust, adjusted P-value. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

KEGG pathway enrichment analysis of the differentially expressed proteins. KEGG pathway enrichment analysis of the differentially expressed proteins in (A) group 3 and group 4 and (B) in group 3 and group 1. GA, gestational age; KEGG, Kyoto Encyclopedia of Genes and Genomes; p.adjust, adjusted P-value. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

Interaction network of differentially expressed proteins. Interaction network of differentially expressed proteins in (A) group 1 and group 2, (B) 3 and group 4 and (C) 3 and group 1. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

RT-qPCR analysis of Smad4, Fabp5, S100a4, S100a8 and S100a9. RT-qPCR detection and amplification of (A) Smad4, (B) Fabp5, (C) S100a4, (D) S100a8 and (E) S100a9. The dissolution curves and relative mRNA expression levels are shown for each target. RT-qPCR, reverse transcription-quantitative PCR; Fabp5, fatty acid binding protein 5; Smad4, Smad family member 4; S100, S100 calcium binding protein. Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. *P<0.05; **P<0.01. GA, gestational age.

Immunofluorescence analysis Smad4, Fabp5, S100a4, S100a8 and S100a9. (A and B) Smad4 staining of upper lip tissue from (A) the GA16.5 group and (B) the GA18.5 group. (C and D) Fabp5 staining of upper lip tissue from (C) the GA16.5 group and (D) the GA18.5 group. (E and F) S100a4 staining of upper lip tissue from (F) the GA16.5 group and (F) the GA18.5 group. (G and H) S100a8 staining of upper lip tissue from (G) the GA16.5 group and (H) the GA18.5 group. (I and J) S100a9 staining of upper lip tissue from (I) the GA16.5 group and (J) the GA18.5 group. (K) Relative IOD values for Smad4, Fabp5, S100a4, S100a8 and S100a9 staining. *P<0.05; **P<0.01; ***P>0.001. Fabp5, fatty acid binding protein 5; Smad4, Smad family member 4; S100, S100 calcium binding protein; IOD, integral optical density; GA, gestational age.

Comparison of differentially expressed protein numbers between samples.

Sample Differentially expressed proteins, n Upregulated proteins, n Downregulated protein, n
Group 1 vs. Group 2 57 37 20
Group 3 vs. Group 4 312 171 141
Group 3 vs. Group 1 312 151 138

Group 1, upper-left lip without scar repair group at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip normal group at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip scar repair group at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip normal group at 72 h after modeling in GA18.5 rats.

Differential protein expression in group 1 and group 2.

Protein ID Gene name Protein name P-value Fold-change
G3V8R3 Hbz Hemoglobin, zeta 0.004 7.788
B2RYS8 Ndufb8 NADH dehydrogenase 0.000 5.024
Q920P6 Ada Adenosine deaminase 0.038 4.831
O88752 Hbe1 Hemoglobin, epsilon 1 0.003 4.801
Q499N7 Ptpn6 Tyrosine-protein phosphatase non-receptor type 6 0.003 4.700
Q4FZU2 Krt6a Keratin 6A 0.001 4.044
P06762 Hmox1 Heme oxygenase 1 0.011 3.902
Q6IFU9 Krt16 Keratin, type I cytoskeletal 16 0.006 3.613
Q99PD6 Tgfb1i1 Transforming growth factor beta-1-induced transcript 1 protein 0.004 3.590
Q6P7S1 Asah1 Acid ceramidase 0.029 3.167
Q63066 Hbg1 Hemoglobin, gamma A 0.002 3.082
Q10758 Krt8 Keratin, type II cytoskeletal 8 0.004 2.901
Q6AYQ4 Tmem109 Transmembrane protein 109 0.048 2.710
Q9Z2Q7 Stx8 Syntaxin-8 0.035 2.660
G3V9M8 Fam50a Protein fam50a 0.034 2.515
M0R9Y3 Nup43 Nucleoporin 43 0.005 2.461
B2GVB9 Fermt3 Fermitin family homolog 3 0.018 2.425
G3V8H Olfml3 Olfactomedin-like protein 3 precursor 0.045 2.383
D4A531 Polr2i Rna polymerase ii subunit i 0.049 2.290
Q68FS1 Nubp2 Cytosolic Fe-S cluster assembly factor 0.028 2.221
D3ZLS5 Hectd1 Hect domain e3 ubiquitin protein ligase 1 0.033 2.112
D4A0M2 Nxn Nucleoredoxin 0.033 2.056
Q6IE17 Stfa2l2 Stefin-3 0.007 1.969
P27139 Ca2 Carbonic anhydrase 2 0.032 1.957
D3ZF44 LOC684499 Protein LOC684499 0.015 1.940
Q6LDZ3 Ptprc Receptor-type tyrosine-protein phosphatase C 0.007 1.878
Q5XI38 Lcp1 Plastin-2 0.018 1.843
Q5PPG2 Lgmn Legumain precursor 0.015 1.820
P06765 Pf4 Platelet factor 4 0.050 1.708
Q9R1T3 Ctsz Cathepsin Z 0.011 1.707
Q5U1Y2 Rac2 Ras-related C3 botulinum toxin substrate 2 0.021 1.669
Q5U2V4 Plbd1 Phospholipase B-like 1 0.028 1.630
Q9EPX0 Hspb8 Heat shock protein beta-8 0.005 1.603
O35532 Msmo1 Methylsterol monooxygenase 1 0.037 1.592
Q91ZN1 Coro1a Coronin-1A 0.013 1.586
O88201 Clec11a C-type lectin domain family 11 member A 0.025 1.547
Q5U329 Slc4a1 Band 3 anion transport protein 0.027 1.512
Q496Z5 Prph Peripherin 0.041 0.626
P19527 Nefl Neurofilament light polypeptide 0.040 0.608
Q9ESI7 Dcx Neuronal migration protein doublecortin 0.003 0.597
Q6AY98 Ube2e2 Ubiquitin conjugating enzyme e2 e2 0.046 0.577
Q7TSX7 Nr3c1;gr Glucocorticoid receptor 0.026 0.560
O70437 Smad4 Mothers against decapentaplegic homolog 4 0.043 0.557
F1M754 Map4k4 Mitogen-activated protein kinase kinase kinase kinase 4 0.022 0.526
D4A2Z8 Dhx36 Probable ATP-dependent RNA helicase DHX36 0.009 0.522
P31430 Dpep1 Dipeptidase 1 0.010 0.513
Q6AXY8 Dhrs1 Dehydrogenase/reductase SDR family member 1 0.019 0.495
D4A414 Cox15 COX15 homolog 0.031 0.476
D4ABV5 Calm1 Calmodulin 1 0.012 0.473
D3ZRN3 Actbl2 Beta-actin-like protein 2 0.048 0.413
Q8CGS4 Chmp3 Charged multivesicular body protein 3 0.022 0.410
D3ZHA7 Myl6b Myosin light chain 6b 0.011 0.390
P70541 Eif2b3 Translation initiation factor eif-2B subunit gamma 0.002 0.324
D3ZX50 Krtap11-1 Uncharacterized protein 0.037 0.287
D3ZD07 Fmo9 Flavin containing monooxygenase 9 pseudogene 0.007 0.277
Q6IFX1 Krt24 Keratin, type I cytoskeletal 24 0.012 0.050
Q6IG02 Krt2 Keratin, type II cytoskeletal 2 0.008 0.021

Group 1: Upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 2, upper-right lip of fetus at 72 h after modeling in GA16.5 rats. GA, gestational age.

Differential protein expression in group 3 and group 4.

Protein ID Gene name Protein name P-value Fold change
D3ZGE2 Mpo Myeloperoxidase 0.000 377.923
Q62714 Np4 Neutrophil antibiotic peptide NP-4 0.000 231.771
D3ZY96 Ngp Neutrophilic granule protein precursor 0.010 226.724
P50115 S100a8 S100 Calcium Binding Protein A8 0.001 92.828
Q7TP54 Fam65b Protein FAM65B 0.000 77.718
D3ZMI6 Olfm4 Olfactomedin-4 precursor 0.001 63.833
D4A081 Setdb1 Histone-lysine N-methyltransferase SETDB1 0.000 47.032
Q9JI30 Itgam Integrin alpha-M precursor 0.011 39.489
B2RYB8 Itgb2 Integrin beta 2 precursor 0.003 34.443
P50116 S100a9 S100 Calcium Binding Protein A9 0.000 30.191
Q920P6 Ada Adenosine deaminase 0.028 27.433
Q499N7 Ptpn6 Tyrosine-protein phosphatase non-receptor type 6 0.003 23.157
Q9ERL1 Cybb Cytochrome b-245, beta polypeptide 0.002 21.484
Q9JKB7 Gda Guanine deaminase 0.019 20.202
Q5U1Y2 Rac2 Ras-related C3 botulinum toxin substrate 2 0.007 19.291
Q6IFU9 Krt16 Keratin, type I cytoskeletal 16 0.001 13.524
O54854 Klk6 Kallikrein-6 precursor 0.000 11.605
B2GVB9 Fermt3 Fermitin family homolog 3 0.012 11.176
Q5PQW8 Gbp2 Interferon-induced guanylate-binding protein 2 0.019 10.854
Q6LDZ3 Ptprc Receptor-type tyrosine-protein phosphatase C 0.015 10.626
Q4G075 Serpinb1a Leukocyte elastase inhibitor A 0.001 9.051
Q6PDV1 Lyz1 Lysozyme C-1 precursor 0.002 9.049
Q6IE17 Stfa2l2 Stefin-3 0.000 8.930
Q5U2V4 Plbd1 Phospholipase B-like 1 0.003 8.669
Q91ZN1 Coro1a Coronin-1A 0.001 8.199
P14669 Anxa3 Annexin A3 0.008 8.100
Q9R0D6 Tcn2 Transcobalamin-2 precursor 0.014 7.286
Q4QQV6 Lsp1 Lymphocyte specific 1 0.004 6.785
P06768 Rbp2 Retinol-binding protein 2 0.006 6.051
Q5XI38 Lcp1 Plastin-2 0.001 5.841
Q91W30 Akr1b8 Aldo-Keto Reductase Family 1 Member B8 0.001 5.492
Q63015 Csap1 Common salivary protein 1 precursor 0.001 5.454
P31720 C1qa Complement C1q subcomponent subunit A 0.008 5.339
G3V904 Pld4 Phospholipase D4 0.005 4.857
D4ADD7 Glrx5 Glutaredoxin-related protein 5 0.002 4.782
P22985 Xdh Xanthine dehydrogenase/oxidase 0.003 4.221
P06866 Hp Haptoglobin Haptoglobin alpha chain Haptoglobin beta chain 0.002 3.945
B2RYS9 Trmt112 Uncharacterized protein 0.016 3.827
P23640 Rab27a Ras-related protein Rab-27A 0.017 3.774
P06762 Hmox1 Heme oxygenase 1 0.008 3.769
Q9WUQ4 Slpi Secretory leukocyte peptidase inhibitor precursor 0.015 3.710
P07150 Anxa1 Annexin A1 0.003 3.449
D3ZX79 Ly6g6c Lymphocyte antigen 6 complex G6C precursor 0.018 3.236
O88752 Hbe1 Hemoglobin, epsilon 1 0.029 3.211
Q9R1T3 Ctsz Cathepsin Z 0.002 3.195
D3ZJH9 Me2 NAD-dependent malic enzyme, mitochondrial 0.034 2.926
P05942 S100a4 S100 Calcium Binding Protein A4 0.002 2.897
Q5XJW6 Cfh Complement factor H precursor 0.008 2.891
O54892 Hk2 Hexokinase-2 0.007 2.876
Q6P7D4 Cyp20a1 Cytochrome P450 20A1 0.013 0.490
D3ZWC6 Sntb1 beta-1-syntrophin 0.025 0.490
Q62997 Gfra1 GDNF family receptor alpha-1 0.043 0.488
P02600 Myl1 Myosin light chain 1/3 0.011 0.482
O35878 Hspb2 Heat shock protein beta-2 0.005 0.481
P17209 Myl4 Myosin light chain 4 0.004 0.475
D4A8H3 Uba6 Ubiquitin-like modifier-activating enzyme 6 0.031 0.471
A1L1K3 Anapc5 Anaphase-promoting complex subunit 5 0.046 0.470
D3ZTW9 Exog Nuclease EXOG 0.028 0.467
D4A3D2 Smyd1 SET and MYND domain-containing protein 1 0.004 0.465
P04466 Mylpf Myosin regulatory light chain 2 0.009 0.464
P12847 Myh3 Myosin-3 0.007 0.461
P13413 Tnni1 Troponin I 0.001 0.460
D4A4Y2 Hsd17b14 17-beta-hydroxysteroid dehydrogenase 14 0.033 0.455
P23928 Cryab Alpha-crystallin B chain 0.020 0.454
Q7TNB2 Tnnt1 Troponin T 0.002 0.451
D3ZCD7 Tp53rk TP53-regulating kinase 0.004 0.450
P00564 Ckm Creatine kinase M-type 0.037 0.450
Q80W59 Hrc Sarcoplasmic reticulum histidine-rich calcium-binding protein precursor 0.019 0.445
P50463 Csrp3 Cysteine and glycine-rich protein 3 0.013 0.444
Q5XIG1 Ldb3 Ldb3 protein 0.017 0.443
D3ZUB7 Anapc4 Anaphase-promoting complex subunit 4 0.030 0.442
Q64578 Atp2a1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 0.032 0.442
Q6P792 Fhl1 Four and a half LIM domains protein 1 0.013 0.431
Q8K4F2 Alox15b Arachidonate 15-lipoxygenase B 0.025 0.428
M0RBL8 Tceal6 Protein LOC679974 0.003 0.427
P51868 Casq2 Calsequestrin-2 precursor 0.008 0.425
B4F789 Apobec2 Probable C->U-editing enzyme APOBEC-2 0.008 0.421
P16290 Pgam2 Phosphoglycerate mutase 2 0.014 0.418
Q9Z2J4 Nexn Nexilin 0.002 0.412
Q9QYU4 Crym Thiomorpholine-carboxylate dehydrogenase 0.017 0.411
D3ZUQ0 Rilpl1 RILP-like protein 1 0.006 0.409
D4A2H6 Rbfox3 Fox-1 homolog C 0.037 0.408
D3ZVM5 Hspa12b Heat shock 70 kDa protein 12B 0.038 0.406
O54747 Pold1 DNA polymerase delta catalytic subunit 0.001 0.403
P52481 Cap2 Adenylyl cyclase-associated protein 2 0.007 0.396
Q63544 Sncg Gamma-synuclein 0.004 0.381
Q496Z5 Prph Peripherin 0.001 0.376
P07483 Fabp3 Fatty acid-binding protein, heart 0.011 0.357
P23565 Ina Alpha-internexin 0.005 0.332
D4ADS4 Mgst3 Microsomal glutathione S-transferase 3 0.024 0.328
P19527 Nefl Neurofilament light polypeptide 0.006 0.326
P12839 Nefm Neurofilament medium polypeptide 0.004 0.326
B2RZ77 Dpt Dermatopontin precursor 0.024 0.320
Q6AYG3 Prune Prune homolog 0.017 0.320
G3V7K1 Myom2 Myomesin 2 0.025 0.299
G3V6V5 Atp1b4 Protein ATP1B4 0.005 0.272
Q9Z2Z8 Dhcr7 7-dehydrocholesterol reductase 0.000 0.270
P19633 Casq1 Calsequestrin-1 0.021 0.201
D3ZX18 Myoz2 Myozenin-2 0.001 0.198
Q812D3 Ppil3 Peptidyl-prolyl cis-trans isomerase-like 3 0.000 0.179

Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats; Group 4, upper-right lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.

Differential protein expression in group 3 and group 1.

Protein ID Gene name Protein name P-value Fold change
Q6jhy3 Smgc Submandibular gland protein c precursor 0.001 753.286
D3zge2 Mpo Myeloperoxidase precursor 0.000 271.832
Q62714 Np4 Neutrophil antibiotic peptide np-4 0.000 244.647
D3zmi6 Olfm4 Olfactomedin-4 precursor 0.000 189.639
D3zy96 Ngp Neutrophilic granule protein precursor 0.014 130.459
B2ryb8 Itgb2 Integrin beta 2 precursor 0.002 44.874
G3v8l7 Itgam Integrin alpha-m precursor 0.012 32.024
Q6ig02 Krt2 Keratin, type ii cytoskeletal 2 epidermal 0.011 26.577
Q63015 Csap1 Common salivary protein 1 precursor 0.003 26.571
P50115 S100a8 S100 calcium binding protein a8 0.002 25.933
P50116 S100a9 S100 calcium binding protein a9 0.001 19.538
Q9jkb7 Gda Guanine deaminase 0.023 19.076
D3zd07 Fmo9 Flavin containing monooxygenase 9 pseudogene 0.003 17.428
Q5u1y2 Rac2 Ras-related c3 botulinum toxin substrate 2 0.011 12.859
O54854 Klk6 Kallikrein-6 precursor 0.000 10.614
Q5u2v4 Plbd1 Phospholipase b-like 1 0.002 10.508
Q6pdv1 Lyz1 Lysozyme c-1 precursor 0.002 8.631
Q4g075 Serpinb1a Leukocyte elastase inhibitor a 0.001 8.260
Q6ldz3 Ptprc Receptor-type tyrosine-protein phosphatase c 0.023 8.077
Q9wuq4 Slpi Secretory leukocyte peptidase inhibitor precursor 0.007 7.093
Q9erl1 Cybb Cytochrome b-245, beta polypeptide 0.009 7.056
E0a3n4 Serpina3n Serine protease inhibitor a3n 0.013 7.049
G3v6k1 Tcn2 Transcobalamin-2 precursor 0.017 6.577
P14669 Anxa3 Annexin a3 0.010 5.005
P22985 Xdh Xanthine dehydrogenase/oxidase 0.003 4.802
Q91zn1 Coro1a Coronin-1a 0.001 4.694
P05982 Nqo1 Nad(p)h quinone dehydrogenase 1 0.001 4.334
P23640 Rab27a Ras-related protein rab-27a 0.019 3.830
Q6ifu9 Krt16 Keratin, type i cytoskeletal 16 0.001 3.797
Q62894 Ecm1 Extracellular matrix protein 1 0.039 3.655
Q5×i38 Lcp1 Plastin-2 0.003 3.469
P07150 Anxa1 Annexin a1 0.003 3.446
Q78zr5 Hopx Homeodomain-only protein 0.002 3.376
P01015 Agt Angiotensinogen angiotensin-1 angiotensin-2 angiotensin-3 0.010 3.300
Q6axy8 Dhrs1 Dehydrogenase/reductase sdr family member 1 0.006 3.287
Q91w30 Akr1b8 Aldose reductase-related protein 2 0.007 3.244
P32755 Hpd 4-hydroxyphenylpyruvate dioxygenase 0.024 3.149
Q499n7 Ptpn6 Tyrosine-protein phosphatase non-receptor type 6 0.038 3.025
G3v755 Sprr1a Cornifin-a 0.002 3.006
Q5×fv4 Fabp4 Fatty acid-binding protein, adipocyte 0.024 2.910
B1wbv8 Pld4 Phospholipase d4 0.011 2.909
D3zpf9 Serpinb12 Serpin b12 0.038 2.880
Q4qqv6 Lsp1 Lymphocyte specific 1 0.001 2.665
P29524 Serpinb2 Plasminogen activator inhibitor 2 type a 0.001 2.653
O55162 Lypd3 Ly6/plaur domain-containing protein 3 0.004 2.551
D4a5u3 Tgm3 Protein-glutamine gamma-glutamyltransferase e protein 0.033 2.547
D3zsh7 Col17a1 Collagen alpha-1(xvii) chain 0.002 2.485
D3zjk2 Serpinb3a Protein serpinb3a 0.038 2.445
Q6ie17 Stfa2l2 Stefin-3 0.005 2.439
Q5u206 Calml3 Calmodulin-like protein 3 0.013 2.429
Q4v885 Colec12 Collectin-12 0.017 0.547
D3zqi1 Gpx7 Glutathione peroxidase 7 precursor 0.036 0.541
D3z9m5 Fkbp7 Peptidyl-prolyl cis-trans isomerase fkbp7 precursor 0.014 0.530
O88201 Clec11a C-type lectin domain family 11 member a 0.047 0.529
D3zrd3 Pde6d Retinal rod rhodopsin-sensitive cgmp 3′,5′-cyclic phosphodiesterase subunit delta 0.009 0.528
P21807 Prph Peripherin 0.008 0.527
G3v6m4 Capn6 Calpain-6 0.024 0.514
D3zg88 Sssca1 Sjogren syndrome/scleroderma autoantigen 1 homolog 0.034 0.502
Q2eja0 Yap1 Yorkie homolog 0.002 0.494
Q3b7u1 Maged2 Melanoma-associated antigen d2 0.003 0.492
O35276 Nrp2 Neuropilin-2 0.015 0.491
D3zun5 Pofut2 Gdp-fucose protein o-fucosyltransferase 2 precursor 0.018 0.490
P70583; d4a6v3 Dut Deoxyuridine 5′-triphosphate nucleotidohydrolase 0.021 0.489
P19527 Nefl Neurofilament light polypeptide 0.026 0.479
M0r649 Exoc4 Exocyst complex component 4 0.031 0.466
Q99pd6 Tgfb1i1 Transforming growth factor beta-1-induced transcript 1 protein 0.048 0.466
P54001 P4ha1 Prolyl 4-hydroxylase subunit alpha-1 0.019 0.461
D3zt07 Sept5 Septin-5 0.046 0.452
P12839; g3v7s2 Nefm Neurofilament medium polypeptide 0.020 0.444
B5df50 Galnt2 Polypeptide n-acetylgalactosaminyltransferase 2 0.038 0.436
D3zuq0 Rilpl1 Rilp-like protein 1 0.041 0.418
D4a8h3 Uba6 Ubiquitin-like modifier-activating enzyme 6 0.024 0.415
D4a9u4 Eln Elastin 0.041 0.409
D4ad75 Dpy19l1 Protein dpy-19 homolog 1 0.014 0.408
Q6p7d4 Cyp20a1 Cytochrome p450 20a1 0.007 0.406
Q5×i28 Raver1 Ribonucleoprotein ptb-binding 1 0.045 0.398
P09117 Aldoc Fructose-bisphosphate aldolase c 0.004 0.396
D3zct5 Pald1 Paladin 0.004 0.395
A1l1k3 Anapc5 Anaphase-promoting complex subunit 5 0.028 0.392
P62966 Crabp1 Cellular retinoic acid-binding protein 1 0.016 0.384
Q569b7 Rwdd4 Rwd domain-containing protein 4 0.040 0.384
Q5hze4 Mri1 Methylthioribose-1-phosphate isomerase 0.011 0.376
F1lqz3 Kif3a Kinesin family member 3a 0.011 0.376
O88752 Hbe1 Hemoglobin, epsilon 1 0.033 0.375
Q5u1z0 Rab3gap2 Rab3 gtpase-activating protein non-catalytic subunit 0.003 0.373
A1a5r1 Rbfox1 Fox-1 homolog c 0.033 0.366
D4a845 Rpa3 Replication protein a 14 kda subunit 0.021 0.366
D3zwc6 Sntb1 Beta-1-syntrophin 0.003 0.365
G3v8m1 Pold1 Dna polymerase delta catalytic subunit 0.003 0.353
P23565 Ina Alpha-internexin 0.039 0.352
Q4klk9 Ssu72 Rna polymerase ii subunit a c-terminal domain phosphatase ssu72 0.025 0.349
F1mah6 Cdh11 Cadherin 11 0.007 0.326
Q6ayg3 Prune Prune homolog (drosophila) (ec:3.6.1.1) 0.001 0.278
P04638 Apoa2 Apolipoprotein a-ii 0.010 0.270
Q9z2z8 Dhcr7 7-dehydrocholesterol reductase 0.000 0.221
Q10758 Krt8 Keratin, type ii cytoskeletal 8 0.002 0.214
Q812d3 Ppil3 Peptidyl-prolyl cis-trans isomerase-like 3 0.000 0.188
G3v8r3 Hbz Hemoglobin, zeta 0.004 0.128
B5dfl9 Sestd1 Sec14 and spectrin domains 1 0.000 0.065
Q9eph1 A1bg Alpha-1b-glycoprotein 0.016 0.033

Group 1, upper-left lip of fetus at 72 h after modeling in GA16.5 rats; Group 3, upper-left lip of fetus at 72 h after modeling in GA18.5 rats. GA, gestational age.