Evolutionary analysis of paired box gene family and biological function exploration of Lr.Pax7 in lamprey (Lethenteron reissneri)

Abstract

Gene regulation refers to the precise regulation of gene expression in an organism, and transcription factors are proteins that bind to DNA and regulate gene expression by promoting or inhibiting the expressions of target genes. Since the late 1980s [1], scientists have studied special genes called Pax genes that control how genes function in organisms as they grow. There are nine Pax genes found in animals such as mice, zebrafish, and humans [2]. Based on the composition domain and homology of the sequence, the Pax family is divided into four subfamilies: Pax1/9, Pax2/5/8, Pax4/6, and Pax3/7 [3]. Pax7 plays a pivotal role in the implementation, protection, and repair of skeletal muscle. Pax7 helps to control the balance between self-renewal and differentiation of satellite cells, ensuring that they can proliferate when needed to generate new muscle cells and differentiate into mature muscle fibers when necessary for muscle development and repair. The expression of the Pax7 gene in nerve cells is critical for dorsal root and sensory ganglia development. The Pax7 gene serves as a primary controlling factor for skeletal muscle development while influencing different biological processes; however, its exact role in jawless vertebrates such as lamprey remains unclear, and extensive research is needed to elucidate the intricate underlying mechanisms involved. Given the unique status of lamprey as an ancient jawless fish, possessing an ancient lineage and distinctive biological features, it is rare to explore gene function across hundreds of millions of years of vertebrate evolution. The use of lamprey as a model system for gene function research represents an innovative approach in the fields of evolutionary and comparative genomics. In this study, we investigated the regulatory mechanism of Pax7 in lamprey via gene cloning, gene expression analysis, gene silencing and transcriptome data analysis. We also explored the interactions between genes with significant differences. Identification of Lr.Pax7 in lamprey tissues began with the retrieval of protein sequences that are similar to those of human Pax family members in sea lamprey (Petromyzon marinus) or zebrafish (Danio rerio) from the NCBI protein database (Supplementary Table S1) and the use of BLAST to identify corresponding homologs (Supplementary Table S2). Subsequently, we extracted the Pax sequences from our library. Lethenteron reissneri specimens were dissected to isolate various tissues. Primers targeting the pax domains were designed based on the Pax7 nucleotide sequence in the Lampreys cDNA library, and the aim was to verify the effectiveness of the Lampreys cDNA as a template for validation (Supplementary Table S3). Lr.Pax7 was successfully amplified via PCR in muscle tissue. Here, a variety of methods were used for bioinformatics analysis. The results showed that the amino acid sequence of Pax7 is highly similar among animals (Figure 1A), with a decreasing trend from higher to lower organisms, as revealed by sequence alignment. It can be observed from the evolutionary tree (Figure 1B) that Pax genes for each subfamily are present in ancestral chordate and that Pax genes are present in amphioxus. Petromyzon marinus, Lethenteron camtschaticum, and Lethenteron reissneri constitute a sister group and have become good models for the study of jawless vertebrates. Pax9, Pax2, Pax6, and Pax7 show high similarity to those of other higher vertebrates. Therefore, these genes were named Lr.Pax9, Lr.Pax2, Lr.Pax7, and Lr.Pax6. The results indicate that the Pax7 gene is significantly preserved across various species, from higher to lower. This suggests that the DNA sequence of the gene is remarkably similar among different species. Lr.Pax7 is positioned between vertebrates and invertebrates and is most closely related to P. marinus Pax7. This finding provides more insight into the original evolutionary position of the lamprey. Crystal structure prediction analysis revealed that Lr.Paxs and Hm.Paxs have highly homologous structures (Figure 1C). The Pax gene has a similar structure (Figure 1D), including a conserved DNA-binding structure called the pair-box domain. This structure contains approximately 128 amino acids and is responsible for binding with specific DNA sequences, regulating gene expression, and interacting with other proteins. To further investigate the evolutionary history of Pax7 in vertebrates, we compared the genetic environment of Pax7 with that of other vertebrates (Figure 1E). In addition, many Pax gene members also contain DNA-binding structures called homeodomains, which play important roles in development.

1. Introduction

Gene regulation refers to the precise regulation of gene expression in an organism, and transcription factors are proteins that bind to DNA and regulate gene expression by promoting or inhibiting the expressions of target genes. Since the late 1980s [1], scientists have studied special genes called Pax genes that control how genes function in organisms as they grow. There are nine Pax genes found in animals such as mice, zebrafish, and humans [2]. Based on the composition domain and homology of the sequence, the Pax family is divided into four subfamilies: Pax1/9, Pax2/5/8, Pax4/6, and Pax3/7 [3].

Pax7 plays a pivotal role in the implementation, protection, and repair of skeletal muscle. Pax7 helps to control the balance between self-renewal and differentiation of satellite cells, ensuring that they can proliferate when needed to generate new muscle cells and differentiate into mature muscle fibers when necessary for muscle development and repair. The expression of the Pax7 gene in nerve cells is critical for dorsal root and sensory ganglia development. The Pax7 gene serves as a primary controlling factor for skeletal muscle development while influencing different biological processes; however, its exact role in jawless vertebrates such as lamprey remains unclear, and extensive research is needed to elucidate the intricate underlying mechanisms involved.

Given the unique status of lamprey as an ancient jawless fish, possessing an ancient lineage and distinctive biological features, it is rare to explore gene function across hundreds of millions of years of vertebrate evolution. The use of lamprey as a model system for gene function research represents an innovative approach in the fields of evolutionary and comparative genomics. In this study, we investigated the regulatory mechanism of Pax7 in lamprey via gene cloning, gene expression analysis, gene silencing and transcriptome data analysis. We also explored the interactions between genes with significant differences.