To gain an understanding of what biological activities are significantly associated with these enriched genes, we performed a gene ontology (GO) analysis using the g:Profiler software (Reimand et al.,2007). GO annotation of chicken genes is relatively poor, we therefore used the human GO annotation and analyzed biological processes that are significantly associated with human orthologues of the enriched chicken genes. In total, 323 of the 378 genes could be annotated this way (Supp. Table S2 for all significantly associated biological processes). Not surprisingly, over 60% of the annotated genes were associated with metabolic processes. Constituent genes associated with lipid (54), carbohydrate (32), and amino acid (35) metabolisms are shown in Supp. Table S2. A few other biological processes were also highly represented. These include body fluid regulation, blood coagulation, nutrient sensing, and liver function (Supp. Table S2), suggesting that the YS endoderm also plays important roles in the global regulation of embryonic development.
The GO profile of highly associated biological processes was confirmed by manual analysis of constituent genes. One example is provided here. Among genes involved in lipid metabolism, we found many key proteins required for binding (e.g., fatty acid binding protein FABP2, vitamin D binding protein GC, and riboflavin binding protein RBP), breakdown (e.g., phospholipases PLA1A, PLA2G12B, and FAAH), biosynthesis (e.g., monoacylglycerol acyltransferase MOGAT1, long chain acyl-CoA synthetase ACSL5, and lecithin-cholesterol acyltransferase LCAT), and export (e.g., apolipoproteins ApoA4, A5, B, and H) of lipids. In addition, of particular interest to us, many enriched genes appear to encode serum proteins, suggesting a non–cell-autonomous role of the YS endoderm. The protein composition of the earliest serum is not known. The entire YS membrane has been shown to be capable of synthesizing some serum proteins (Kram and Klein,1976) at peri-circulation stages; and later during embryogenesis, it has been suggested that the YS endoderm participates in the synthesis and secretion of transferrin, alpha globulins and albumin into the circulation (Amin,1961; Kram and Klein,1976; Young et al.,1980; Young and Klein,1983). In our screen, we detected many enriched genes with a clear functional implication as constituent serum proteins, and many minor ones which may be involved in growth or hormonal regulations. Among the major proteins, prealbumin, alpha-2-macroglobulin, and transferrin were highly expressed, confirming these earlier studies. The apolipoproteins, ApoA4, ApoA5, ApoB, and ApoH, constitute another group of genes with high and enriched expression in E2–E4 YS endoderm. ApoA1 is highly expressed in both E1 and E2–E4 from our gene chip and in situ data, confirming the data from a previous report (Bertocchini and Stern,2008). A large group of additional enriched genes appear to play a role in the embryonic vasculature. These include complement proteins CF1, C1R, C5, C8A, and C8G; coagulation regulators F2, F5, F7, TFPI, PROC, and PROZ; as well as several antitrypsins, antithrombin, COL3A1, COL6A1, FGA, FGG, PLG, and VTN. These data suggest that YS endoderm produces a whole range of major and minor serum proteins similar to those produced in adult liver. A third group of genes of interest to us is the transcription factors. They include SOX5, ARH, NR3C2, TFCP2L1, PROX1, RXRG, and IRF7.
Combining both the GO and manual annotation approaches, these enriched YS endoderm genes can be grouped in five broad categories (Fig. 3): enzymes, serum proteins, transcription factors, and others (annotated and nonannotated). A large proportion, 42% (159), of the 378 enriched genes belong to the group with catalytic activities, 8% (30) to the serum proteins, and 2% (7) to the transcription factors. The remaining 48% (182), including both annotated (126) and nonannotated (56), contain genes with various other functions.