Cloning and expression of Hoxc6 gene from Pampus argenteus and its relationship with pelvic fin absence

Highlights

• The Hoxc6 full-length cDNA from P. argenteus was cloned.

• Hoxc6 levels were higher at embryonic stages than that in silver pomfret larvae.

• Hoxc6 levels were more abundant in fin tissues than brain and eye tissues.

• Hoxc6 transcript was strongly expressed in the anterior part of body axis in 1-day-old larvae.

Abstract

Hoxc6 gene can be described as having roles in axial patterning in early embryogenesis, and in at least some species, having a contribution to limb positioning. In this study, we cloned and characterised Pampus argenteus Hoxc6. The highly conserved HOXC6 protein sequence contains a homeodomain and a low-complexity region. Expression of Hoxc6 mRNA was measured at different developmental stages and in different tissues by real-time PCR (p < 0.05), and was high during eye capsule and brain differentiation stages, but low in 7 and 13-day-old larvae. Hoxc6 mRNA was more abundant in fin tissue than brain and eye tissues. Western blotting showed that HOXC6 protein levels were high at embryonic stages, but decreased significantly in 7, 13, 16 and 19-day-old larvae, and levels were essentially consistent with those of mRNA measured by real-time PCR in different tissues. In situ hybridisation showed that the Hoxc6 transcript was strongly expressed in the whole brain and anterior part of the body axis in 1-day-old larvae, but in the hindbrain, pectoral fin, mandible and hypothetical pelvic fin region in 7, 13, 16 and 19-day-old organisms. These results clarify the expression and localisation characteristics of Hoxc6 gene in P. argenteus, and provide a theoretical basis for the molecular mechanism of pelvic fin loss in silver pomfret.

Introduction

Silver pomfret (Pampus argenteus), belonging to Perciformes, Stromateidae, Pampus, is an economically important marine fish species. To date, most research on this species has mainly focused on ecological resource assessment (Liu and Zhan, 1999), reproductive characteristics (Almatar et al., 2004; Shi et al., 2009), nutritional composition (Gu, 2012; Wang et al., 2015; Xu et al., 2012; Yan et al., 2015) and immunology (Gao et al., 2018; Li et al., 2019). Silver pomfret is oval in shape, flattened laterally, with similar shaped dorsal and anal fins, a bifurcated caudal fin, a pectoral fin, but no pelvic fin. According to classical ichthyology research, the pelvic fin can help the dorsal fin and anal fin to maintain balance, and can assist rise-fall and turning, and poor balance is experienced by fish without a pelvic fin. Under artificial feeding conditions, this species constantly circles and cruises in groups, resulting in bruising caused by rubbing against the pool wall. Additionally, silver pomfret has a flat body, and its dorsal and anal fins are not very well developed, hence swimming mainly depends on the powerful pectoral and caudal fins to provide forward power and maintain body balance. Thus, what on earth causes the pelvic fin loss? The problem urges us to study in depth.

From the perspective of skeletal development and evolution, the pectoral and pelvic fins of fish are homologous to the forelimb and hindlimb of tetrapods (Ruvinsky and Gibson-Brown, 2000), and they are regulated by the same developmental pattern and gene signalling pathway (Eckalbar et al., 2016; Montavon et al., 2011). All fins undergo three developmental stages; limb positioning, limb bud initiation and limb bud outgrowth (Tanaka et al., 2005). Specifically, in somitic mesoderm and lateral plate mesoderm (LPM), Hox genes expression defines the position of the body axis, and participates in determining the locations of the forelimb and hindlimb. Subsequently, Tbx4 and Tbx5 activate the Fgf10 signal in LPM, causing Fgf8 to be expressed in the surrounding ectoderm (SE), forming limb buds. Ectodermal cells then grow and develop along the edge of the limb bud and induce the formation of the apical ectodermal ridge (AER), which secretes fibroblast growth factors (FGFs) that instruct mesenchymal cells to facilitate the further growth of limb buds (Sun et al., 2002).

Hox genes in metazoans contain a homeobox domain. All Hox genes have a homeobox of ~180 bp encoding a polypeptide of ~60 amino acids (Burglin, 2011). In mammals, Hox genes are divided into four chromosomal (HOXA-D) clusters. According to sequence similarity and the position within the cluster, they can also be organised into 13 paralogous groups (HOX1-13) (Alexander et al., 2009; Mallo and Alonso, 2013). However, in teleost fish, Hox genes are arranged into seven or eight clusters, possibly evolve via the whole genome duplication that occur at the origin of teleosts (the 3R WGD) (Hurley et al., 2005).

Hox genes are important developmental regulatory genes that participate widely in a variety of biological processes, including regulation of the body axis (Wellik, 2007), limbs (Zakany and Duboule, 2007) and nervous system (Rogulja-Ortmann and Technau, 2008). Consistent with this, studies have shown that mutations in a single Hox gene can lead to subtle limb defects. For example, Hoxa10/Hoxc10/Hoxd10 mutants cause truncation of hindlimb femurs (Wellik and Capecchi, 2003), and defects in Hoxb5 can lead to a more anterior repositioning of forelimbs (Rancourt et al., 1995). Mutation of the Hox8 gene produces a tail-shifted hindlimb (van Den Akker et al., 2001). These studies indicate a key role for Hox genes in limb positioning. In addition, Hox genes dysregulation occurs in many human cancers (Abate-Shen, 2002), such as breast cancer, colon cancer, lung cancer and kidney cancer.

It is known that HoxA and HoxD contribute to forelimb formation, while the HoxC cluster is primarily responsible for hindlimb formation (Raines et al., 2015). Hoxc6 belongs to the HoxC cluster, and if Hoxc6 function is lost in Xenopus, the body axis is truncated (Zhu et al., 2017). In addition, Hoxc6 also can promote the osteogenic differentiation of adult mesenchymal stem cells (Simeone et al., 1987). To date, research on Hoxc6 has mainly focused on axis formation (Prince et al., 1998a, 1998b), the occurrence of various cancers (McCabe et al., 2008; Zhou et al., 2019), and the appendage development (Ahn and Gibson, 1999; Tanaka et al., 2005). Cloning and expression of Hoxc6 gene have also been reported in some fish such as zebrafish, threespine stickleback and pufferfish (Ahn and Gibson, 1999; Aparicio et al., 1997; Prince et al., 1998b).

In this paper, we explored the relationship between Hoxc6 and pelvic fin loss in silver pomfret by cloning and characterising the full-length cDNA sequence, and evaluating Hoxc6 mRNA and protein expression during different developmental stages and in different tissues. Probes for Hoxc6 were synthesised to investigate localisation by whole-mount in situ hybridisation. The findings clarify the expression characteristics of the Hoxc6 gene, and provide a theoretical basis for the molecular mechanism of pelvic fin loss in silver pomfret.