Transcriptome analysis of pigeon milk production – role of cornification and triglyceride synthesis genes (2014)

Background The pigeon crop is specially adapted to produce milk that is fed to newly hatched young. The process of pigeon milk production begins when the germinal cell layer of the crop rapidly proliferates in response to prolactin, which results in a mass of epithelial cells that are sloughed from the crop and regurgitated to the young. We proposed that the evolution of pigeon milk built upon the ability of avian keratinocytes to accumulate intracellular neutral lipids during the cornification of the epidermis. However, this cornification process in the pigeon crop has not been characterised. Results We identified the epidermal differentiation complex in the draft pigeon genome scaffold and found that, like the chicken, it contained beta-keratin genes. These beta-keratin genes can be classified, based on sequence similarity, into several clusters including feather, scale and claw keratins. The cornified cells of the pigeon crop express several cornification-associated genes including cornulin, S100-A9 and A16-like, transglutaminase 6-like and the pigeon âlactatingâ crop-specific annexin cp35. Beta-keratins play an important role in âlactatingâ crop, with several claw and scale keratins up-regulated. Additionally, transglutaminase 5 and differential splice variants of transglutaminase 4 are up-regulated along with S100-A10. Conclusions This study of global gene expression in the crop has expanded our knowledge of pigeon milk production, in particular, the mechanism of cornification and lipid production. It is a highly specialised process that utilises the normal keratinocyte cellular processes to produce a targeted nutrient solution for the young at a very high turnover. Background Pigeon lactation was first noted in the literature in 1786 when John Hunter described pigeon milk as being like â..granulated white curdâ [1]. This curd-like substance is produced in the crop of male and female pigeons and regurgitated to the young. Like the mammary gland, the pigeon crop undergoes significant changes to the tissue structure during lactation. Several histological studies have characterised these changes and determined that pigeon milk consists of desquamated, sloughed crop epithelial cells [2, 3]. The process of pigeon milk production begins when the germinal cell layer of the crop rapidly proliferates in response to prolactin [4, 5], and this results in a convoluted, highly folded epithelial structure that then coalesces as it out-grows the vasculature, to form the nutritive cell layer that is sloughed off to produce the milk. This nutritive cell layer contains lipid-filled vacuoles [2, 3, 5, 6]. The lipid content of pigeon milk consists mainly of triglycerides, along with phospholipids, cholesterol, free fatty acids, cholesterol esters and diglycerides [7]. The triglyceride content decreases across the lactation period, from 81.2% of total lipid at day one, to 62.7% at day 19, whereas the other lipids increase, which suggests the cellular lipid content decreases towards the end of the lactation period, but the cell membrane-associated lipids remain constant [7]. Several studies have investigated the differences in gene expression between âlactatingâ pigeon crop tissue and non-âlactatingâ crop tissue [6, 8, 9]. Nearly three decades ago, Horseman and Pukac were the first to identify that mRNA species differ in response to prolactin injection in the crop [8]. Specifically, they identified and characterised gene expression and protein translation of the prolactin-responsive mRNA anxI cp35 and the non-prolactin-responsive isoform, anxI cp37 [9, 10]. In addition, a recent global gene expression study in our laboratory [6] showed that genes encoding products involved in triglyceride synthesis and tissue signalling were up-regulated in the âlactatingâ crop. We proposed that the evolution of the processes that result in the production of pigeon milk has built upon the more general ability of avian keratinocytes to accumulate intracellular neutral lipids during the cornification of the epidermis [11] in order to produce a nutritive substance for their young [6]. The mechanism of avian epidermal cornification and lipid accumulation is not well-characterised. However, studies have shown that antibodies against mammalian cornification proteins, which are relatively well-characterised, can cross-react with avian and reptilian species [12, 13], which suggests similarities in cornification proteins amongst vertebrate species. Cultured chicken keratinocytes have been shown to express beta-keratins (feather, scale and claw keratins), alpha-keratins (type I and II cytokeratins) and the cornified envelope precursor genes envoplakin and periplakin, as well as accumulating neutral lipids [11]. Mammalian keratinocytes differ from avian keratinocytes in that they are unable to accumulate intracellular neutral lipids [11], and can express alpha-keratins (cytokeratins) but not beta-keratins, which expanded from early archosaurians [14]. There are many cornification-associated proteins characterised from mammalian epidermal tissues. The proteins that form the cornified envelope include keratins, S100 proteins, small proline-rich proteins (SPRRs), late cornified envelope (LCE) proteins, annexins, involucrin, loricrin, filaggrin, desmoplakin, envoplakin, periplakin, trichohyalin, cystatin A, elafin and repetin [15]. Trans-glutaminase enzymes, some of which require cleavage by proteases and an increase in intracellular calcium concentration to become active, cross-link the cornified envelope proteins to form a ceramide lipid-coated protective barrier to the epidermis [16]. Many of the cornified envelope genes are present in the âepidermal differentiation complexâ (EDC) which was first identified on chromosome 1q21 in humans [17]. Interestingly, the EDC region has been identified in an avian species (chicken), and is linked to the genes for beta-keratins, but lacks the LCE proteins [18]. Here we present an analysis of the pigeon crop transcriptome to show that pigeon milk production involves a specialised cornification process and de novo synthesis of lipids that accumulate intracellularly.