Review Article

Journal of Innate J Innate Immun 2019;11:111–124 Received: June 25, 2018 DOI: 10.1159/000493719 Accepted after revision: September 9, 2018 Immunity Published online: November 2, 2018

Dynamics of Structural Barriers and Innate Immune Components during Incubation of the Avian : Critical Interplay between Autonomous Embryonic Development and Maternal Anticipation

a–d d d d Maxwell T. Hincke Mylène Da Silva Nicolas Guyot Joël Gautron e f d Marc D. McKee Rodrigo Guabiraba-Brito Sophie Réhault-Godbert a b Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; Department of c Innovation in Medical Education, University of Ottawa, Ottawa, ON, Canada; LE STUDIUM Research Consortium, d Loire Valley Institute for Advanced Studies, Orléans-Tours, Nouzilly, France; BOA, INRA, Val de Loire Centre, e Université de Tours, Nouzilly, France; Department of Anatomy and Cell Biology and Faculty of Dentistry, McGill f University, Montreal, QC, Canada; ISP, INRA, Université de Tours, Nouzilly, France

Keywords gressive transformation of egg innate immunity by embryo- Chorioallantoic membrane · Chick embryo · Innate generated structures and mechanisms over the 21-day immunity · Antimicrobial peptides · Avian β-defensins · course of egg incubation, and also discusses the critical in- Toll-like receptors · Eggshell terplay between autonomous development and maternal anticipation. © 2018 The Author(s) Published by S. Karger AG, Basel Abstract The integrated innate immune features of the calcareous egg and its contents are a critical underpinning of the re- Introduction markable evolutionary success of the Aves clade. Beginning at the time of laying, the initial protective structures of the Avian are continuously exposed to microbes. egg, i.e., the biomineralized eggshell, egg-white antimicro- They are challenged with high numbers of potentially bial peptides, and vitelline membrane, are rapidly and dra- pathogenic agents from the laying hen during oviposi- matically altered during embryonic development. The em- tion, through the air and litter, and during natural incu- bryo-generated extra-embryonic tissues (chorioallantoic/ bation. Despite this exposure, most eggs remain viable up amniotic membranes, yolk sac, and associated chambers) to hatching. The foremost reason for this is the highly ef- are all critical to counteract degradation of primary egg de- ficient early defense response of the egg’s innate immune fenses during development. With a focus on the chick em- system. In , there are 2 main, complementary types bryo (Gallus gallus domesticus), this review describes the pro- of immune defense: (1) nonspecific mechanisms, which

Dr. Maxwell T. Hincke M.T.H. and M.D.S. contributed equally. Faculty of Medicine, University of Ottawa 451 Smyth Road Ottawa, ON K1H 8M5 (Canada) E-Mail mhincke @ uottawa.ca

© 2018 The Author(s) Dr. Sophie Réhault-Godbert Published by S. Karger AG, Basel INRA-Centre Val de Loire UMR0083 Biologie des Oiseaux et Aviculture (BOA) E-Mail [email protected] This article is licensed under the Creative Commons Attribution- FR–37380 Nouzilly (France) www.karger.com/jin NonCommercial-NoDerivatives 4.0 International License (CC BY- NC-ND) (http://www.karger.com/Services/OpenAccessLicense). E-Mail sophie.rehault-godbert @ inra.fr Usage and distribution for commercial purposes as well as any dis- tribution of modified material requires written permission. act on pathogens in a nontargeted manner (physical and transfers nutrients and immunoglobulin (Ig)Y from the chemical barriers, and components of innate immunity egg yolk to the developing embryo or the newly hatched including antimicrobial molecules and cellular mecha- chick. Therefore, during the first week after hatching, be- nisms, i.e., heterophils and macrophages), and (2) adap- fore the is mature enough to produce its tive mechanisms, which target specific pathogens (anti- own B lymphocytes, a chick’s depends bodies and lymphocytes). The innate immune responses on maternal (IgY) received from the egg yolk. can directly control the replication or spread of patho- gens by induction of phagocytosis or antimicrobial prod- ucts. This review covers studies on the well-documented Innate Immune Receptors and Antimicrobial chick embryo (Gallus gallus domesticus), from which Peptides most of our knowledge on avian embryogenesis is de- rived [1]. TLRs recognize microbes by binding to pathogen-as- The first line of defense against pathogenic microor- sociated molecular patterns (PAMPs) such as lipopoly- ganisms is formed by physical barriers such as the skin saccharide (LPS), lipoteichoic acid, bacterial flagellin, li- or, in the egg, the eggshell (ES), as well as by chemical poproteins, peptidoglycans, glycophosphatidylinositol, innate immune protective mechanisms; together, these and bacterial DNA, in addition to single- and double- resist pathogen invasion from a contaminated environ- stranded viral RNA. At this time, 10 TLRs have been iden- ment. Because the development of an avian embryo oc- tified in ; most of them still require better char- curs in an egg chamber that is physically separated from acterization on ligand-receptor interactions and the as- the hen, the egg contains all the required elements to sociated downstream signaling pathways involved in nourish and protect the developing embryo during the their immune effector functions [7]. However, it is clear entire cycle of its development prior to hatching. How- that chick embryonic tissues express TLRs from ED3 on- ever, the innate defenses initially present within the egg wards, recognizing viral ligands and responding to them, disappear gradually during incubation; therefore, to pre- thereby exhibiting an innate preparedness [4, 8, 9]. Avian vent the penetration and growth of in the egg β-defensins (AvBDs) and cathelicidins (CTHLs) are ma- during embryonic development, new defense systems jor classes of antimicrobial peptides with distinctive ex- (not yet fully characterized) are required [2, 3]. Defensive pression patterns during early embryonic development responses also involve the recognition of pathogens by [9]. There are 14 AvBD genes (AvBD1–14) and 4 mem- Toll-like receptors (TLRs) present in blood vessels, and bers of the CTHL gene family (CTHL1, CTHLB1, CTHL2, by leukocytes that develop within the embryo (hetero- and CTHL3) [9]. Antimicrobial peptides such as AvBDs phils and macrophages) [4]. While there are good indica- have a broad spectrum of activity against Gram-negative tors that an innate immune response can be triggered, it and Gram-positive bacteria, as well as fungi [10]. The is not yet clear how an inflammatory response in the em- ovodefensin OvoDA1/gallin is a novel β-defensin-related bryonated egg would be controlled, as the cellular and antimicrobial peptide which appears to be expressed spe- molecular checkpoints in such a process are completely cifically in the avian oviduct and possesses anti-Escherich- unknown. ia coli activity [11–13]. Much less is known about the an- The developing embryo is able to trigger an tibacterial peptide natural killer (NK)-lysin (the chicken immune response to a pathogen just prior to hatching, a ortholog of human granulysin), which is a novel effector characteristic that is routinely exploited in modern, large- of cytotoxic T cells and NK cells [14, 15]. The chicken scale poultry production with the administration of ortholog of -expressed antimicrobial peptide-2 in ovo for multiple pathogens, including (cLEAP-2) is a cationic antimicrobial peptide (CAMP) Marek’s disease (MD) and infectious bursal disease (IBD). that is expressed in chicken epithelial tissues and upregu- Embryonic development takes 21 days, and the first signs lated in response to Salmonella enterica serovar Enteriti- of a developing immune system are observed by the 10th dis infection [16, 17]. NK-lysin and cLEAP-2 have not day (ED10). On ED11 and ED12, T cells and B cells are been detected in the egg or within extra-embryonic struc- developed, respectively, with differentiation occur- tures to date, but are expressed in the chick embryo [14, ring after ED15. By ED18, the chicken embryo is immu- 16]. Maternal stimulation with TLR ligands was observed nocompetent and capable of producing both an innate to modulate oviduct expression of components of innate and an adaptive response to pathogens [5, 6]. During in- immunity such as proinflammatory , AvBDs, cubation and after hatching, the yolk sac (YS) membrane and CTHLs [18, 19].

112 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 ED0 Eggshell Physical barrier and Egg white antimicrobial molecules Physicochemical barrier (pH, (OCX36, LYZ, TF) viscosity) and antimicrobial molecules (LYZ, TF, antiproteases)

Egg yolk Vitelline membrane Maternal IgY Physical barrier and antimicrobial molecules (AvBD11, VMO1, LYZ)

Amniotic sac Physical barrier (membrane) Protection against dehydration and antimicrobial molecules (LYZ, TF, AvBD11, BMSP, ED8 Chorioallantoic membrane etc.) /allantoic sac Physical barrier (membrane) Acid pH (allantoic fluid) Immune cells Toll-like receptors/interferons Yolk sac Antimicrobial molecules Physical barrier/assimilation of IgY

Fig. 1. Schema contrasting the basal innate defenses of the egg between ED0 (or unfertilized) and ED8 (developed extra-embryonic membranes).

Egg Basic Structures and Innate Immunity tion of the egg microbiome assembly and a link with the developing immune system after they hatch. Bacteria can The egg is formed as it traverses the oviduct of the sexu- infect the egg in 2 possible ways: by vertical transmission, ally mature hen, and it consists of 4 basic structures: yolk, directly from hen reproductive tissues to the egg during its vitelline membrane (VM), egg white (EW), and ES (Fig. 1). formation; or horizontally, by contact with the environ- These acellular structures serve as a source of nutrients and ment once the egg has been laid, through a defective shell energy as well as physical, chemical, and molecular defens- or incomplete cuticle [25, 26]. These protective systems are es to protect the chicken embryo against physical shock very effective against most pathogens except S. enterica se- and microbial infection in the course of its 21-day develop- rovar Enteritidis, a Gram-negative bacterium responsible ment [20–22] (Fig. 2). The freshly laid egg is therefore an for food-borne illness, that is able to survive and grow in enclosure that must remain free of any microorganisms in the EW because it can evade most egg antimicrobial mech- spite of the surface microbiota of the ES, where a variety of anisms [25, 27, 28]. However, bacteria that reach the yolk bacterial species coexist [23, 24]. The microbiome is essen- can easily proliferate, thanks to the abundance and neces- tial for development, health, and homeostasis throughout sarily complete diversity of all the yolk nutrients required an animal’s life. However, the origin and transmission pro- for chick development and growth in the absence of a ma- cesses governing animal microbiomes remain elusive for ternal blood supply. This remarkable yolk is maintained in nonhuman vertebrates, and oviparous vertebrates in par- the center of the egg by 2 EW-derived “suspensory liga- ticular. Eggs may function as transgenerational carriers of ments” (chalazae) and is surrounded by various protective the maternal microbiome, thus warranting characteriza- layers (the VM, EW, and ES).

Innate Immunity Development in J Innate Immun 2019;11:111–124 113 Embryonated Avian Egg DOI: 10.1159/000493719 ED11/ED12 and B cell development, ED13, B cell differentiation

ED4 Onset of circulating macrophages

ED3 Expression of TLRs

Embryogenesis Growth 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

ED12–ED17 Yolk sac Chorioallantoic Egg white transfer into membrane the amniotic sac Amniotic and allantoic sacs ED13–ED18 ED19 -Oral absorption of amniotic Allantoic and fluid/egg white mixture amniotic -Ion and water absorption membranes by the CAM from the degenerate allantoic fluid

ED17-2 days Yolk sac abdominal resorption

Fig. 2. Timeline contrasting the evolution of innate and adaptive immune systems between ED0 and ED21, over the course of embryogenesis and growth (ED0 to ED21). TLR, Toll-like receptor; CAM, chorioallantoic mem- brane.

Egg Yolk not produce antibodies or mount an effective adaptive The yolk accumulates during the process of vitellogen- response [35]. esis in the of the hen [29]. With the exception of The immunoglobulins in the blood of the hen at the maternal immunoglobulins, yolk compounds are largely time of egg formation are transferred to the yolk by endo- secreted by the liver and transported to the ovary via the cytosis via the Fcγ receptor (FcγR). Their specificity blood, mainly in the form of very low-density lipopro- therefore represents a snapshot at a given time of the ma- teins [30]. Yolk proteomic studies have identified over ternal immune system, reflecting the microbiota to which 200 proteins [31–34], with the most abundant including the hen has been exposed [33, 34]. In the yolk, IgY is the IgY, avidin (AVD), ovotransferrin (TF), transthyretin dominant immunoglobulin, with a singular structure that (TTR), cystatin (CST3), α-2 macroglobulin (A2M), apo- is similar to that of mammalian IgG. However, the hinge lipoprotein A1 (ApoA1) and a protein predicted to be a region of IgG is larger, which makes it more flexible than β-microseminoprotein (BMSP). Some antimicrobial IgY [36]. Indeed, the limited flexibility of avian IgY may molecules, e.g., lysozyme (LYZ) or TF, are found in the account for some unique biochemical properties, such as yolk but, overall, this fluid is rich in nutrients and pro- the inability to precipitate antigens at physiological salt vides a favorable environment for bacterial growth. The concentrations seen in chickens and ducks. For example, maternal immunoglobulins concentrated in this com- the 2 arms may be so closely aligned that they preclude partment are mainly used by the embryo at the time of crosslinking of epitopes on large antigens, and IgY recog- hatching, and within the following few days. In parallel, nition is biased towards short sequences compared to the the B lymphocytes first emigrate from the bursa to seed mammalian [37, 38]. secondary lymphoid organs (tonsils, etc.) about 3 days The selective transport of IgY from the yolk to the em- before hatching (ED18) whereas the first population of T bryonic circulation through the YS begins slowly on ED7 cells leaves the around ED6, with the second and (< 100 μg/day) [39] and involves YS receptors whose IgY- third waves of migration taking place on about ED12 and binding is pH-dependent [40]. IgY is then detected in around the time of hatching. Therefore, the embryo can- other compartments such as the EW, and the amniotic

114 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 and allantoic sacs [41–43]. Transport accelerates 3 days tion to its role in nutrition of the embryo, its strategic before hatching (> 600 μg/day), to strengthen the defens- position around the embryo and the yolk as well as its es of the future chick at the time of hatching, but also dur- unique physicochemical properties (pH and viscosity) ing the following weeks, pending the activation of B lym- and complement of antibacterial proteins make it an ef- phocytes and antibody synthesis by the embryo [44]. fective barrier against pathogens. More than 300 proteins Only 10% of total yolk IgY will be absorbed by the em- have been identified in the EW, 8 of which account for bryo, corresponding to YS resorption in the gut of the approximately 90% of the protein content: OVAL (54%), embryo [22, 30]. It is likely that residual IgY from the YS TF (12%), ovomucoid (SPINK7, 11%), ovoglobulin G2 provides local enteric protection [45, 46]. Overall, the (BPIFB2/G2/TENP, 4%), ovomucin (3.5%), LYZ (3.4%), contribution of the yolk to the protection of the embryo ovoinhibitor (SPINK5, 1.5%), and ovoglycoprotein is minimal before hatching. (ORM1, 1%) [55, 57–62]. OVAL likely serves as a nutri- tional source of amino acids for the embryo [63]. Ovo- Vitelline Membrane DA1/gallin, AvBD11, TF, LYZ, ovomucin, SPINK5, and The VM forms an extracellular protein matrix around SPINK7 participate in the antimicrobial activity of EW the oocyte and yolk and provides a physical separation [64–67]. In addition, SPINK5 and SPINK7 are major EW from the EW. It consists mainly of fibrous structural pro- protease inhibitors [68], which could protect EW pro- teins and antibacterial polypeptides. Its inner layer is teins from proteolysis until their assimilation by the em- equivalent to the mammalian zona pellucida, and its con- bryo [69, 70]. stituents are secreted by the ovarian granulosa cells and the The antimicrobial nature of unfertilized EW and its liver [47, 48]. The inner membrane contains mainly zona proteins encompass at least 4 distinct mechanisms: (1) pellucida proteins, which are critical for the adherence of the chelation of compounds essential for survival and spermatozoa to the oocyte during fertilization [47–49]. bacterial growth, (2) direct interaction with the bacterial Conversely, the outer membrane is formed after ovulation wall, (3) inhibition of invasive proteases, and (4) limiting in the infundibulum, the first segment of the oviduct and bacterial adherence [54, 64, 69, 70]. Although maternal the site of fertilization, which also provides both physical exposure to environmental microbes can selectively in- and molecular protection to prevent pathogens from crease EW antibacterial activity, the molecular details of reaching the yolk [50]. A proteomic study of total VM this phenomenon have not yet been deciphered [71]. IgA identified 137 proteins, including 4 major ones: ovalbumin and IgM are found in EW [59, 60], but their role and (OVAL, approx. 75%), LYZ (approx. 21%), VM outer mechanism of action during embryonic development re- membrane 1 (VMO1, approx. 1%), and AvBD11 (approx. main unclear. 1%) [50, 51]. The disulfide bridges formed between ovo- During incubation, water from the EW is transferred mucins are essential to maintain the integrity of the fibrous to egg yolk (76% by ED10) [72, 73]. On ED12, EW is VM network, even after the solubilization of the 2 major transferred to the amniotic sac. The mixture of amniotic outer membrane proteins LYZ and VMO1 [52, 53]. At fluid (AmF) and EW is then orally absorbed by the em- least 35 of the proteins identified in the VM have con- bryo from ED13 onwards, to accompany the intensive firmed or predicted antibacterial activity [51]. Three of the phase of growth of the embryo body and organs during major proteins, LYZ, VMO1, and AvBD11, can act direct- the second half of incubation [22, 74, 75]. Part of the EW ly against S. enterica serovar Enteritidis [54]. The total con- protein is absorbed across the gut epithelium and redi- centrations of ovomucin and LYZ are 17 times higher in rected to the organs of the embryo [76, 77], and OVAL the outer membrane than in the EW, indicating the strong can be detected in the brain, spinal cord, and muscle tis- antibacterial potential, both bacteriostatic and bacteriolyt- sues [69, 70]. Other proteins are transported to the YS [74, ic, of the VM. However, the integrity of the membrane is 78, 79], to be digested with the other yolk compounds severely impacted by the age of the hen, and also affected before transfer to the embryo. The amino acids, peptides, by the duration and temperature of egg storage [55]. This or proteins resulting from embryonic metabolism are se- deterioration is partially due to the solubilization of mem- creted into the allantoic sac [80, 81], from where they can brane proteins such as VMO1 and AvBD11 [56]. be reabsorbed by the chorioallantoic membrane (CAM) [82]. Direct transfer of gut proteins to the allantoic sac is Egg White also likely to occur [83]. EW anti-Listeria monocytogenes The EW is deposited around the VM during the pas- and anti-Streptococcus uberis activities are maintained sage of the forming egg through the magnum. In addi- during embryonic development up to ED12 [84], and EW

Innate Immunity Development in J Innate Immun 2019;11:111–124 115 Embryonated Avian Egg DOI: 10.1159/000493719 antibacterial proteins have been shown to be active even cified membranes that enclose the EW and provide a scaf- after their transfer into the amniotic sac [20]. fold for the nucleation and growth of the calcitic mam- In addition to the activity of antibacterial proteins and millary cones and palisade layer (Fig. 3). Respiratory peptides, the physicochemical properties of the EW also pores traverse the ES to regulate the exchange of water play a major role in preventing the proliferation of invad- and respiratory gases, and partial dissolution of its inner ing pathogens. In a freshly laid egg, whether fertilized or portion with its calcium-rich composition provides an es- not, the EW pH increases rapidly from 7.6 to 9.0, attribut- sential component for skeleton mineralization during the able to the diffusion of CO2 through the ES [84]. This latter half of development [22, 91]. phenomenon not only affects the survival and growth of Regulation of ES mineralization within the acellular bacteria, but also their flagellar mobility and the oxidative uterine fluid is a poorly understood process; however, the stress experienced by bacteria [85]. It also modulates the organic matrix plays a key role [92–94]. More than 700 activity of certain antibacterial proteins, such as TF, proteins have been identified in this organic matrix [93, which chelates iron better, an element essential to certain 95]. Some are also associated with other egg structures bacteria such as S. enterica serovar Enteritidis [86], or (OVAL, LYZ, and TF), while others are relatively ES-spe- LYZ which loses its N-acetylmuramide glycanhydrolase cific (ovocleidin [OC]17 and OC116; ovocalyxin activity at an alkaline pH [87]. Overall, the bactericidal [OCX]21, OCX25, OCX32, and OCX36) and involved in and bacteriostatic activities of EW are enhanced at elevat- the biomineralization of the ES [92, 96]. The ES protein ed pH values [88]. The pH of the fertilized EW then grad- osteopontin has recently been shown to control ES nano- ually decreases to 7.5 by ED12. Conversely, the pH of EW structure, where the dimensions of the calcite mineral in incubated, unfertilized eggs continues to increase, structure correlate with ES hardness [97]. The outermost reaching 9.7 on the ED12 [84]. This difference is ex- layer of protein-rich cuticle contains antimicrobial pro- plained by the respiratory metabolism of the embryo, teins such as LYZ [98, 99]. which causes a decrease in the EW pH in embryonated The ES protects the contents of the egg and the embryo eggs and modifies the antibacterial properties (increased from physical assaults and is an impervious barrier to hydrolase activity of LYZ and decreased iron chelation penetration by microorganisms. Its strength is related not by TF). only to its thickness (300–400 μm), but also to its ultra- The mobility of pathogens is modified by fluid viscos- structure [92, 100]. Indeed, the morphology, size, num- ity [89], and EW will therefore slow pathogens and limit ber, and orientation of calcite crystals provide the ES with their access to yolk nutrients. The gelled structure of EW remarkable and unique mechanical properties. The final is directly related to the presence of ovomucin, a highly layer of vertical crystals (perpendicular to the ES) as well glycosylated protein composed of 2 subunits: an α sub- as the cuticle that covers the entire shell, prevent not only unit (MUC5B), low in carbohydrates, and a β subunit, water loss but also bacterial penetration. Cuticle quality with a higher carbohydrate content (MUC6) [55]. The (thickness and completeness of coverage) is highly heri- formation of complexes with LYZ reinforces this struc- table and strongly linked to resistance to bacterial pene- ture. However, the viscosity of EW is affected by many tration [27, 101]. parameters, such as storage conditions and time [90]. In- Interactions between inorganic mineral and organic deed, the increase in the pH of the EW during storage matrix proteins establish the unique ES architecture that causes dissociation of the complex formed between ovo- prevents most pathogens from accessing the egg interior. mucin and LYZ, which liquefies the EW and promotes This organic matrix contains a number of antibacterial the mobility of invasive bacteria. However, this liquefac- proteins such as LYZ, TF, OCX36, OC17, OCX32, CST3, tion may solubilize some proteins that were initially en- and the AvBDs [93, 95, 99, 102]. The mechanism of action trapped in the gel structure, resulting in the release of of these molecules within the ES remains unclear, but it their intrinsic antimicrobial activity. is likely that they are solubilized during limited ES disso- lution that occurs during embryonic development and Eggshell could also provide local protection at the interface be- The final step in egg formation is shell deposition, a tween the ES and extra-embryonic structures (Fig. 3). process that lasts about 18 h. The ES surrounds all the Nevertheless, some pathogens can reach the egg interior other structures of the egg and forms the first physical because of irregularities in the ES (a nonhomogeneous barrier protecting the egg against physical and microbial cuticle, an abnormal mammillary layer, microcracks re- aggression. The ES has different layers, including noncal- lated to physical damage, etc.) [103].

116 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 Unfertilized ED17

Palisades

Mammillae

Ground section Resorbed mammillary tips, Membranes membranes detached

Palisades

Mammillae Cross-section SEM

Membranes

Fig. 3. a Ground and polished sections and * scanning electron microscopy (SEM) of the eggshell (ES) and its membranes, showing dissolution of the innermost portion of the ES and detachment of the membranes dur- * ing incubation. Brackets, dissolved mam- millary tips; asterisks, intact mammillary tips; arrows, residual membrane fibers. face SEM Interior en Scale bars, 10 µm. b Cartoon depicting the * Membranes and mammillae Mammillae impact of the chorioallantoic membrane a on ES dissolution and its predicted role in innate defense. (Figure continued on next page.)

From the second half of incubation (ED10/ED11) to During the ES dissolution process, the tips of mammil- hatching (ED21), the ES is progressively degraded and lary cones are gradually dissolved, resulting in detach- used as a source of calcium by the embryo [82] (Fig. 3). ment of the shell membranes from the mineralized ES While most of the solubilized calcium is transported to (Fig. 3) [104]. Solubilization and calcium transport in- the embryo through the CAM for skeletal mineralization, volve a number of mechanisms, including a proton pump, a certain proportion is redirected to the YS for storage, to calcium-binding CAM proteins, calcium ATPases, and meet the nutritional needs of the chick after hatching, i.e., carbonic anhydrases [105, 106]. The process releases oc- at the time when the immune system starts to develop. In cluded antimicrobial proteins from the organic matrix this way, calcium may also be mobilized for biological (Fig. 3b), which, we hypothesize, are liberated to act lo- functions that start to gain prominence, such as hemato- cally. By the end of incubation, the thinning of the ES fa- poiesis and the development of lymphoid organs. The cilitates the emergence of the hatchling from the egg contribution of calcium to these processes in ovo remains [104]. However, this weakened ES could also more easily unknown. result in contamination of the egg and embryo in the lat-

Innate Immunity Development in J Innate Immun 2019;11:111–124 117 Embryonated Avian Egg DOI: 10.1159/000493719 ED10 ED16

Dissolution/resorption

Eggshell Mammillary knobs

H+ Antimicrobials Ca2+ Eggshell membrane Secretion Transport Chorionic epithelium

Mesoderm membrane Chorioallantoic

Allantoic epithelium b 3

er stages of development, and new developing structures tion, digestion, and respiration. These are the YS (sur- are necessary to resist such threats. rounding the yolk), the amniotic sac (amniotic mem- Microorganisms in close interaction with eggs may brane and AmF), and the allantoic sac (CAM and allan- act as a selective force on avian hatching success [107, toic fluid [AlF]). The adaptive defense mechanisms of the 108]. In this earliest stage of life, they may be harmful embryo become functional only in the first few weeks af- because of their potential pathogenicity against embryos. ter hatching [44, 112]. Indeed, during embryonic devel- However, only a small subset of bacterial species might opment, the primary (, thymus) and actually be pathogenic to the embryo, and an increase in secondary (, cecal tonsils [CT], and Peyer’s patches the number of nonpathogenic bacteria during incuba- [PP], i.e, the lymphoid organs in the gut intestine) organs tion could be seen as a complementary parental approach of the immune system are formed, but no antibodies are to avoid colonization by pathogenic bacteria through di- secreted. The development of PP and CT starts during rect inhibition or competitive exclusion [109]. Under- late embryogenesis (ED13), at the same time as the follicle standing which factors drive the microbial communities of the bursa. The appearance of surface IgM-positive cells on the ES may lead to a better comprehension of the in PP and CT is independent of the development of the evolutionary strategies that improve embryo survival follicle of the bursa [113]. Mature lymphocytes capable of [110, 111]. secreting immunoglobulins are not detected in the chick until 6 days after hatching [39]. Therefore, before hatch- ing, the IgY, IgA, and IgM present in the egg originate Embryonic Structures and Their Role in Egg Defense solely from maternal sources. The yolk IgY may defend the embryo during and after hatching, functioning simi- During the development of the embryo, extra-embry- lar to colostrum and breast milk immunoglobulins in vi- onic compartments and their contents are elaborated to viparous species; the IgA and IgM contained in the EW support the vital functions of the embryo, such as circula- are absorbed by the embryo during incubation (trans-

118 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 ferred from the albumen to yolk at ED12) and come into Yolk Sac contact with the intestinal mucosa, and could thus pro- During incubation, the cell structure of the YS gradu- vide enteric protection [83, 114]. ally replaces the VM, thus forming a new barrier. Analy- Concomitant with the progressive alteration/modifi- sis of its transcriptome has revealed that the YS synthe- cation of basic egg structures, new defense structures es- sizes many lipid metabolism proteins, but no antibacte- sentially composed of supporting tissues are put in place rial effectors [120]. However, the YS serves as a support quickly during incubation. Intestinal epithelial cells at for the cells of the , including ED17 have the capacity to take up and process bacteria, monocytes and macrophages, from ED10 and ED12, re- and they respond to bacterial products such as LPS and spectively [121]. The generation of (e.g., lipoteichoic acid with enhanced expression of proinflam- macrophages) in the YS and their infiltration of the em- matory genes (interleukin [IL]-6 and IL-18), acute-phase bryo have been demonstrated in the chick and in the proteins (AVD and LYZ) and secretory components from mouse. YS-derived macrophages in the chick were shown the polymeric immunoglobulin receptor [115]. Immune to enter the developing central nervous system indepen- cells such as macrophages enter the liver and kidneys at dently of vascularization. Their origin was confirmed ED12 and ED16, respectively [116]. Already at ED4/ED5, through the use of quail-chick YS chimeras. However, embryonic macrophages are observed in the blood vessels the interspecific chimera system used did not permit full and at perivascular locations; chicken embryonic macro- development, so there was no evidence from the chick phages are not recruited to incisional wounds, but they system as to whether the YS-derived cells were retained are able to recognize and phagocytose microbial antigens after hatching [121]. [117]. These immune cells can also be found in extra- embryonic membranes, particularly in the chorionic sur- Amniotic Membrane face of the CAM in response to an immune challenge The amniotic membrane represents the last physical [118]. Moreover, macrophages are active in embryonic barrier before reaching the embryo. Initially, it contains tissues; they can recognize and engulf microbes associ- only a few cells, but becomes more complex with the ap- ated with blood vessel walls and respond to chemotactic pearance of epithelial cells on its surface, as well as a fi- signaling [117]. brous matrix that seems to strengthen the structure [22]. A general trend of increased expression of certain However, from ED14, the membrane no longer grows AvBDs and CTHLs, as well as cLEAP-2 and NK-lysin, and, therefore, to support the growth of the embryo, it during embryogenesis is observed; notably, the expres- stretches and becomes weaker. The plasticity of this mem- sion of AvBD9 and AvBD10 increases considerably by brane is essential to support the progressive but massive ED9 and ED12, respectively, during normal development inflow of water from the beginning of development, and [9]. From ED12 onwards, AvBD9 is predominantly found in EW during the second half of incubation. During the in enteroendocrine cells throughout the gut, while CTHL2 first half of incubation, the water contained in EW is re- is exclusively found in heterophils [119]. Embryonic ex- directed to the extra-embryonic compartments, passing pression of cLEAP-2 is maximal at ED7 [16], while NK- through the yolk before accumulating in the amniotic and lysin expression by thymic T cells increases continuously allantoic compartments to form the AmF and AlF. Dur- from ED16 [14]. ing the second half of incubation, these fluids will be re- A comprehensive assessment of the role of the extra- absorbed directly or indirectly by the embryo, to support embryonic structures in the defense of the embryo is not the acceleration of its metabolism during the growth yet available. Their strategic position around the embryo phase (Fig. 2) [72, 122]. and their role in the assimilation of the compounds of the In humans, proteome analysis of AmF has identified

ES, yolk, and EW, however, give these compartments pri- > 900 proteins, 25% of which are predicted to be involved ority for the relocation of the molecular defenses initially in the immune response [123, 124]. However, unlike in present in the egg and the implementation of new defense , chicken AmF does not collect excretory prod- systems. The appearance of extra-embryonic membranes ucts and its protein composition strikingly changes at at the beginning of incubation adds an additional level in mid-development due to the massive inflow of EW pro- the defense of the egg and the embryo: the CAM envelops teins, which are thereafter swallowed by the embryo to all the internal structures of the egg, the amniotic mem- support its growth. Proteomics has identified 91 nonre- brane isolates the embryo from the other compartments, dundant proteins delineating the chicken AmF proteome and the YS surrounds the egg yolk [1] (Fig. 1). at ED11, before EW transfer [20]. These proteins are es-

Innate Immunity Development in J Innate Immun 2019;11:111–124 119 Embryonated Avian Egg DOI: 10.1159/000493719 sentially associated with the metabolism of nutrients, im- Chorioallantoic Membrane mune response, and developmental processes. Forty- The CAM lines the ES and covers all the structures of eight proteins are common to both chicken and human the egg by ED11/ED12 [105]. Because its epithelium is in AmF, including serum albumin, ApoA1, and α-fetoprotein. direct contact with ES membranes, it is the second physi- Chicken AmF exhibits antibacterial activity on ED11, cal barrier after the ES during development (Fig. 2). This which is greatly enhanced after EW transfer, presumably strategic position allows the cells of innate immunity to due to the activity of LYZ, AvBD11, VMO1, and BMSP as act locally, in case of bacterial penetration through the ES. the most likely antibacterial candidates. Following EW In fact, a bacterial infection simulated by the deposition transfer, its antibacterial proteins may also contribute to of LPS (an essential component of the outer membrane protect the embryo before and after hatching, like the ver- of Gram-negative bacteria) directly on the CAM induces nix caseosa in humans [125]. Interestingly, several pro- a significant inflammatory response, resulting in hetero- teins recovered in the AmF prior and after EW transfer phil and monocyte recruitment to the stimulated site are uniquely found in birds (OVAL and its related pro- [118]. The CAM was the first tissue to reveal the presence teins OVAX and OVAY, and also AvBD11) or oviparous and action of interferon γ following viral infection [128], species (vitellogenin [VTG]1, VTG2, and riboflavin- and a functioning response to TLR ligands is present by binding protein [RBP]) [20]. ED10–14 [4]. CAM proteomic analysis at ED19 identified 2 bacte- Allantoic Fluid riostatic proteins, TF and RBP [129], which are poten- The AlF is a product of embryonic metabolism, spe- tially expressed by the CAM cells or arrive via the blood- cifically the metabolism of the main digestive organ, the stream. Because of their proximity, it is likely that other YS. The composition of the fluid is rich in nitrogen me- proteins of the EW or ES are found in the CAM, espe- tabolites such as uric acid and ammonia, a difficult me- cially since it has been demonstrated in quail that the dium for the survival and growth of bacteria. As the vol- CAM can endocytose large quantities of EW by fluid- ume of AlF decreases from ED13, these compounds are phase endocytosis [130]. Further analysis by proteomics concentrated and eventually precipitate to form urate and transcriptomics would provide a better understand- crystals [80]. From the second half of incubation, the im- ing of the development of CAM defense mechanisms that portant secretion of protons into the compartment, fol- protect the egg from pathogens. Moreover, live imaging lowing the acceleration of embryonic metabolism, induc- of the CAM to gain insight into leukocyte recruitment, es a drop in pH of about 2 units (pH 7 on ED8 to pH 5.5 penetration, and response to pathogens, using, for exam- on ED18) [126]. This phenomenon can induce function- ple, colony-stimulating factor 1 receptor (CSF1R) report- al changes at the protein level. er chicken immune cells [117], would be an exciting re- Major AlF proteins are mostly yolk proteins involved search avenue to explore. in lipid and vitamin metabolism, and metal ion trans- port, with few changes in this profile between ED8 and ED16. Characterization of proteolytic enzymes in these Conclusions embryonic fluids has identified 12 proteases in the AlF, compared to 5 in the AmF [127]. AlF appears to concen- The calcareous egg of birds and reptiles, and previ- trate digestive proteases such as aminopeptidase N ously dinosaurs, is a successful reproductive adaptation (ANPEP), dipeptidyl peptidase-4 (DPP4), meprin A to the desiccating terrestrial environment. Embryonic de- (MEP1A), and 72-kDa type IV collagenase prepropro- velopment within this autonomous chamber has been tein (MMP2), while proteases identified in both fluids shaped through evolution to resist physical and patho- could have a specific role in morphogenesis (hepatocyte genic challenges, while satisfying the metabolic and nu- growth factor activator [HGFA], suppressor of tumori- tritional needs of the developing embryo. The evolution genicity 14 [ST14], and astacin-like metalloendopepti- of the oviparous reproduction model to the viviparous dase [ASTL]) and hemostasis (prothrombin [F2] and co- model has led to important distinctions between corre- agulation factor X [F10]). However, aside from the acid- sponding extra-embryonic structures, particularly con- ic pH that may challenge bacterial survival in AlF if cerning the placenta. In humans, for example, the allan- contaminated, there is, to date, no clear evidence that AlF toic sac is not an independent structure as in the avian plays a significant a role in antibacterial defense during embryo, but forms part of the umbilical cord. The urine incubation. of the human embryo is therefore secreted directly into

120 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 the amniotic sac whereas the chicken embryo secretes likened to the mammalian placenta) has many unex- metabolic waste into the allantoic sac, thus forming the plored functions and plays a major role in the develop- AIF. However, the important presence of proteins and ment and protection of the living avian embryo. peptides in connection with the immune response and defense in human AmF is mirrored by the demonstration of such molecules in the avian AIF and AmF. In contrast, Acknowledgements much less is known about the specific molecules that in- tervene in the mobilization of calcium (decalcification) M.T.H. is grateful to LE STUDIUM for support during the preparation of this manuscript, and is a LE STUDIUM Research from the avian ES, and the potential upregulation of in- Fellow, Loire Valley Institute for Advanced Studies, Orléans- nate immune genes at this critical site. Tours, and BOA, INRA, Val de Loire Centre, Nouzilly, France. Transcriptomic and proteomic studies of the develop- M.T.H. and M.D.M. acknowledge funding from the Natural Sci- mental changes that occur in the CAM, a structure that ences and Engineering Research Council of Canada. embraces both the embryo and all associated extra-em- bryonic structures, and that constitutes the first barrier against ES-penetrating pathogens, will be fruitful, partic- Disclosure Statement ularly if augmented with functional studies of cellular and The authors declare no conflicts of interest. molecular changes. We believe that the CAM (that can be

References

1 Bellairs R, Osmond M. The Atlas of Chick De- chicken embryological development. Dev and the bacterial membrane. Biochem Bio-

velopment, 3rd ed. Oxford: Academic Press; Comp Immunol. 2009 Apr; 33(4): 516–24. phys Res Commun. 2009 Sep; 387(3): 500–3. 2014. 10 Cuperus T, Coorens M, van Dijk A, Haags- 18 Abdel-Mageed AM, Isobe N, Yoshimura Y. 2 Bornelöv S, Seroussi E, Yosefi S, Pendavis K, man HP. Avian host defense peptides. Dev Effects of different TLR ligands on the expres-

Burgess SC, Grabherr M, et al. Correspon- Comp Immunol. 2013 Nov; 41(3): 352–69. sion of proinflammatory cytokines and avian dence on Lovell et al.: identification of chick- 11 Gong D, Wilson PW, Bain MM, McDade K, β-defensins in the uterine and vaginal tissues en genes previously assumed to be evolution- Kalina J, Hervé-Grépinet V, et al. Gallin; an of laying hens. Vet Immunol Immunopathol.

arily lost. Genome Biol. 2017 Jun; 18(1): 112. antimicrobial peptide member of a new avian 2014 Dec; 162(3-4): 132–41. 3 Magor KE, Miranzo Navarro D, Barber MR, defensin family, the ovodefensins, has been 19 Abdel-Mageed AM, Nii T, Isobe N, Yoshimu- Petkau K, Fleming-Canepa X, Blyth GA, et al. subject to recent gene duplication. BMC Im- ra Y. Modulatory roles of proinflammatory

Defense genes missing from the flight divi- munol. 2010 Mar; 11(1): 12. cytokines on the expression of cathelicidins in

sion. Dev Comp Immunol. 2013 Nov; 41(3): 12 Hervé V, Meudal H, Labas V, Réhault-God- the lower regions of the oviduct of laying

377–88. bert S, Gautron J, Berges M, et al. Three-di- hens. . 2017 Nov; 99: 66–72. 4 Barjesteh N, Brisbin JT, Behboudi S, Nagy É, mensional NMR structure of Hen Egg Gallin 20 Da Silva M, Dombre C, Brionne A, Monget P, Sharif S. Induction of antiviral responses (Chicken Ovodefensin) reveals a new varia- Chesse M, De Pauw M, et al. The unique fea- against in embryonated tion of the β-defensin fold. J Biol Chem. 2014 tures of proteins depicting the chicken amni-

chicken eggs with toll-like receptor ligands. Mar; 289(10): 7211–20. otic fluid. Mol Cell Proteomics. DOI: 10.1074/

Viral Immunol. 2015 May; 28(4): 192–200. 13 Whenham N, Lu TC, Maidin MB, Wilson mcp.RA117.000459. 5 Davison TF. The immunologists’ debt to the PW, Bain MM, Stevenson ML, et al. Ovode- 21 Rehault-Godbert S, Herve-Grepinet V, Gau-

chicken. Br Poult Sci. 2003 Mar; 44(1): 6–21. fensins, an Oviduct-Specific Antimicrobial tron J, Cabau C, Nys Y, Hincke M. Molecules 6 Schilling MA, Katani R, Memari S, Cavana- Gene Family, Have Evolved in Birds and Rep- involved in chemical defence of the chicken ugh M, Buza J, Radzio-Basu J, et al. Transcrip- tiles to Protect the Egg by Both Sequence and egg. In: Nys Y, Bain M, Van Immerseel F, ed- tional Innate Immune Response of the Devel- Intra-Six-Cysteine Sequence Motif Spacing. itors. Improving the Safety and Quality of

oping Chicken Embryo to Newcastle Disease Biol Reprod. 2015 Jun; 92(6): 154. Eggs and Egg Products, vol 1: Egg Chemistry,

Virus Infection. Front Genet. 2018 Feb; 9: 61. 14 Cui J, Sofer L, Cloud SS, Burnside J. Patterns Production and Consumption. Cambridge: 7 St Paul M, Brisbin JT, Abdul-Careem MF, of gene expression in the developing chick Woodhead; 2011, pp. 183–208.

Sharif S. Immunostimulatory properties of thymus. Dev Dyn. 2004 Mar; 229(3): 480–8. 22 Romanoff AL. The Avian Embryo. Structural Toll-like receptor ligands in chickens. Vet Im- 15 Lee MO, Jang HJ, Han JY, Womack JE. Chick- and functional development. New York,

munol Immunopathol. 2013 Apr; 152(3-4): en NK-lysin is an alpha-helical cationic pep- USA: The Macmillan Compagny; 1960. 191–9. tide that exerts its antibacterial activity 23 Musgrove MT. Microbiology and safety of ta- 8 Kannaki TR, Reddy MR, Verma PC, Shanmu- through damage of bacterial cell membranes. ble eggs. In: Van Immerseel F, Nys Y, Bain M,

gam M. Differential Toll-like receptor (TLR) Poult Sci. 2014 Apr; 93(4): 864–70. editors. Improving the Safety and Quality of mRNA expression patterns during chicken 16 Michailidis G. Expression of chicken LEAP-2 Eggs and Egg Products, vol 2: Egg Safety and embryological development. Anim Biotech- in the reproductive organs and embryos and Nutritional Quality. Cambridge: Woodhead;

nol. 2015; 26(2): 130–5. in response to Salmonella enterica infection. 2011, pp 3–33.

9 Meade KG, Higgs R, Lloyd AT, Giles S, Vet Res Commun. 2010 Jun; 34(5): 459–71. 24 Wilson PB. Recent advances in avian egg sci-

O’Farrelly C. Differential antimicrobial pep- 17 Townes CL, Michailidis G, Hall J. The inter- ence: A review. Poult Sci. 2017 Oct; 96(10): tide gene expression patterns during early action of the antimicrobial peptide cLEAP-2 3747–54.

Innate Immunity Development in J Innate Immun 2019;11:111–124 121 Embryonated Avian Egg DOI: 10.1159/000493719 25 Gantois I, Ducatelle R, Pasmans F, Haeseb- 40 West AP Jr, Herr AB, Bjorkman PJ. The 54 Guyot N, Labas V, Harichaux G, Chessé M, rouck F, Gast R, Humphrey TJ, et al. Mecha- chicken yolk sac IgY receptor, a functional Poirier JC, Nys Y, et al. Proteomic analysis of nisms of egg contamination by Salmonella equivalent of the mammalian MHC-related egg white heparin-binding proteins: towards

Enteritidis. FEMS Microbiol Rev. 2009 Jul; Fc receptor, is a phospholipase A2 receptor the identification of natural antibacterial mol-

33(4): 718–38. homolog. Immunity. 2004 May; 20(5): 601– ecules. Sci Rep. 2016 Jun; 6(1): 27974. 26 Messens W, Grijspeerdt K, Herman L. Egg- 10. 55 Li-Chan EC, Kim HO. Structure and chemical shell penetration by Salmonella: a review. 41 Bencina D, Narat M, Bidovec A, Zorman-Ro- compositions of eggs. In: Mine Y, editor. Egg

Worlds Poult Sci J. 2005; 61(01): 71–85. js O. Transfer of maternal immunoglobulins Bioscience and Biotechnology. New Jersey: 27 De Reu K, Grijspeerdt K, Messens W, Heyn- and antibodies to Mycoplasma gallisepticum John Wiley and Sons; 2008. https://doi. drickx M, Uyttendaele M, Debevere J, et al. and Mycoplasma synoviae to the allantoic and org/10.1002/9780470181249.ch1. Eggshell factors influencing eggshell penetra- amniotic fluid of chicken embryos. Avian 56 Schäfer A, Drewes W, Schwagele F. Analysis

tion and whole egg contamination by different Pathol. 2005; 34: 463–472. of vitelline membrane proteins of fresh and bacteria, including Salmonella enteritidis. Int 42 Kramer TT, Cho HC. Transfer of immuno- stored eggs via HPLC. Z Lebensm Unters

J Food Microbiol. 2006 Dec; 112(3): 253–60. globulins and antibodies in the hen’s egg. Im- Forsch A. 1998; 206(5): 329–32.

28 Howard ZR, O’Bryan CA, Crandall PG, Ricke munology. 1970 Jul; 19(1): 157–67. 57 D’Ambrosio C, Arena S, Scaloni A, Guerrier SC. Salmonella Enteritidis in shell eggs: cur- 43 Rose ME, Orlans E, Buttress N. Immunoglob- L, Boschetti E, Mendieta ME, et al. Exploring rent issues and prospects for control. Food ulin classes in the hen’s egg: their segregation the chicken egg white proteome with combi-

Res Int. 2012; 45(2): 755–64. in yolk and white. Eur J Immunol. 1974 Jul; natorial peptide ligand libraries. J Proteome

29 Réhault-Godbert S, Guyot N. Vitellogenesis 4(7): 521–3. Res. 2008 Aug; 7(8): 3461–74. and Yolk Proteins, Birds. In: Skinner M, edi- 44 Fellah JS, Jaffredo T, Dunon D. Development 58 Liu Y, Qiu N, Ma M. Comparative proteomic tor. Encyclopedia of Reproduction. Amster- of the avian immune system. In: Davidson F, analysis of hen egg white proteins during ear- dam, London: Elsevier; 2018. https://doi. Kaspers B, Schat KA, editors. Avian Immu- ly phase of embryonic development by com- org/10.1016/B978-0-12-809633-8.20568-2. nol. London, UK: Elsevier; 2008. https://doi. binatorial peptide ligand library and matrix- 30 Noble RC, Cocchi M. Lipid metabolism and org/10.1016/B978-012370634-8.50006-8. assisted laser desorption ionization-time of

the neonatal chicken. Prog Lipid Res. 1990; 45 Beal RK, Powers C, Davison TF, Smith AL. flight. Poult Sci. 2013 Jul; 92(7): 1897–904.

29(2): 107–40. Avian gut function in health and disease. In: 59 Mann K. The chicken egg white proteome.

31 Farinazzo A, Restuccia U, Bachi A, Guerrier Perry GC, editor. Avian gut function in Proteomics. 2007 Oct; 7(19): 3558–68. L, Fortis F, Boschetti E, et al. Chicken egg yolk Health and Disease. Bristol, UK: CABI; 2006. 60 Mann K, Mann M. In-depth analysis of the cytoplasmic proteome, mined via combinato- 46 Engberg RM, Kaspers B, Schranner I, Kosters chicken egg white proteome using an LTQ

rial peptide ligand libraries. J Chromatogr A. J, Losch U. Quantification of the immuno- Orbitrap Velos. Proteome Sci. 2011 Feb; 9(1):

2009 Feb; 1216(8): 1241–52. globulin classes IgG and IgA in the young and 7. 32 Mann K, Mann M. The chicken egg yolk plas- adult pigeon (Columbia livia). Avian Pathol. 61 Omana DA, Liang Y, Kav NN, Wu J. Pro-

ma and granule proteomes. Proteomics. 2008 1992; 21: 409–420. teomic analysis of egg white proteins during

Jan; 8(1): 178–91. 47 Takeuchi Y, Nishimura K, Aoki N, Adachi T, storage. Proteomics. 2011 Jan; 11(1): 144–53. 33 Hamal KR, Burgess SC, Pevzner IY, Erf GF. Sato C, Kitajima K, et al. A 42-kDa glycopro- 62 Qiu N, Liu W, Ma M, Zhao L, Li Y. Differ- Maternal antibody transfer from dams to tein from chicken egg-envelope, an avian ho- ences between fertilized and unfertilized their egg yolks, egg whites, and chicks in meat molog of the ZPC family glycoproteins in chicken egg white proteins revealed by 2-di-

lines of chickens. Poult Sci. 2006 Aug; 85(8): mammalian Zona pellucida. Its first identifi- mensional gel electrophoresis-based pro-

1364–72. cation, cDNA cloning and granulosa cell-spe- teomic analysis. Poult Sci. 2013 Mar; 92(3):

34 Smith NC, Wallach M, Miller CM, Braun R, cific expression. Eur J Biochem. 1999 Mar; 782–6.

Eckert J. Maternal transmission of immunity 260(3): 736–42. 63 Da Silva M, Beauclercq S, Harichaux G, Labas to Eimeria maxima: western blot analysis of 48 Waclawek M, Foisner R, Nimpf J, Schneider V, Guyot N, Gautron J, et al. The Family Se- protective antibodies induced by infection. WJ. The chicken homologue of zona pellucida crets of Avian Egg-Specific Ovalbumin and

Infect Immun. 1994 Nov; 62(11): 4811–7. protein-3 is synthesized by granulosa cells. Its Related Proteins Y and X. Biol Reprod.

35 Janković BD, Isaković K, Lukić ML, Vujanović Biol Reprod. 1998 Nov; 59(5): 1230–9. 2015 Sep; 93(3): 71. NL, Petrović S, Marković BM. Immunological 49 Okumura H, Kohno Y, Iwata Y, Mori H, Aoki 64 Baron F, Jan S, Gonnet F, Pasco M, Jardin J, capacity of the chicken embryo. I. Relation- N, Sato C, et al. A newly identified zona pel- Giudici B, et al. Ovotransferrin plays a major ship between the maturation of lymphoid tis- lucida glycoprotein, ZPD, and dimeric ZP1 of role in the strong bactericidal effect of egg sues and the occurrence of cell-mediated im- chicken egg envelope are involved in sperm white against the Bacillus cereus group. J

munity in the developing chicken embryo. activation on sperm-egg interaction. Bio- Food Prot. 2014 Jun; 77(6): 955–62.

Immunology. 1975 Sep; 29(3): 497–508. chem J. 2004 Nov; 384(Pt 1): 191–9. 65 Bourin M, Gautron J, Berges M, Attucci S, Le 36 Müller S, Schubert A, Zajac J, Dyck T, Oelk- 50 Chung WH, Lai KM, Hsu KC. Comparative Blay G, Labas V, et al. Antimicrobial potential rug C. IgY antibodies in human nutrition for study on histological structures of the vitelline of egg yolk ovoinhibitor, a multidomain Ka-

disease prevention. Nutr J. 2015 Oct; 14(1): membrane of hen and duck egg observed by zal-like inhibitor of chicken egg. J Agric Food

109. cryo-scanning electron microscopy. J Agric Chem. 2011 Dec; 59(23): 12368–74.

37 Schade R, Henklein P, Hlinak A, de Vente J, Food Chem. 2010 Feb; 58(3): 1794–9. 66 Chen J, Shallo Thesmar H, Kerr WL. Out- Steinbusch H. Specificity of Chicken (IgY) 51 Mann K. Proteomic analysis of the chicken growth of Salmonellae and the physical prop- versus Rabbit (IgG) Antibodies Raised against egg vitelline membrane. Proteomics. 2008 erty of albumen and vitelline membrane as

Cholecystokinin Octapeptide (CCK-8). AL- Jun; 8(11): 2322–32. influenced by egg storage conditions. J Food

TEX. 1996; 13(5): 80–5. 52 Back JF, Bain JM, Vadehra DV, Burley RW. Prot. 2005 Dec; 68(12): 2553–8. 38 Warr GW, Magor KE, Higgins DA. IgY: clues Proteins of the outer layer of the vitelline 67 Nakimbugwe D, Masschalck B, Deckers D, to the origins of modern antibodies. Immunol membrane of hen’s eggs. Biochim Biophys Callewaert L, Aertsen A, Michiels CW. Cell

Today. 1995 Aug; 16(8): 392–8. Acta. 1982 Jul; 705(1): 12–9. wall substrate specificity of six different lyso- 39 Kowalczyk K, Daiss J, Halpern J, Roth TF. 53 Kido S, Doi Y. Separation and properties of zymes and lysozyme inhibitory activity of Quantitation of maternal-fetal IgG transport the inner and outer layers of the vitelline bacterial extracts. FEMS Microbiol Lett. 2006

in the chicken. Immunology. 1985 Apr; 54(4): membrane of hen’s eggs. Poult Sci. 1988; Jun; 259(1): 41–6.

755–62. 67(3): 476–86.

122 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719 68 Saxena I, Tayyab S. Protein proteinase inhibi- 82 Moran ET Jr. Nutrition of the developing em- 95 Mann K, Macek B, Olsen JV. Proteomic

tors from avian egg whites. Cell Mol Life Sci. bryo and hatchling. Poult Sci. 2007 May; analysis of the acid-soluble organic matrix of

1997 Jan; 53(1): 13–23. 86(5): 1043–9. the chicken calcified eggshell layer. Pro-

69 Shinohara H, Iwasaki T, Miyazaki Y, Matsuo 83 Kaspers B, Bondl H, Göbel TW. Transfer of teomics. 2006 Jul; 6(13): 3801–10. K, Aoki T, Matsumoto M, et al. Thermostabi- IgA from albumen into the yolk sac during 96 Tian X, Gautron J, Monget P, Pascal G. What lized ovalbumin that occurs naturally during embryonic development in the chicken. makes an egg unique? Clues from evolution-

development accumulates in embryonic tis- Zentralbl Veterinarmed A. 1996 Jun; 43(4): ary scenarios of egg-specific genes. Biol Re-

sues. Biochim Biophys Acta. 2005 May; 225–31. prod. 2010 Dec; 83(6): 893–900.

1723(1-3): 106–13. 84 Guyot N, Réhault-Godbert S, Slugocki C, Ha- 97 Athanasiadou D, Jiang W, Goldbaum D, Sal- 70 Sugimoto Y, Sanuki S, Ohsako S, Higashimo- richaux G, Labas V, Helloin E, et al. Charac- eem A, Basu K, Pacella MS, et al. Nanostruc- to Y, Kondo M, Kurawaki J, et al. Ovalbumin terization of egg white antibacterial proper- ture, osteopontin, and mechanical proper-

in developing chicken eggs migrates from egg ties during the first half of incubation: A com- ties of calcitic avian eggshell. Sci Adv. 2018; white to embryonic organs while changing its parative study between embryonated and 4:eaar3219.

conformation and thermal stability. J Biol unfertilized eggs. Poult Sci. 2016 Dec; 95(12): 98 Kulshreshtha G, Rodriguez-Navarro A, San-

Chem. 1999 Apr; 274(16): 11030–7. 2956–70. chez-Rodriguez E, Diep T, Hincke MT. Cu- 71 Bedrani L, Helloin E, Guyot N, Réhault-God- 85 Guyot N, Rehault-Godbert S, Nys Y, Baron F. ticle and pore plug properties in the table

bert S, Nys Y. Passive maternal exposure to Understanding the natural antibacterial de- egg. Poult Sci. 2018 Apr; 97(4): 1382–90. environmental microbes selectively modu- fences of egg white and their regulation. In: 99 Rose-Martel M, Du J, Hincke MT. Pro- lates the innate defences of chicken egg white Roberts J, editor. Achieving sustainable pro- teomic analysis provides new insight into by increasing some of its antibacterial activi- duction of eggs. New England. Australia: Bur- the chicken eggshell cuticle. J Proteomics.

ties. BMC Microbiol. 2013 Jun; 13(1): 128. leigh Dodds Science Publishing Limited; 2012 May; 75(9): 2697–706. 72 Baggott GK, Deeming DC, Latter GV. Elec- 2017. https://doi.org/10.19103/AS.2016. 100 Nys Y, Gautron J, Garcia-Ruiz JM, Hincke trolyte and Water Balance of the Early Avian 0012.07. MT. Avian eggshell mineralization: bio- Embryo: Effects of Egg Turning. Sci Rev (Sin- 86 Tranter HS, Board RG. The influence of incu- chemical and functional characterization of

gap). 2000; 2002: 105–19. bation temperature and pH on the antimicro- matrix proteins. C R Palevol. 2004; 3(6-7): 73 Malik GK, Sinha SM, Saksena PN, Kapoor bial properties of hen egg albumen. J Appl 549–62.

AK, Mehra P, Bagchi M, et al. Amniotic fluid Bacteriol. 1984 Feb; 56(1): 53–61. 101 Bain MM, McDade K, Burchmore R, Law A, proteins in relation to fetal maturity. Indian J 87 Banerjee P, Keener KM, Lukito VD. Influence Wilson PW, Schmutz M, et al. Enhancing

Pediatr. 1981 Mar-Apr; 48(391): 149–52. of carbon dioxide on the activity of chicken the egg’s natural defence against bacterial

74 Carinci P, Manzoli-Guidotti L. Albumen ab- egg white lysozyme. Poult Sci. 2011 Apr; penetration by increasing cuticle deposition.

sorption during chick embryogenesis. J Em- 90(4): 889–95. Anim Genet. 2013 Dec; 44(6): 661–8.

bryol Exp Morphol. 1968 Aug; 20(1): 107–18. 88 Alabdeh M, Lechevalier V, Nau F, Gautier M, 102 Wellman-Labadie O, Lakshminarayanan R, 75 Geelhoed SE, Conklin JL. An electrophoretic Cochet MF, Gonnet F, et al. Role of incuba- Hincke MT. Antimicrobial properties of avi- study of proteins in chick embryonic fluids. J tion conditions and protein fraction on the an eggshell-specific C-type lectin-like pro-

Exp Zool. 1966; 162(3): 257–61. antimicrobial activity of egg white against Sal- teins. FEBS Lett. 2008 Mar; 582(5): 699–704. 76 Miska KB, Fetterer RH. The mRNA expres- monella Enteritidis and Escherichia coli. J 103 Nascimento VP, Solomon SE. The transfer

sion of amino acid and sugar transporters, Food Prot. 2011 Jan; 74(1): 24–31. of bacteria (Salmonella enteritidis) accross aminopeptidase, as well as the di- and tri-pep- 89 Schneider WR, Doetsch RN. Effect of viscos- the eggshell wall of eggs classified as “poor”

tide transporter PepT1 in the intestines of Ei- ity on bacterial motility. J Bacteriol. 1974 Feb; quality. Anim Technol. 1991; 42: 157–66.

meria infected broiler chickens. Poult Sci. 117(2): 696–701. 104 Chien YC, Hincke MT, McKee MD. Ultra-

2017 Feb; 96(2): 465–73. 90 Silversides FG, Scott TA. Effect of storage and structure of avian eggshell during resorption 77 Speier JS, Yadgary L, Uni Z, Wong EA. Gene layer age on quality of eggs from two lines of following egg fertilization. J Struct Biol. 2009

expression of nutrient transporters and diges- hens. Poult Sci. 2001 Aug; 80(8): 1240–5. Dec; 168(3): 527–38. tive enzymes in the yolk sac membrane and 91 Freeman BM, Vince MA. Development of the 105 Gabrielli MG, Accili D. The chick chorioal- small intestine of the developing embryonic Avian Embryo. Dordrecht: Springer Nether- lantoic membrane: a model of molecular,

chick. Poult Sci. 2012 Aug; 91(8): 1941–9. lands; 1974. https://doi.org/10.1007/978-94- structural, and functional adaptation to 78 Baintner K Jr, Fehér G. Fate of egg white tryp- 009-5710-7. transepithelial ion transport and barrier sin inhibitor and start of proteolysis in devel- 92 Hincke MT, Nys Y, Gautron J, Mann K, Rod­ function during embryonic development. J

oping chick embryo and newly hatched chick. riguez-Navarro AB, McKee MD. The egg- Biomed Biotechnol. 2010; 2010: 940741.

Dev Biol. 1974 Feb; 36(2): 272–8. shell: structure, composition and mineraliza- 106 Narbaitz R, Bastani B, Galvin NJ, Kapal VK,

79 Sugimoto Y, Saito A, Kusakabe T, Hori K, tion. Front Biosci. 2012 Jan; 17(1): 1266–80. Levine DZ. Ultrastructural and immunocy- Koga K. Flow of egg white ovalbumin into the 93 Marie P, Labas V, Brionne A, Harichaux G, tochemical evidence for the presence of po- yolk sac during embryogenesis. Biochim Bio- Hennequet-Antier C, Rodriguez-Navarro larised plasma membrane H(+)-ATPase in

phys Acta. 1989 Sep; 992(3): 400–3. AB, et al. Quantitative proteomics provides two specialised cell types in the chick em- 80 Bolin G, Burggren WW. Metanephric new insights into chicken eggshell matrix bryo chorioallantoic membrane. J Anat.

development in the chicken embryo: glomer- protein functions during the primary events 1995 Apr; 186(Pt 2): 245–52. ular numbers, characteristics and perfusion. of mineralisation and the active calcification 107 Beissinger SR, Cook MI, Arendt WJ. The

Comp Biochem Physiol A Mol Integr Physiol. phase. J Proteomics. 2015 Aug; 126: 140–54. shelf life of eggs: testing egg viability us-

2013; 166: 343–350. 94 Rodríguez-Navarro AB, Marie P, Nys Y, ing a tropical climate gradient. Ecology.

81 Bolin G, Dubansky B, Burggren WW. Incuba- Hincke MT, Gautron J. Amorphous calcium 2005; 86(8): 2164–75. tion relative humidity induces renal morpho- carbonate controls avian eggshell mineraliza- 108 Cook MI, Beissinger SR, Toranzos GA, Rod­ logical and physiological remodeling in the tion: A new paradigm for understanding rap- riguez RA, Arendt WJ. Microbial infection embryo of the chicken (Gallus gallus domes- id eggshell calcification. J Struct Biol. 2015 affects egg viability and incubation behavior

ticus). Comp Biochem Physiol A Mol Integr Jun; 190(3): 291–303. in a tropical passerine. Behav Ecol. 2005;

Physiol 2017; 204: 185–196. 16(1): 30–6.

Innate Immunity Development in J Innate Immun 2019;11:111–124 123 Embryonated Avian Egg DOI: 10.1159/000493719 109 Cook MI, Beissinger SR, Toranzos GA, Ar- 116 Qureshi MA, Heggen CL, Hussain I. Avian 123 Cho CK, Shan SJ, Winsor EJ, Diamandis EP. endt WJ. Incubation reduces microbial macrophage: effector functions in health Proteomics analysis of human amniotic flu-

growth on eggshells and the opportunity for and disease. Dev Comp Immunol. 2000 id. Mol Cell Proteomics. 2007 Aug; 6(8):

trans-shell infection. Ecol Lett. 2005 May; Mar-Apr; 24(2-3): 103–19. 1406–15.

8(5): 532–7. 117 Balic A, Garcia-Morales C, Vervelde L, Gil- 124 Michaels JE, Dasari S, Pereira L, Reddy AP, 110 Grizard S, Dini-Andreote F, Tieleman BI, hooley H, Sherman A, Garceau V, et al. Vi- Lapidus JA, Lu X, et al. Comprehensive pro- Salles JF. Dynamics of bacterial and fungal sualisation of chicken macrophages using teomic analysis of the human amniotic fluid communities associated with eggshells dur- transgenic reporter genes: insights into the proteome: gestational age-dependent

ing incubation. Ecol Evol. 2014 Apr; 4(7): development of the avian macrophage lin- changes. J Proteome Res. 2007 Apr; 6(4):

1140–57. eage. Development. 2014 Aug; 141(16): 1277–85. 111 van Veelen HP, Salles JF, Tieleman BI. Mi- 3255–65. 125 Tollin M, Jägerbrink T, Haraldsson A, Ager- crobiome assembly of avian eggshells and 118 Valdes TI, Kreutzer D, Moussy F. The chick berth B, Jörnvall H. Proteome analysis of

their potential as transgenerational carriers chorioallantoic membrane as a novel in vivo vernix caseosa. Pediatr Res. 2006 Oct; 60(4):

of maternal microbiota. ISME J. 2018 May; model for the testing of biomaterials. J 430–4.

12(5): 1375–88. Biomed Mater Res. 2002 Nov; 62(2): 273–82. 126 Boutilier RG, Gibson MA, Toews DP, An- 112 Bar-Shira E, Sklan D, Friedman A. Establish- 119 Cuperus T, van Dijk A, Dwars RM, Haags- derson W. Gas exchange and acid-base regu- ment of immune competence in the avian man HP. Localization and developmental lation in the blood and extraembryonic flu- GALT during the immediate post-hatch pe- expression of two chicken host defense pep- ids of the developing chicken embryo. Respir

riod. Dev Comp Immunol. 2003 Feb; 27(2): tides: cathelicidin-2 and avian β-defensin 9. Physiol. 1977 Sep; 31(1): 81–9.

147–57. Dev Comp Immunol. 2016 Aug; 61: 48–59. 127 Da Silva M, Labas V, Nys Y, Réhault-God- 113 Kajiwara E, Shigeta A, Horiuchi H, Matsuda 120 Yadgary L, Wong EA, Uni Z. Temporal tran- bert S. Investigating proteins and proteases H, Furusawa S. Development of Peyer’s scriptome analysis of the chicken embryo composing amniotic and allantoic fluids

patch and cecal tonsil in gut-associated lym- yolk sac. BMC Genomics. 2014 Aug; 15(1): during chicken embryonic development.

phoid tissues in the chicken embryo. J Vet 690. Poult Sci. 2017 Aug; 96(8): 2931–41.

Med Sci. 2003 May; 65(5): 607–14. 121 Janse EM, Jeurissen SH. Ontogeny and func- 128 Isaacs A, Lindenmann J. Virus interference: 114 Bar-Shira E, Cohen I, Elad O, Friedman A. tion of two non-lymphoid cell populations I. The interferon. CA Cancer J Clin. 1988

Role of goblet cells and mucin layer in pro- in the chicken embryo. Immunobiology. Sep-Oct; 38(5): 280–90.

tecting maternal IgA in precocious birds. 1991 Aug; 182(5): 472–81. 129 Cordeiro CM, Hincke MT. Quantitative

Dev Comp Immunol. 2014 May; 44(1): 186– 122 Baggott GK. Development of extra-embry- proteomics analysis of eggshell membrane 94. onic membranes and fluid compartments. proteins during chick embryonic develop-

115 Bar Shira E, Friedman A. Innate immune Avian Biol Res. 2009; 2(1): 21–6. ment. J Proteomics. 2016 Jan; 130: 11–25. functions of avian intestinal epithelial cells: 130 Yoshizaki N, Ito Y, Hori H, Saito H, Iwasawa response to bacterial stimuli and localization A. Absorption, transportation and digestion of responding cells in the developing avian of egg white in quail embryos. Dev Growth

digestive tract. PLoS One. 2018 Jul; Differ. 2002 Feb; 44(1): 11–22. 13(7):e0200393.

124 J Innate Immun 2019;11:111–124 Hincke et al. DOI: 10.1159/000493719