Let a Virus Paint Your Eggs in Blue

ResearchBlogging.orgConsidering it is Easter I though it would be very appropriate to write something on an egg-related topic and, as a personal bonus, to mix some viruses in as well ☺ .
I suppose most of us are used to seeing eggs that have either white or brown shells. However, those are just the types that are sold in supermarkets, but people who grow different types of chickens will note that sometimes the eggshells can be blue or green as well. Were do all these colours come from? Eggshell colours are determined by the pigments that are secreted when eggs develop in uterus of a hen. Pigment protoporphyrin, for example, is derived from blood haemoglobin and contributes to brown eggshell coloration. By contrast, blue eggs predominantly have billiverdin pigment in their shells. Billiverdin is a component of bile salts and acts as an antioxidant, which is why it has been proposed that blue egg coloration could act as a signal of female genetic fitness (figure 1).

Figure 1
Figure 1

An oocyan locus has been for a long time linked to blue shell coloration, however, it was not clear what genes exactly induce the production for blue shells? To answer this question scientists used genetic linkage analysis to map the site of blue colour-linked genes to a 120kb region on chicken chromosome 1. Analysis of this region has showed that it codes for 4 genes called, SLCO1C1, SLCO1B3, LOC418189 and SLCO1A2 (figure 2).

Figure 2. Oocyan  locus on chromosome 1 in chicken
Figure 2. Oocyan locus on chromosome 1 in chicken

As eggshell colour is determined in uterus the study next looked at the expression patterns of these 4 genes in the uterus of hens that lay either blue or brown eggs. Surprisingly, scientists found that hens which lay blue eggs specifically express SLCO1B3 gene whereas, brown egg laying hens do not. The same results were confirmed with in situ hybridization (a technique used to visually observe mRNAs produced from a gene of interest), where only blue-shelled hens were observed to express SLCO1B3 transcripts (figure 3).

In situ hybridization for SLCO1B3 mRNA in blue and brown uterus from blue or brown egg laying hens
Figure 3. In situ hybridization for SLCO1B3 mRNA in uterus from blue or brown egg laying hens. SLCO1B3 mRNA in green and cell nuclei in blue

Next, to determine the cause of SLCO1B3 expression in blue but not brown egg laying hens, the sequences of SLCO1B3 gene and its flanking regions were analysed. No significant changes in the sequence of the gene itself (i.e. single nucleotide polymorphisms) were found to be linked with blue egg phenotype, however the sequences that came from blue-egg laying hens had a 4.3kb insertion upstream of the SLCO1B3 gene. This insertion was not just a random sequence but an endogenous retrovirus sequence. It is perhaps not a great surprise to find a retrovirus integrated into a genome, after all 8% of human genome is made up of retroviral sequences. The standard retrovirus genome sequence can be depicted as 5′LTR-gag-pol-env-LTR3′ , where LTRs are Lon regulatory regionsl Repeats (sequences containing elements that regulate expression of viral genes) and gag, pol and env are genes required for replication and formation of virus particle. The retroviruses that are permanently integrated into a genome, however, are usually crippled because they lack some parts of their genes. The retrovirus that was found to be integrated upstream of SLCO1B3 gene, for example, lacks pol and some parts of its gag and env genes and therefore cannot itself replicate (figure 4). The sequence of this integrated virus most closely matched to that of EAV-HP avian retrovirus. While integrated EAV-HP cannot itself replicate its LTRs still contain regulatory elements that could potentially be driving the expression of SLCO1B3 gene.

Insertion of  EAV-HP upstream of SLCO1B3  gene
Figure 4. Insertion of EAV-HP upstream of SLCO1B3 gene

SLCO1B3 gene codes for OATP1B3 protein, which is has been previously described as a liver-specific transporter of bile salts. As billiverdin (pigment that causes blue colour) itself is a bile salt it is possible that EAV-HP integration upstream of SLCO1B3 gene leads to increased production of OATP1B3 in uterus which in turn increases the amount of billiverdin deposited in eggshells, colouring them in blue. Amazingly EAV-HP insertion has been identified in 705 chickens from 12 different breeds worldwide and has been exclusively linked to blue eggshell production. Moreover, the insertion sites of EAV-HP in breeds of blue-egg-laying chickens from China and Chile are slightly different suggesting that the EAV-HP integration in these breeds happened in two independent events rather than a result of a descend from a common ancestor.

So that is my Easter science story for you. I should make a mental note on writing something more about endogenous retroviruses because they are quite a fascinating topic. Yes, colouring eggshells in blue may not seem to be something very significant, however, for example, a retroviral gene called syncytin is expressed in placenta of mammals including humans. This gene plays an essential role in placental morphogenesis and has potentially contributed to evolution of Mammalia clade .

Wang Z, Qu L, Yao J, Yang X, Li G, Zhang Y, Li J, Wang X, Bai J, Xu G, Deng X, Yang N, & Wu C (2013). An EAV-HP insertion in 5′ Flanking region of SLCO1B3 causes blue eggshell in the chicken. PLoS genetics, 9 (1) PMID: 23359636

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