Genome-Wide Analysis of the KLF Gene Family in Chicken: Characterization and Expression Profile

Simple Summary Broilers provide nutrition for people all around the world, and their muscle and fat contribute major roles. However, little is known about the relationships between the kruppel-like factor (KLF) and the development of muscle and fat. In recent years, studies have shown that KLFs are involved in many physiological processes such as cell development, adipocyte differentiation and neurodevelopment. In our study, we aim to learn the functions KLFs play in the muscle and fat development of chickens. Various bioinformatics analyses were used to illustrate the genomic information of KLFs. A qPCR was performed to show the relative expression level of KLFs. In addition, we collected RNA-seq data and we found that KLFs have different expression levels in the same tissues at different points in time. This study helps explore the regulation mechanism of KLFs in skeletal muscle and fat, and provides a theoretical basis for broiler breeding. Abstract The kruppel-like factor (KLF) gene family is a group of transcription factors containing highly conserved zinc-finger motifs, which play a crucial role in cell proliferation and differentiation. Chicken has been widely used as a model animal for analyzing gene function, however, little is known about the function of the KLF gene family in chickens. In this study, we performed genome-wide studies of chicken KLF genes and analyzed their biological and expression characteristics. We identified 13 KLF genes from chickens. Our phylogenetic, motif, and conserved domain analyses indicate that the KLF gene family has remained conserved through evolution. Synteny analysis showed the collinear relationship among KLFs, which indicated that they had related biomolecular functions. Interaction network analysis revealed that KLFs worked with 20 genes in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that KLF2 was involved in Apelin and Forkhead Box O (FOXO) signaling pathways. Moreover, qPCR showed that 13 KLF genes were expressed in the nine selected tissues and displayed various gene expression patterns in chickens. RNA-seq showed that KLF3 and KLF10 genes were differentially expressed in the normal and high-fat diet fed groups, and KLF4, KLF5, KLF6, KLF7, KLF9, KLF12, and KLF13 genes were differentially expressed between undifferentiated and differentiated chicken preadipocytes. Besides, RNA-seq also showed that KLF genes displayed different expression patterns in muscle at 11 and 16 embryonic days old, and in 1-day-old chickens. These results indicated that the KLF genes were involved in the development of muscle and fat in chickens. Our findings provide some valuable reference points for the subsequent study of the function of KLF genes.


Introduction
Chicken meat is a good source of protein, with lower fat and cholesterol than other kinds of meats [1]. Ensuring the stability of chicken production is crucial in promoting socio-economic development in the face of population growth. Biological and pathological processes and other genetic factors are the main limiting factors affecting chicken growth construct multiple alignment models using hidden Markov model software (HMM) to identify putative KLF genes in chickens using the presence of the zf-H2C2 domain [31].

Protein Alignment, Phylogenetic Analysis, and Chromosome Location Analysis of KLF Genes
To understand the evolutionary relationship of the KLF genes from these four species, multiple sequence alignments of identified KLF genes were performed using MUSCLE analysis [32]. The neighbor-joining (NJ) tree was constructed by Molecular Evolutionary Genetics Analysis (MEGA) 7.0 software with pairwise deletion of bootstrap value 1000 [33]. Conserved domains of KLF proteins were identified using CDD method in Batch software [34]. The SMART software also detected those domains found by a HMM model to confirm the final results [35]. The chromosomal locations of these KLF genes were derived from the genome annotation files downloaded from the Ensembl database [36]. Furthermore, the chromosome location of the KLF genes was displayed by TBtools [37].

Motif Identification and Conserved Domain Analysis of KLF Genes
The motifs of the KLF protein domain were identified using the Multiple EM (Expectation Maximization) for Motif Elicitation (MEME) program using the default parameters [38]. The number of motifs was set to 15. The minimum motif and maximum motif lengths were 6 and 50, respectively. The conserved domains of KLFs were searched in the Conserved Domain Database and displayed with TBtools [37].

Synteny Analysis and Interaction Network Analysis of KLF Genes in Chickens
The syntenic relationships between the KLF genes and gene density of KLF genes were analyzed by using TBtools. The interaction networks of chicken KLF genes were identified using the STRING database and the predicted interaction network was optimized by Cytoscape software [39,40].

GO Enrichment Analysis and KEGG Pathway Analysis of KLF Genes in Chicken
Gene Ontology enrichment analysis was used to identify the putative biological processes, cellular components, and molecular functions of KLF genes in chickens [41]. KEGG enrichment analysis was performed by KOBAS software to identify the functions of KLF genes in signaling pathways [42].

RNA-seq Results Analysis of the G. gallus KLF Genes
The first RNA-Seq data of 40 samples of testis, liver, lung, brain, kidney, intestine, muscle, and heart were obtained from the GEO DataSets and its GEO accession was GSE133401. The second RNA-Seq data derived from 12 samples of livers and abdominal fat in dwarf broilers were also obtained from the GEO DataSets and its GEO accession was GSE129840. The broilers began to be fed with a normal or a high-fat diet at 1 week old and they were executed to get livers and abdominal fat for extracting total RNAs at 8 weeks old [43]. The heatmap was made based on the sequencing data by R software [44].
The RNA-seq data of thigh muscle in Xinghua chicken was collected from GEO dataset and its GEO accession was GSE91060. Chicken leg muscles of three different developmental stages (11-day-old embryos, 16-day-old embryos, and 1-day post-hatch) were used to assess lncRNA and mRNA expression during chicken skeletal muscle development [45]. The sequencing data of preadipocytes that had not differentiated and six-day differentiated preadipocytes were obtained with this study.

Animals, Tissue Collections, RNA Extraction, and Quantitative Real-Time PCR (qPCR)
Heying Black chickens used in this study were provided by Jiangsu Heying Poultry Group Co., Ltd. (Sheyang, China). After artificial insemination, the eggs were collected and hatched at 37 • C and 60% humidity. The experiment group contained four male chickens and they were euthanized at 18 embryonic days (the sex was determined by the development of the gonad). Thigh muscle, pectoral muscle, heart, lung, glandular stomach, hypothalamus, liver, spleen and abdominal fat were collected. Subsequently, these tissues were snap-frozen in liquid nitrogen, and stored at −80 • C for RNA extraction.
Total RNA samples were extracted with TRIzol. The quality, concentration, and integrity of RNA samples were tested by spectrophotometer (Thermo, Waltham, MA, USA). Then 1 ug RNA of each sample was reverse-transcribed to cDNA by using HiScript QRT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The forward primers and reverse primers of KLFs were designed by Primer Premier 5.0 (Table S1). These primers were synthesized by Sangon (Shanghai, China). The qPCR experiments were conducted on Applied Biosystems 7500 (ABI, Los Angeles, California, USA) using SYBR qPCR Master Mix (Vazyme, Nanjing, China), and β-actin was used as housekeeping gene [46], with three technical repetitions for each sample. The 2 −∆∆CT method was used to calculate the relative expression of KLFs [47].

Statistics Analysis
Statistical analysis was performed by using SPSS18.0 software (SPSS Inc., Chicago, IL, USA). A one-way ANOVA was used for multiple-group comparison analysis. Duncan's multiple range test was used to determine significance. Unpaired Student's t-test was used for a two-group comparison analysis. The data were considered statistically significant when p less than 0.05.

Identification and Phylogenetic Analysis of KLF Genes
Based on the presence of the zf-H2C2 domain, we obtained 56 KLF genes in four species, including 18 H. sapiens genes, 8 M. musculus genes, 17 S. scrofa genes, and 13 G. gallus genes, respectively. To explore the evolutionary relationship among the members of the KLF families, an NJ tree based on protein sequences of these 56 genes from four species was constructed ( Figure 1). Based on the alignment of the sequences and the phylogenetic analysis, 56 genes were divided into three groups, named groups I to III. Group I contained 12 KLFs, group II contained 6 KLFs, and group III contained 38 KLFs. We saw from the phylogenetic tree that Ss-KLF13, Hs-KLF13, Gg-KLF13, Gg-KLF9, Ss-KLF9, Hs-KLF9, Ss-KLF16, Mm-KLF16, Hs-KLF16, Hs-KLF14, Ss-KLF14, and Mm-KLF14 are clustered into Group I. Gg-KLF11, Ss-KLF11, Hs-KLF11, Gg-KLF10, Ss-KLF10, and Hs-KLF10 are clustered into Group II, and the remaining 38 KLFs are clustered into Group III.

Chromosomal Locations of G. gallus KLF Genes
The 13 KLF genes were distributed unevenly on ten G. gallus chromosomes ( Figure 2). The accession numbers of the gene sequence, protein sequence, and coding sequence of the 13 KLF genes and their genomic locations were listed in Table 1. Chromosome 1 contained two genes, Gg-KLF5 and Gg-KLF12. Chromosome 2 also had two genes, Gg-KLF6 and Gg-KLF10, but these two genes were at different positions on the same chromosome. Like chromosome 2, chromosome 4 contained two genes, Gg-KLF3 and Gg-KLF8. The remaining seven genes were distributed on the other seven chromosomes, meaning each had one KLF gene. Gg-KLF11, Gg-KLF7, Gg-KLF13, Gg-KLF4, Gg-KLF2, Gg-KLF15, and Gg-KLF9 were found on chromosome 3, chromosome 7, chromosome 10, chromosome 8, chromosome 28, chromosome 12, and chromosome Z, respectively.

Motif Identification and Conserved Domain Analysis
The phylogenetic analysis ( Figure 3A) was combined with gene structures and motif analysis to indicate the relationship among KLF genes of H. sapiens, M. musculus, S. scrofa, and G. gallus. Using the HMMER program, we discovered that the KLF genes have 15 motifs. Motif 1 and motif 2 were present in all 56 KLF genes ( Figure 3B). The most closely related genes in the same subfamilies shared common motif compositions, which may be indicative of similar functions. The conserved domain analysis illustrated that 27 conserved domains could be found in these 56 genes ( Figure 3C). The genes clustered in the same group shared similar conserved domains. The sequences of those 56 proteins were used to construct a neighbor-joining (NJ) tree. The tree was divided into three groups (I-III). "H. sapiens" was abbreviated to "Hs". "M. musculus" was abbreviated to "Mm". "S. scrofa" was abbreviated to "Ss". "G. gallus" was abbreviated to "Gg".

Chromosomal Locations of G. gallus KLF Genes
The 13 KLF genes were distributed unevenly on ten G. gallus chromosomes ( Figure  2). The accession numbers of the gene sequence, protein sequence, and coding sequence of the 13 KLF genes and their genomic locations were listed in Table 1. Chromosome 1 contained two genes, Gg-KLF5 and Gg-KLF12. Chromosome 2 also had two genes, Gg-KLF6 and Gg-KLF10, but these two genes were at different positions on the same chromosome. Like chromosome 2, chromosome 4 contained two genes, Gg-KLF3 and Gg-KLF8. The remaining seven genes were distributed on the other seven chromosomes, meaning each had one KLF gene. Gg-KLF11, Gg-KLF7, Gg-KLF13, Gg-KLF4, Gg-KLF2, Gg-KLF15, and Gg-KLF9 were found on chromosome 3, chromosome 7, chromosome 10, chromosome 8, chromosome 28, chromosome 12, and chromosome Z, respectively. The sequences of those 56 proteins were used to construct a neighbor-joining (NJ) tree. The tree was divided into three groups (I-III). "H. sapiens" was abbreviated to "Hs". "M. musculus" was abbreviated to "Mm". "S. scrofa" was abbreviated to "Ss". "G. gallus" was abbreviated to "Gg".

Motif Identification and Conserved Domain Analysis
The phylogenetic analysis ( Figure 3A) was combined with gene structures and motif analysis to indicate the relationship among KLF genes of H. sapiens, M. musculus, S. scrofa, and G. gallus. Using the HMMER program, we discovered that the KLF genes have 15 motifs. Motif 1 and motif 2 were present in all 56 KLF genes ( Figure 3B). The most closely related genes in the same subfamilies shared common motif compositions, which may be indicative of similar functions. The conserved domain analysis illustrated that 27 conserved domains could be found in these 56 genes ( Figure 3C). The genes clustered in the same group shared similar conserved domains.

Synteny Analysis and Interaction Network Analysis
In Figure 4A, the middle and outermost circles showed low gene density of all 13 KLFs in chicken. According to curves of different colors, we found that KLF2 had a collinear relationship with KLF13. KLF3 and KLF8 had collinear relationships with KLF5 and

Synteny Analysis and Interaction Network Analysis
In Figure 4A, the middle and outermost circles showed low gene density of all 13 KLFs in chicken. According to curves of different colors, we found that KLF2 had a collinear relationship with KLF13. KLF3 and KLF8 had collinear relationships with KLF5 and KLF12. KLF6 had a collinear relationship with KLF7. KLF10 had a collinear relationship with KLF11. Other KLFs didn't have collinear relationships.   Figure 5A shows nine KLF proteins located at the nucleoplasm, nuclear lumen, membrane-enclosed lumen, organelle lumen, intracellular organelle lumen, and nuclear part, respectively, which were the top six GO Enrichment categories (Table S2). Figure 5B illustrates that 11 KLFs took part in biological processes like the regulation of transcription by RNA polymerase II, transcription by RNA polymerase II, regulation of gene expression, and so on (Table S3). According to the KEGG pathway analysis, we found only KLF2 was involved in Apelin signaling pathway and FOXO signaling pathway ( Figure S1) [48]. Using KEGG, the 12 putative KLF proteins were not assigned to any signaling pathway. To better understand the biological function and the regulatory network of KLFs, their protein-protein interactions (PPI) were predicted ( Figure 4B). The results showed that 11 KLFs had putative interaction relationships and 20 corresponding functional genes were found. Figure 5A shows nine KLF proteins located at the nucleoplasm, nuclear lumen, membrane-enclosed lumen, organelle lumen, intracellular organelle lumen, and nuclear part, respectively, which were the top six GO Enrichment categories (Table S2). Figure 5B illustrates that 11 KLFs took part in biological processes like the regulation of transcription by RNA polymerase II, transcription by RNA polymerase II, regulation of gene expression, and so on (Table S3). According to the KEGG pathway analysis, we found only KLF2 was involved in Apelin signaling pathway and FOXO signaling pathway ( Figure S1) [48]. Using KEGG, the 12 putative KLF proteins were not assigned to any signaling pathway.

Expression Profile Analysis of G. gallus KLF Genes
The KLF genes can be classified into four groups ( Figure 6A) according to their differential expression patterns in tissues. Moreover, the eight chicken tissue types can also be clustered into eight main clades based on the expression patterns of all 13 genes. It could be seen from the expression profile that KLF2, KLF3, KLF4, KLF6, KLF7, and KLF13 were expressed highly in the lungs. Further, KLF9 was expressed highly in muscle. In Figure 6B, the expression levels of KLF genes were significantly higher in fat than liver, except for KLF15. The expression level of KLF3 was significantly higher in the normal diet-fed group than the high-fat diet-fed group, while the expression level of KLF10 was significantly lower in the normal diet-fed group than the high-fat diet-fed group. Figure 5A shows nine KLF proteins located at the nucleoplasm, nuclear lumen, membrane-enclosed lumen, organelle lumen, intracellular organelle lumen, and nuclear part, respectively, which were the top six GO Enrichment categories (Table S2). Figure 5B illustrates that 11 KLFs took part in biological processes like the regulation of transcription by RNA polymerase II, transcription by RNA polymerase II, regulation of gene expression, and so on (Table S3). According to the KEGG pathway analysis, we found only KLF2 was involved in Apelin signaling pathway and FOXO signaling pathway ( Figure S1) [48]. Using KEGG, the 12 putative KLF proteins were not assigned to any signaling pathway.

Expression Profile Analysis of G. gallus KLF Genes
The KLF genes can be classified into four groups ( Figure 6A) according to their differential expression patterns in tissues. Moreover, the eight chicken tissue types can also be clustered into eight main clades based on the expression patterns of all 13 genes. It could be seen from the expression profile that KLF2, KLF3, KLF4, KLF6, KLF7, and KLF13 were expressed highly in the lungs. Further, KLF9 was expressed highly in muscle. In Figure 7B, the expression levels of KLF genes were significantly higher in fat than liver, except for KLF15. The expression level of KLF3 was significantly higher in the normal diet-fed group than the high-fat diet-fed group, while the expression level of KLF10 was significantly lower in the normal diet-fed group than the high-fat diet-fed group. tissues from five adult roosters: testis, liver, lung, brain, kidney, intestine, muscle, and heart. Colors red, white, blue represent high expression, medium expression and low expression levels, respectively. (B) KLF genes in livers and abdominal fats between normal-and high-fat diet-fed dwarf broilers. The colors yellow, white, and blue represent high expression, medium expression, and low expression levels, respectively. tissues from five adult roosters: testis, liver, lung, brain, kidney, intestine, muscle, and heart. Colors red, white, blue represent high expression, medium expression and low expression levels, respectively. (B) KLF genes in livers and abdominal fats between normal-and high-fat diet-fed dwarf broilers. The colors yellow, white, and blue represent high expression, medium expression, and low expression levels, respectively.

QPCR Results of the G. gallus KLF Genes
QPCR data was normalized with the same and unique normalizer gene, β-actin. The qPCR results showed that KLF genes had quite different expression patterns (Figure 7). The expression levels of KLF2, KLF5, KLF6, KLF10 and KLF15 were highest in thigh muscle, pectoral muscle and abdominal fat. KLF4, KLF8, and KLF13 were most highly expressed in thigh muscle and pectoral muscle. We also discovered that the expression level of KLF3 was highest in liver, followed by heart, pectoral muscle, lungs, thigh muscle, abdominal fat

QPCR Results of the G. gallus KLF Genes
QPCR data was normalized with the same and unique normalizer gene, β-actin. The qPCR results showed that KLF genes had quite different expression patterns (Figure 7). The expression levels of KLF2, KLF5, KLF6, KLF10 and KLF15 were highest in thigh muscle, pectoral muscle and abdominal fat. KLF4, KLF8, and KLF13 were most highly expressed in thigh muscle and pectoral muscle. We also discovered that the expression level of KLF3 was highest in liver, followed by heart, pectoral muscle, lungs, thigh muscle, abdominal fat and other organs. The expression levels of KLF7 were highest in the hearts and lungs, while the expression level of KLF9 was highest in the hypothalamus. KLF11 expressed highest in pectoral muscle but much lower in other organs. Like KLF3, the expression level of KLF12 was highest in liver.

RNA-seq Result Analysis of the G. gallus KLF Genes
The expression level of KLF2, KLF3, KLF9, KLF10, KLF11, KLF13 and KLF15 increased in samples from 11 embryonic-day-old to 1-day-old chicken ( Figure 8A). The expression level of KLF4 decreased between samples from 11 embryonic-day-old to 16 embryonicday-old chicken, but it increased quickly from 16-embryonic-day-old to 1-day-old

RNA-seq Result Analysis of the G. gallus KLF Genes
The expression level of KLF2, KLF3, KLF9, KLF10, KLF11, KLF13 and KLF15 increased in samples from 11 embryonic-day-old to 1-day-old chicken ( Figure 8A). The expression level of KLF4 decreased between samples from 11 embryonic-day-old to 16 embryonicday-old chicken, but it increased quickly from 16-embryonic-day-old to 1-day-old chicken. Contrary to KLF4, the expression levels of KLF5 and KLF6 rose between samples from 11 embryonic-day-old to 16 embryonic-day-old chickens, but it decreased quickly from 16-embryonic-day-old to 1-day-old chickens. The expression levels of KLF7 and KLF12 decreased between samples from 11 embryonic-day-old to 1-day-old chickens. As to KLF8, it was not included in the RNA-seq data and the expression of KLF8 was absent. As Figure 8B showed, the expression levels of KLF9 and KLF12 increased significantly in differentiated preadipocytes, while the expression levels of KLF4, KLF5, KLF6, KLF7, and KLF13 all decreased significantly after preadipocyte differentiation. from 16-embryonic-day-old to 1-day-old chickens. The expression levels of KLF7 and KLF12 decreased between samples from 11 embryonic-day-old to 1-day-old chickens. As to KLF8, it was not included in the RNA-seq data and the expression of KLF8 was absent. As Figure 8B showed, the expression levels of KLF9 and KLF12 increased significantly in differentiated preadipocytes, while the expression levels of KLF4, KLF5, KLF6, KLF7, and KLF13 all decreased significantly after preadipocyte differentiation. KLF mRNA relative expression levels in preadipocytes which had not differentiated and six-daydifferentiated preadipocytes. P0 referred to preadipocytes which had not differentiated. P6 referred to six-day-differentiated preadipocytes. "ns" represents no significance. "*" represents p < 0.05.

Discussion
It is known that KLFs are a family of transcription factors characterized by zinc-finger structures at the C-terminal end, and they play critical roles in cell proliferation, differentiation, and migration [49]. Using BLAST and HMM, we identified 56 KLF genes from four species and then a total of 13 KLF genes with specific conserved-domain zf-H2C2 genes were identified in G. gallus. The NJ tree was constructed with 56 gene sequences to compare chicken KLF genes with sequences from the three other species. The results demonstrated that the 56 KLF genes were divided into three groups. Comparative analysis suggested that chickens, mice, and humans have similar numbers of gene families [50]. Gene structure is an important factor in the evolution of gene families [51]. We found that the KLF gene structure of chickens is quite similar to that of humans, pigs, and mice. Motif analysis revealed that KLFs clustered in groups based on the similarity of their subdomains. The results of conserved-domain analysis showed that KLF genes are relatively conserved among these species. Among 20 genes predicted by interaction network analysis, NCOA3 was found to participate in regulating porcine skeletal muscle satellite cell proliferation [52]. Furthermore, differential expression of NCOA3 was proven to be regulated by miR-17-5p in pig intramuscular and subcutaneous adipose tissues [53]. (B) KLF mRNA relative expression levels in preadipocytes which had not differentiated and six-daydifferentiated preadipocytes. P0 referred to preadipocytes which had not differentiated. P6 referred to six-day-differentiated preadipocytes. "ns" represents no significance. "*" represents p < 0.05.

Discussion
It is known that KLFs are a family of transcription factors characterized by zincfinger structures at the C-terminal end, and they play critical roles in cell proliferation, differentiation, and migration [49]. Using BLAST and HMM, we identified 56 KLF genes from four species and then a total of 13 KLF genes with specific conserved-domain zf-H2C2 genes were identified in G. gallus. The NJ tree was constructed with 56 gene sequences to compare chicken KLF genes with sequences from the three other species. The results demonstrated that the 56 KLF genes were divided into three groups. Comparative analysis suggested that chickens, mice, and humans have similar numbers of gene families [50]. Gene structure is an important factor in the evolution of gene families [51]. We found that the KLF gene structure of chickens is quite similar to that of humans, pigs, and mice. Motif analysis revealed that KLFs clustered in groups based on the similarity of their subdomains. The results of conserved-domain analysis showed that KLF genes are relatively conserved among these species. Among 20 genes predicted by interaction network analysis, NCOA3 was found to participate in regulating porcine skeletal muscle satellite cell proliferation [52]. Furthermore, differential expression of NCOA3 was proven to be regulated by miR-17-5p in pig intramuscular and subcutaneous adipose tissues [53].
Excessive fat deposition is considered an undesirable factor which affects feed efficiency, meat production costs, meat quality, and consumer health [54]. Hence, studies have been done to explore the relationship between KLF genes and fat deposition. A previous experiment proved that Gg-KLF2 was highly expressed in abdominal adipose tissue, and its transcripts fluctuated during adipose tissue development [55]. Our research showed that the relative expression level of KLF2 was highest in abdominal fat in male chickens. Genetic variants in the promoter region of the KLF3 gene were associated with fat deposition in Qinchuan cattle [56]. KLF3 was also demonstrated to be negatively correlated with intramuscular fat content in the longissimus dorsi muscle of Erhualian pigs [57]. The expression profile analysis found that KLF3 might play important roles in the fat development process.
In our research, the expression level of KLF3 in abdominal fat was higher than that in the glandular stomach, hypothalamus, and spleen. However, there was no significant difference between the expression level of KLF3 in differentiated and undifferentiated preadipocytes. Sometimes, KLF genes work together to produce a marked effect. Guo et al. found the KLF15 gene was overexpressed through adenoviral vector (Ad-KLF15) in bovine adipocytes, and the expression level of the KLF3 gene was increased, which indicated that KLF15 promoted the transcription of the KLF3 gene in bovine adipocytes [58]. Our synteny analysis showed that KLF3 had collinear relationships with KLF5 and KLF12, while interaction network analysis showed that 11 KLF genes had interaction relationships, including KLF3 and KLF15. Besides, our research showed that KLF15 was expressed at relatively high levels in abdominal fat. KLF4 was proven to be one of potential therapeutic targets for obesity-induced cardiac injuries [59]. There was a significant difference between the expression level of KLF4 in differentiated and undifferentiated preadipocytes in our research. Cardiomyocyte-specific KLF5 knockout mice were found to have accelerated diet-induced obesity associated with increased white adipose tissue [60]. A qPCR showed that the expression level of KLF5 was second highest in abdominal fat. The expression levels of KLF5 decreased significantly after preadipocyte differentiation.
Iqbal et al. discovered that KLF6 regulated fat synthesis in bovine mammary epithelial cells (BMECs) by targeting the PPARA, the PPARG pathway, and other fat marker genes [61]. Our research showed that KLF6 expression was highest in abdominal fat. In our study, a significant difference was found between undifferentiated and differentiated adipocytes. RNA-seq result revealed that the down-regulation of the KLF6 gene significantly up-regulated the genes that regulate adipogenesis, differentiation and regulation of adipocytes, and the homeostasis of bovine adipocytes, which proved that KLF6 has a role in regulating lipid metabolism in bovine adipocytes [62]. KLF7 was revealed to modulate the differentiation and proliferation of chicken preadipocytes [63]. We also found the expression level of KLF7 significantly decreased after differentiation. KLF8 was targeted by miR-10a-5p to inhibit the differentiation of goat intramuscular preadipocytes [64]. In our study, the expression level of KLF8 decreased after their differentiation in chicken preadipocytes. KLF9 was found to inhibit chicken intramuscular preadipocyte differentiation [65]. However, the expression level of KLF9 increased significantly after chicken preadipocyte differentiation. KLF10, KLF11, KLF12, and KLF13 function as positive organizers by a variety of different mechanisms, such as crosstalk with C/EBP and PPARγ, to regulate adipogenesis and associated pathways in cattle [66]. The expression profile analysis revealed that KLF10 might play important roles in the fat development process. Our research showed that the expression of KLF10 in abdominal fat was the second-most abundant of all the tissues tested, while no significant difference was detected between the expression level of KLF10 in differentiated and un-differentiated preadipocytes. There was also no significant difference between the expression level of KLF11 in differentiated and undifferentiated preadipocytes. However, we found the expression level of KLF12 increased significantly and the expression level of KLF13 decreased significantly in chicken preadipocytes.
KLF genes play not only important roles in fat deposition but also have great effects on skeletal muscle development. Manoharan et al. [67] identified KLF2 as a key regulator of myeloid cell functions in mouse skeletal muscle regeneration. In our study, KLF2 expression level increased in samples from 11 embryonic-day-old chicks to 1-day-old chicks. Moreover, KLF2 was found to be involved in the FOXO signaling pathway. This FOXO signaling pathway played an important role in the pathogenesis of skeletal muscle atrophy by regulating E3 ubiquitin ligases and some autophagy factors [68]. This indicated that KLF2 played an important role in chickens' skeletal muscle development. Zhang et al. [69] confirmed that miR-21-5p promoted the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) by targeting KLF3. The increasing expression level of KLF3 in 1-dayold chicks might prove the function of KLF3 for promoting muscle development. The KLF4 gene was found to significantly promote the proliferation and differentiation of chicken primary myoblasts (CPMs) [70]. The expression level of KLF4 was extremely high in chicken thigh muscle and pectoral muscle in samples at 18 embryonic days old in our research. A study suggested that KLF5 may regulate the atrophy of chicken skeletal muscle through the Wnt/β-catenin signaling pathway [71], while KLF5 expression levels decreased when chickens were hatched in our study. These results meant that KLF5 displayed a vital role in the development of chicken muscle.
KLF6 was confirmed as a new potential target gene of miR-148a-3p, and miR-148a-3p impeded bovine myoblast cell proliferation and promoted apoptosis through the posttranscriptional down-regulation of KLF6 [72]. This conclusion corresponded with the downtrend of KLF6 expression level in samples from 16 embryonic-day-old to 1-day-old chicks in our study. KLF6 was also found to play a crucial role in regulating cell differentiation, proliferation, and muscle development, so it could be used as a potential candidate marker gene for the improvement of the Qinchuan cattle breed [73]. There is no report on the relationship between muscle development and KLF7 or KLF8. In our research, the results in Figure 8A also showed that KLF7 and KLF8 might have a relationship with the development of muscle. RNA-Seq exploration of meat quality in Spanish goats illustrated that KLF9 was associated with the effects of stress on skeletal muscle proteins [74]. We also found that KLF9 expression levels were very high in the thigh muscle of one-day-old chicken. KLF10 was known to control numerous genes in many cell types that are involved in various key biological processes like differentiation, proliferation, and apoptosis [75]. KLF10 was expressed highly in the thigh muscle of Heying Black chickens and the expression level increased in samples from 11-embryonic-day-old to 1-day-old chicken, indicating that KLF10 was involved in chicken muscle proliferation and differentiation. Furthermore, the sequencing result was used to show that KLF11 was related to muscle development [76]. In our experiment, we also found the expression level of KLF11 was high in pectoral muscle and that KLF11 was also related to chicken muscle development. According to the RNA-seq results from the embryonic days samples and 1-day-old samples ( Figure 8A), KLF12 and KLF13 might have the opposite effect on muscle development in chickens. KLF15 was found to be a key regulator of branched-chain amino acid metabolism in the skeletal muscle of fish (tilapia) [77]. Both the RNA-seq and qPCR results showed that the expression level of KLF15 was high in muscle, and its expression increased in samples from 11 embryonicday-old to 1-day-old chicken, which indicated that KLF15 was involved in the development of chicken muscle.

Conclusions
In this study, we identified 13 KLF genes in G. gallus. Phylogenetic analysis showed that the KLF genes could be divided into three groups. Chromosomal locations showed that the 13 Gg-KLFs were distributed on ten different chromosomes. Motif analysis and conserved domain analysis showed KLFs shared similar conserved domains in four species. Synteny analysis and interaction network analysis illustrated the collinear relationships and the relationships among KLFs and 20 corresponding genes. KEGG pathway analysis predicted that KLF2 was involved in the Apelin-signaling pathway and the FOXO-signaling pathway. Our expression profile analysis revealed that the KLF genes displayed diverse expression patterns in chickens. This study provides some evidence that the KLF gene family plays a critical role in the development of chicken muscle and fat, which may give some suggestions for future research in the KLF gene family.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13091429/s1, Figure S1: Apelin signaling pathway and FOXO signaling pathway; Table S1: The forward primers and reverse primers of KLFs designed by Primer Premier 5.0; Table S2: The Go enrichment analysis of the cellular components of KLF genes in chicken; Table S3: The Go enrichment analysis of biological processes of KLF genes in chicken.
Author Contributions: X.L. wrote the original manuscript. Q.W., J.Z. and G.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.