Morphology of the avian yolk sac

The avian yolk sac is a multifunctional extraembryonic organ that serves not only as a site of nutrient (yolk) absorption, but also for early hemopoiesis, and formation of blood vessels. Although the yolk sac membrane being specialized to function as an extraembryonic absorptive organ, it is neither morphologically nor functionally part of the embryonic gut. Yolk absorption is by the phagocytic activity of the extraembryonic endoderm. I used cryohistology and resin embedding histology of complete developmental series of Japanese quail to document the development of the avian yolk sac and changes of the microscopic anatomy throughout development. This material is complemented by complete series of MRT‐scans of live ostrich embryos from beginning of incubation through hatching. Considerable changes of size and shape of the yolk mass are documented and discussed as resulting from water flux from albumen to yolk associated with the biochemical activation of yolk sac proteins. During embryogenesis, the yolk sac endoderm forms villi that increase the absorptive surface and reach into the yolk ball. The histology of the absorptive epithelium is specialized for phagocytic absorption of yolk. During early developmental stages, the extraembryonic endoderm is single layered, but it eventually becomes several layers thick during later stages. The extraembryonic mesoderm forms an extensive layer of hematopoietic tissue; deep in this tissue lie the yolk sac vessels. During late stages of development, the erythropoietic tissue disappears, blood vessels are obliterated, and the yolk sac epithelium becomes apoptotic. Results are discussed in the light of the evolutionary history and phylogeny of the amniote egg.


| INTRODUCTION
The avian egg is an almost paradigmatic model of amniote eggs. However, diverging evolutionary pathways have led to different functional, developmental, and morphological features of the eggs in the diverse groups of amniotes (e.g. Packard & Packard, 1980;Elinson, Stewart, Bonneau, & Blackburn, 2015;Starck, Stewart & Blackburn, this issue of JMOR). However, knowledge of the amniote egg is scattered through the published literature, incomplete for morphology and clades providing the necessary phylogenetic coverage, and rarely comprehensive and integrative in an evolutionary context. While the eggshell and the chorioallantois have been covered to some degree by morphological studies, the analysis of the morphology of the yolk mass and the cellular yolk sac has been neglected. Therefore, this paper focuses on the morphology and histology of the avian yolk sac so that comparisons with other amniote eggs may be made and placed in a phylogenetic and evolutionary framework.
The avian egg is a cleidoic egg; that is, a protective layer of eggshell membranes, and a calcified, but semi-conductive eggshell (only water vapor and respiratory gases diffuse across shell membranes and shell) enclose proportionally large volumes of yolk and albumen. Yolk, albumen, and eggshell are the main constituents of the egg. The ovary and glandular oviduct tissues deposit them, and consequently, size, composition, and quality of these components depend on the females' physiological condition, that is, ultimately maternal fitness (e.g. Krist, 2011). Maternal condition together with the cleidoic nature of the egg determines and, ultimately, constrains avian embryonic and post-hatching development. Maternal effects like energy content, hormone levels, and immune globulin concentration of yolk affect the post-hatching life history parameters of the hatched chick and reach even into adulthood (e.g. Williams & Groothuis, 2015).
Among bird species, the yolk content of eggs ranges between 15% and 70% of the egg fresh weight, depending on egg size, length of incubation period, phylogeny, and mode of post-hatching development (Carey, Rahn, & Parisi, 1980;Ricklefs, 1977;Sotherland & Rahn, 1987). The yolk content of eggs of altricial birds is ca. 15%, in most precocial birds it ranges around 35%. In species with extended incubation periods, like megapodes (Megapodiidae), the yolk content may reach up to 50% of the egg fresh weight; in the Kiwi (Apteryx australis) the yolk content of the egg may reach 70% of fresh weight.
At laying, the yolk is a sphere, centered in the middle of the egg.
A thin, noncellular vitelline membrane surrounds it. The yolk contains almost all energy and other components necessary for the development of the bird, from first cleavage to hatching; only respiratory gases and water vapor pass through the eggshell. About 50% of the yolk is water, 16% is protein, 32% is fat, and about 2% is ash (Romanoff & Romanoff 1949;Mehner 1983). The yolk also contains vitamins (A, B-complex, C, E), minerals (Ca, Cu, Fe, K, Mg, Mn, Na, P, Sr, Zn;Hopcroft, Cowieson, Muir, & Groves, 2019), hormones (e.g. Merrill, Chiavacci, Paitz, & Benson, 2019), enzymes, antibodies, and carotenoids. Proteomic analysis revealed 119 different proteins from (unincubated) chicken egg yolk (Mann & Mann, 2008). More recent datasets list a total of 260 distinct proteins for egg yolk (and 148 for egg white, respectively). Proteomics and interactomics point to yolk proteins being functional in cell development and proliferation, cell-cell interaction and hematopoiesis, egg white proteins were mainly related to cell migration (D'Alessandro, Righetti, Fasoli, & Zolla, 2010).
All components contained in the yolk need to be absorbed, processed, and transferred to the developing embryo. The cellular yolk sac that serves these functions develops during early stages of embryogenesis. It continuously grows and adjusts to the increasing (energy) requirements of the developing embryo. Being functionally similar to the intestine, but distinct in histology, morphological origin, and time of functioning, the cellular yolk sac essentially functions as a non-gut absorptive organ. The mesodermal component of the cellular yolk sac also is the site of primary hemopoiesis and immune cells (review in Sheng, 2010).
Changes of size and shape of the yolk ball during the initial period of incubation have received little attention. However, they are not only universal among birds, but also indicative of functional and compositional processes concerning the yolk that occur even before the yolk sac membrane starts growing as an extraembryonic organ. Much later, before hatching, when the embryo prepares for independent living outside the egg, the cellular yolk sac regresses and yolk residues are incorporated into the body cavity of the young bird. Basic research on the avian yolk sac has been reported/summarized by Lillie (1908), Patten (1929), Raginosa (1961), Mossmann (1987), and Bellairs and Osmond (2005). Lambson (1970) has studied the ultrastructure of the endodermal cells of the yolk sac, for chicken, and Yoshizaki et al. (2004) that of Japanese quail. They provided convincing ultrastructural evidence for phagocytotic yolk uptake by the extraembryonic endoderm. McMillan (1979, 1981) studied the early period of chick embryogenesis showing that the hypoblast endoderm cells actively phagocytose yolk. Ectodermal cells were not involved in yolk phagocytosis. Physiological and biochemical evidence support phagocytotic yolk uptake by endodermal cells of the yolk sac (e.g. Sheng & Foley, 2012).
Many papers focus on specific detailed questions (e.g. protein composition: Mann & Mann, 2008;hemopoiesis: Sheng, 2010), or noninvasive imaging (Duce, Morrison, Welten, Baggott, & Tickle, 2011), but few efforts have been made at bringing morphological data together to allow reconstructing the evolutionary history of the amniote egg. However, the changing microscopic anatomy of the extraembryonic tissues throughout development will be essential information for any comparative analysis of the evolution of the amniote egg. Therefore, this paper looks at details of the development of the avian yolk sac throughout embryonic development. This paper aims at providing knowledge of the types of tissues and cells of the avian yolk sac (and their possible functioning), and how they change during development. To reach that goal, this report documents and reviews the changing histology of the avian yolk sac from the earliest embryonic stages through hatching and compares these data with that of other sauropsids in an evolutionary framework. Results of this study will be included in a phylogenetic analysis at the end of this issue of the Journal of Morphology (Starck et al. this issue of JMOR).

| MATERIALS AND METHODS
Fertilized eggs of Japanese quail (Coturnix japonica Temminck and Schlegel, 1849) were obtained from the animal livestock facility at the University of Halle. Eggs were incubated in an automated incubator at 38 C and 78% humidity, turned automatically during intervals of 11 hr with a 1 hr break, and were allowed to cool for 1 hr per day. Eggs were collected every day and either preserved by (a) freezing, by placing them in a commercial freezer (at −25 C) in natural position, or (b) by tissue samples of yolk sac dissected and fixed in 4% paraformaldehyde in 0.1 mol L −1 phosphate buffer. Staging of embryos was according to Starck (1989). Frozen eggs were peeled, mounted on stubs and sectioned (whole egg) using a cryostat. Sections were collected on permafrost slides, dried, and stained with Sudan black and basic fuchsin (Table 1). Paraformaldehyde preserved tissue samples of the yolk sac membrane were dehydrated through graded series of ethanol, embedded in hydroxyethyl methacrylate (Historesin; Leica Microsystems, Wetzlar, Germany), and thin sectioned at 2 μm section thickness using an AO Spencer No. 820 rotary microtome. Sections were mounted on slides and stained with Rüdeberg solution (0.1% methylene blue, 0.1% thionin and 0.1 mol L −1 Na 2 HPO 4 in distilled water; Rüdeberg, 1967; Table 2).
Microphotographs were taken using either an Olympus dot slide scanner microscope or a Zeiss Axiophot equipped with a Plan-Apochromat (63×1.4 oil DC, ∞/0.17) and an Axiocam ERc 5 s. Microphotographs taken at the Axiophot were processed (removing background shade) using Image J software (Rasband, 1997(Rasband, -2018, and stitched using Micro- Fertilized ostrich eggs (Struthio camelus Linnaeus, 1758) were purchased from a local farmer near Tübingen, Germany. Egg were incubated in an artificial incubator (same settings as given above), and submitted to magnetic resonance imaging once a week. After hatching, chicks were returned to the farmer. Magnetic resonance imaging was performed using a Vision Siemens Megatron 1.5 T, 64F tomograph at the Department of Neuroradiology, Section for Experimental Nuclear Resonance of the Central Nervous System, University of Tübingen. Images were taken as T1-or T2-weighted images (details given in figures captions).
Data were collected between 1996 at University of Tübingen and 1998 at University of Jena. Despite the long time since material collection and slide preparation, results of this study have not been published, except for two conference abstracts (Starck, 1999(Starck, , 2019.

| Shape and size of the yolk ball
At egg laying, the yolk has the form of a sphere, centered in the middle of the egg (Figure 1a). A thin vitelline membrane (not documented) covers the yolk ball superficially. At an equatorial position, two spiral bands, that is, chalazae, extend from the vitelline membrane reaching into albumen. They ultimately connect with the inner eggshell membrane at the blunt pole and the pointed pole of the egg. The yolk is deposited in layers. It has a core of liquid yolk (high water content), that is, nucleus of Pander (Figure 1a), which continues into the latebra ( Figure 2a), that is, a stalk of liquid yolk that reaches to the surface of the yolk ball, right below the blastodisc.
With beginning incubation, the yolk ball moves into an eccentric position under the eggshell (Figure 2a) with the blastodisc being closest to the eggshell. Connected to the chalazae, the yolk ball always turns so that the blastodisc is up, that is, at the highest position within the egg, and closest to the eggshell. Also, the air cell develops as an air filled space between inner and outer shell membrane at the blunt pole of the egg (Figure 2a, b). The primary function of the air cell is to compensate for volume changes due to water loss during incubation; thus, throughout incubation, it continuously changes its shape. The size of the air cell increases continuously during development until the end of incubation.
Within a few days after incubation, the yolk sac increases considerably in size (Figure 3a), and changes its shape, now expanding through the egg, forming several horizontal layers of yolk (Figures 1b,c,. For the quail, three layers were recognized: judged by the staining intensity, Y1 is dense yolk, similar to the original yolk ball, Y2 appears diluted, and Y3 is a highly diluted, watery yolk right under the embryo. These layers are transitory and disappear as the embryo grows and demands more space in the egg. For later stages, it is not possible to describe the shape of the yolk ball precisely, because it is largely liquefied, and its shape becomes During the growth period, the cellular yolk sac develops folds that reach into the superficial layers of the yolk mass (Figure 1d, f, h).
When the cellular yolk sac has fully overgrown the yolk mass, the development of the cellular yolk sac continues by extending the folds deeper into the yolk. These folds, however, never penetrate the entire yolk sphere; they always remain as a superficial layer of folds. In older quail embryos (e.g. 12-13 days of incubation; Figure 1g and h), they reach about 3-4 mm deep into the yolk mass, the more central parts of the yolk mass remain noncellularized.
As the embryo grows during development, it sinks deeper and deeper into the yolk, which, in the beginning, appears like a cushion, but later fills available space between the embryo and the eggshell.
There is no predictable pattern of how the embryo sinks into the yolk sac, and the distribution of the yolk in the egg during the latter part of the incubation appears random. During the last period of incubation, between aeriation of the lungs and actual hatching, the residual yolk sac is incorporated into the body cavity ( Figure 1g and 2e, f). It remains connected with the small intestine through the yolk stalk (not documented). showing folds of the endodermal cellular yolk sac reaching into the superficial layer of yolk. ab, albumen; ce, cerebellum; ey, eye; f, feathers; Fe, Femur; H, heart; Li, liver; lt, latebra; Lu, lung; mSt, muscular stomach; Si, small intestine; Y, yolk; Y1-3 layers of yolk that were observed a few days after incubation. Arrows point to folds of the yolk sac 3.2.2 | Histology and cytological differentiation of the yolk sac membrane The yolk sac membrane develops from the area opaca of the blastoderm. At its early stages, it is a bilaminar omphalopleure consisting of a thin, superficial layer of extraembryonic ectoderm, and a deep layer of relatively large cells filled with yolk droplets (Figure 4a), which is derived from the hypoblast. (i.e. "area opaca endoderm" as described in Bellairs & Osmond, 2005). The bilaminar omphalopleure is a transitory structure and represents the growing margin (precisely, the leading F I G U R E 2 Legend on next page.
edge is ectoderm only, the endoderm follows a few cells later [Yoshizaki et al., 2004

| Development of the cellular yolk sac
The cellular yolk sac is an extraembryonic organ designed to absorb yolk from the yolk sphere. As such, it overgrows the yolk sphere and finally surrounds the yolk. The closure of the yolk sac at the abembryonic pole of the egg was not studied here, but was subject of a detailed description by Lillie (1908;p. 217, figures 123, 129) and was referenced in numerous later publications (although unclear if repeated or just referenced Patten, 1929;Witschi, 1956;Mossmann, 1987;Raginosa, 1961). Lillie (1908) reported that an albumen-sac were established outside of the yolk-sac, when the expanding allantois reached the lower pole of the egg, and yolk sac and allantois were united by the undivided portion of the mesoblast around the yolk-sac umbilicus. This connection was never severed, and consequently the residuals of the albumen sac were incorporated into the body cavity together with the yolk sac before hatching.
However, these epithelial configurations have never been documented using histological methods, thus the microscopic anatomical arrangement of the epithelia around the yolk sac umbilicus remains to be investigated.

| Histological development of the yolk sac
Here, I documented the histological and cellular development of the yolk sac in detail throughout development. Some of the histology confirms earlier descriptions. In particular the consecutive developmental steps of bilaminar and trilaminar omphalopleure, the stratified organization of the yolk sac endoderm, and the phagocytotic uptake of yolk by those endoderm cells that are in immediate contact with the yolk. Lambson (1970) and Yoshizaki et al. (2004) already provided detailed ultrastructural evidence for phagocytotic yolk uptake in chick and quail embryos, respectively. McMillan (1979, 1981) used transmission electron microscopy, and injection and tracing experiments to document the endocytotic uptake of the yolk through endodermal cells of the early area vasculosa. More recently, Sheng and Foley (2012) provided biochemical evidence for phagocytotic yolk uptake while Bauer et al. (2013) studied differentiation processes that provide nutrient transport competence to yolk sac endodermal cells.
The formation of the folds of the yolk sac has been described here in detail. Although occasionally mentioned, their detailed microscopic anatomy has not, yet, been reported (to my knowledge). The important point here is that the folds reach only into the superficial layers of the yolk mass, and that they carry a blood vessel at their apical end. During the course of development, the folds reach deeper into the yolk, but never cellularize the entire yolk mass. The blood vessels are increasingly surrounded by hemopoietic tissue, so that at about mid-development a thick layer of hemopoietic tissue surrounds the blood vessels. Any nutrient transport from the yolk through the extraembryonic endoderm into the blood stream must pass through this layer of hemopoietic tissue. It is only towards the end of the incubation period that the hemopoietic tissue regresses from the yolk sac.
At the end of incubation, that is, around aeration of the lungs, the blood vessels in the yolk sac folds cease functioning and the cells of but, her drawings were not to the cellular level.  studied the yolk stalk of 2-week-old posthatching chicken using standard histology and showed that the tunica muscularis of the intestine forms a sphincter like thickening at the beginning of the yolk stalk.
This might explain some of the contrasting reports about the connection between yolk sac and intestine. While the residues of the yolk mass are slowly absorbed during early posthatching life, the residues of the yolk stalk gain importance as extramedullary myelopoietic tissue (Meckel's diverticulum) during later life of the bird (Olah, Glick, & Taylor, 1984). A modern documentation of the connection is missing.
How the residual yolk is absorbed remains largely unclear. From a physiological point of view, Romanoff (1944) showed that the residual F I G U R E 3 Dynamic changes of the yolk sac volume and yolk composition in Struthio camelus. (a) Volume changes (measured from serial MRT-images, throughout the development) during development. After beginning of incubation the volume of the yolk mass increases until week 3, then it continuously declines until hatching at week 7. (b) Magnetic resonance spectroscopy of the yolk at beginning of incubation (see Figure 2a). Measurements are relative and indicate the relative portions of water and lipid in the yolk. The white square in the right hand MRT-images indicates site of measurement (measured volume 1 cm 3 ). (c) Magnetic resonance spectroscopy of the yolk after 2 weeks of incubation (see Figure 2c). Measurement of the lower layer of yolk (= Y1 in Figure 2c). (d) Same egg at same time as in (C), but measurement was taken in the upper layer of yolk (= Y2 in Figure 2c). Comparing the chemical shift spectra in b, c, and d shows a change from almost equal contribution of water and lipids to a high water content in the upper yolk layer yolk in freshly hatched birds is quickly absorbed when birds are fed ad libitum, but its absorption is delayed when birds are fasted.
This indicates that yolk absorption is an active process that requires energy and, in contrast to prevailing paradigms, does not supplement periodically poor feeding conditions in newly hatched birds.
These findings were orchestrated by Noy and Sklan (2001) who showed that feeding chicks stimulated the release of yolk through the yolk stalk.

| Comparison of the avian yolk and cellular yolk sac with that of nonavian sauropsids
The nucleus of Pander and the Chalazae are typically avian features that have not been described for any nonavian sauropsid. Although it is generally problematic finding statements that structures are missing, Rathke (1866) and Reese (1908)  , incorporated cellular yolk sac. The cells of the folds of the cellular yolk sac appear largely necrotic, blood vessels of the yolk sac folds have ceased functioning, and the hemopoietic tissue has completely disappeared. Only few larger blood vessels in the outer wall of the cellular yolk sac are retained. Residual yolk fills the space between the folds. bv, blood vessel; eEc, extraembryonic ectoderm; nft, necrotic fold tissue; rY, residual yolk; *, artifact omphalopleure appears to overgrow the yolk mass, but the liquid yolk mass is invaded by proliferating endodermal cells, which phagocytose (and process?) the yolk material. These cells form clumps that progressively fill the entire yolk mass. Later, small blood vessels derived from the yolk sac vasculature invade the yolk sac cavity. Finally, the endodermal cells arrange in monolayers around these vessels (forming "spaghetti bands" as termed by Blackburn, this issue of the Journal of Morphology). This is substantially different from the avian pattern as described here, which progresses first as a bilaminar omphalopleure, then trilaminar omphalopleure, then the blood vessels move into folds of the extraembryonic endoderm, which ultimately becomes a stratified epithelium. The folds carrying the blood vessels reach only in the peripheral regions of the yolk while the center of the yolk mass remains uncellularized. The intensive development of hemopoietic tissue surrounding the blood vessels during most of the embryonic period, which regresses a few days before hatching, also is a feature that has not been described to non-avian sauropsids. There, blood islands are located on the external layers of the yolk sac, but appear not to form the circumvascular hemopoietic tissue. A detailed phylogenetic analysis is presented at the end of this issue if the Journal of Morphology.

| CONCLUSION
The avian yolk mass and the cellular yolk sac represent a number of clade specific features, that is, autapomorphic characters in a Open access funding enabled and organized by Projekt DEAL.

AUTHOR CONTRIBUTIONS
This is a single author contribution. Conception of the study, analyses, and writing of the manuscript was by the author. Open access funding enabled and organized by Projekt DEAL.

DATA AVAILABILITY STATEMENT
Histological slides are deposited at the Department of Biology II at Ludwig-Maximilians-University. They are available from the author upon request. CT-Image series will be made available at....