As a model animal for developmental biology, chick embryo is easy to control and observe during embryo development period and therefore it is widely used in the study of cardiac development. The application of proteomics has opened the door for large-scale studies to dissect both protein expression, regulation and function during chick heart developing stages. The proteomics study requires to quickly separate a large number of chick heart samples with the same developing stage. However, the traditional morphological standards based on Hamburger-Hamilton and Witschi stages are difficult to fulfill this requirement. Herein, we suppose a new standard for distinguishing chick heart morphology in different developing stages based on the relationship between the embryonic heart development and the amnion folding in chick. Based on this standard, we can accelerate the speed of embryonic heart sample separation and guarantee the quantity and quality of the sample more reliably.
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The heart is the first organ to perform its function during vertebrate embryo development (Wu et al., 2003). Understanding of molecular mechanisms for heart development has been a topic of inquiry. While untill now a large number of genes and their functions remain to be identified, although much progress in clarifying the genetic pathway of heart development has been made.
The chick is one of the ideal model in vertebrate research. It is a representative of polyyelk-egg animals, such as birds and reptiles and also is a transitional type between the simple development course, such as fish and amphibian animals and the complicated development course, such as mammalian. The study on chick development will not only establish a good basis in understanding the mammal embryology, but also offer information about the mammal early development, which is hard to be expounded, especially in the case of heart development (Lamers et al., 1991; Levin et al., 1995).
Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. It has been widely used in developmental study (Stults et al., 2005). The identification of protein molecules during different developing stages with proteomic technology will help to understand the development mechanisms and thus facilitate to discovery new strategies for the therapeutic and clinical management (Gallego-Delgado et al., 2005).
In order to utilize proteomics to study the proteins involved in chick heart at different development stages, it is required to distinguish the subtle developmental stages of embryo heart rapidly and accurately. However, the two traditional morphological standards from Hamburger-Hamilton (HH) and Witschi stages (Hamburger et al., 1951; Harh et al., 1975), which based on the number and size of somite, are not suitable for the proteomic studies, because it is difficult in practical operation to rapidly and accurately separating a large number of heart tissues from embryos at different stages to meet the requirement. In order to settle the problem, we studied the relationship between the embryonic heart development and the amnion folding in chick and appraised the subtle events of every cardiac developmental stage based on the amnion folding during the chick embryo development. As a result, a new morphology standard is established to distinguish heart developmental stages rapidly and accurately, which may provide a basis for the subsequential analysis on proteomics level during cardiac development.
Materials and Methods
White fertilized chick eggs of Hainan were bought from the animal husbandry place of Hunan Province.
Incubation of the Chick Embryos
Put the fresh fertilized egg in 37°C damp-regulating temperature testing incubator. The freshness of eggs will influence the experimental result directly, so dont keep eggs at room temperature exceeding 48 h, otherwise it will effect the normal development of the embryo seriously.
Micro-observation and Separation of the Embryos
According to the scheduled hatch time, take out the eggs after proper time of incubation and prepare a clean culture dish. Open the eggs carefully and transfer the yolk and albumin into the culture dish. Pay attention not to break the yolk membrane, otherwise the flowing-out yolk will probably conceal chick embryo, which is affixed to yolk membrane. Then, cut down the yolk membrane with dissecting scissors, ladle out chick embryo carefully and clean it in 1xPBS for two times, transfer it to the culture dish which is filled with the distilled water and observe it under the stereoscope.
Separation of Embryo Heart
Firstly, using dissecting needles get rid of membrane tissues around the embryo, we can judge the developmental stage and the position and form of the heart in the center of the embryo clearly. Then cut apart the portion with the heart, move it to the ultra-pure water and strip other tissues adhering with heart. It is important to get rid of additional tissues and to keep the heart tissue untouched, which ensure the dependability of the cardiac proteins extracted in the following treatment.
Photography of the Embryos and the Hearts
Take a photograph of each chick embryo and the heart with a Nikon 3 million digital camera.
RESULTS AND DISCUSSION
A New Method to Confirm the Chick Embryo Developmental Stage
In order to use the proteomics method to confirm embryo development incident at each developmental stage, we should first understand and describe the true events during chick heart development accurately. The traditional standards, HH and Witschi stages, are not easy to ascertain the developmental stage quickly and accurately, especially from HH14 to HH19.
|Table 1:||The morphology standard of the developmental stages of chick embryo|
During these stages, many important incidents emerge, such as looping of the cardiac tube, formation and expanding of the atrium and ventricle, initial beat of the heart, etc. (Garcia-Martinez et al., 1993; Manning et al., 1990; Martinsen, 2005).
To guarantee the consistency of embryo development stage and the repetitiveness and dependability of the results of proteomics assay, we have set up a new morphology standard to distinguish the developmental stages of chick embryo, especially the heart, which is mainly based on the cicatrisation degree of the amniotic border during the development stages from 48 to 75 h. The compartmentalization of this standard, the main incidents of heart development and the difference from that of HH are shown in Table 1.
The Relationship Between the Change of the Amniotic Fold and the Heart Development at Different Stages
It is undoubted that the amniotic fold change has close relationship with the key period of heart development (Eyal-Giladi et al., 1976), so it can be used as an important observation standard to divide the embryonic development stage and will be more accurate. Their relation is summarized as following.
At 48 h of embryo development, the cardiac tube begins to fold and loop and the amniotic edge appears in the top region adjacent to cardiac region (Fig. 1A). With further developing, the amnion begins to close gradually and to shift down below cardiac region (Fig. 1B). At 52 h, the heart looping is obvious, venous sinus and atrium appears in dorsal area and the ventricle and arterial conus appear in ventral area. Meanwhile, amniotic fold appears at the position that is ½ of the distance from heart to omphalomesenteric artery (Fig. 1C). At 54 h, amniotic fold shifts down to the position that is 1/3 of the distance from heart to omphalomesenteric artery (Fig. 1D). Then, the amniotic fold shifts down continually to the position above the omphalomesenteric artery (Fig. 1E). At 56 h, the upper limb buds appear, the amniotic fold heals near the omphalomesenteric artery. The heart becomes more obvious and larger compared to earlier stages. Once the cardiac tube closes completely, the venous sinus acts as the pacemaker and the heart begins to beat (Fig. 1F). At 65 h, the whole embryo bends further and the heart beats more vigorously (Fig. 1G). At about 75 h, the amniotic folds merges to an aperture, the truck crooks more, the atrium expands to the left (the back of embryo) and will be separated into right and left atriums soon. The venous sinus, which will develop into the right atrium, is located under the right side of atrium. The development of artery cone continues, which will divide into the main artery (Fig. 1H).
The Relation Between Different Embryo Developmental Stages and the Heart Morphological Characteristics
The cell movement at the beginning of the mesoderm formation forms a tiny-white trace at the caudal end of blastoderm at the beginning of embryo development. It is about 0.3-0.5 mm and is the earliest evidence of plumular axis. At 20 h, the primitive streak forms finally, which is 2.00 mm. At about 22 h head fold appears (Fig. 2A). At stage 24 h, the neural fold is not coalesced and the whole neural groove keeps open and 4 individual somites appear. In the mesoderm, a pair of incrassation regions appears, which will be a primitive heart forming region (Fig. 2B). At 30 h, the neural fold is coalesced. Both sides of endocardium get close to each other gradually and form the primitive cardiac tube (Fig. 2C).
|Fig. 1:||The relation between the change of amniotic fold and cardiac development at different stages|
At 34 h, the cardiac tube starts to bend as U shape. Meanwhile, 3 primitive brain vesicles can be observed and a pair of optic vesicles appear in the front of brain (Fig. 2D). At 38 h, the embryo bends to the right side and the three primitive brain vesicles start to divide into five brain districts, endbrain, interbrain, midbrain, hindbrain and myelencephalon (Fig. 2E). At 48 h, amniotic edge coalesces to the edge of anterior intestine portal; heart loops and folds; ventricle, atrium, artery sinus, artery conus and the aorta arch III can be observed clearly (Fig. 2F). At 65 h, amniotic edge coalesces close to the artery of the omphalomesenteric artery; the ventricle and artery conus moves to the front (abdomen) and the atrium and artery sinus fold to the rear (back ) (Fig. 2G). At 75 h, amniotic edge coalesces to the tail fold, forminga small aperture and the atrium expands. At 85 h, amnion edge coalesces completely; the heart tip appears; the atrium expands to the right and although no obvious demarcation line, the contraction area appears, which can be distinguished from ventricle; in this period, ventricle trabecula appears. After 100 h or later, the allantois becomes bigger; the heart with 4 chambers basically forms and the cardiac development is completed in essence (Fig. 2H).
Assuring accuracy and repeatability in the analysis is not only the precondition, but also the key point of the successful proteomics study. The two traditional standards, Hamburger-Hamilton (HH) and Witschi stages (Hamburger et al., 1951; Harh et al., 1975), are not suitable for the sample preparation for proteomic study because of the following reasons. Firstly, the dyeing will possibly influence the purity of proteins and thus will influence the results of the two-dimensional electrophoresis; secondly, the proteins are very easy to degrade in the course of dyeing and the only way to keep their integrality is to stripped the heart quickly and to preserved samples under -80°C; thirdly, the external development results in the individual difference of chick embryos, even if they are incubated for a same period under same condition. In order to set up a practical method to overcome these shortcomings, we dissected 2,000 chick embryos, separated and got about 1,500 of hearts and observed the morphologic characteristics of chick embryo in each stage.
|Fig. 2:||The morphological characters of chick embryo heart at different stages. The small image on the left corner is the heart of the corresponding stage. A. At 22 h, the head fold appears. B. At 24 h, the neural fold keeps open. C. At 30 h, The neural fold closes and the primitive cardiac tube forms. D. At 34 h, the cardiac tube curves as U shape. E. Developmental stage 38 h. F. At 48 h, the heart loops and folds; ventricle, atrium, artery sinus, artery conus and the arch of aorta III can be observed clearly. G. At 65 h, the left and right atriums and artery sinus are folded to the back. H. After 100 h, the allantois becomes bigger; the heart with 4 chambers basically forms and the cardiac development is almost completed|
We specially paid attention on the embryos of the stages between 48 to 100 h, because this is the important morphagenetic period of heart, during which a number of critical events of heart development happen (Garcia-Martinez et al., 1993). When the embryo developments to 48 h, the cardiac tube loops; to 56 h, the cardiac tube merges totally and begins to beat; to 65 h, heart looping shows its maximal curve; to 75 h, the atrium expands towards right and then the ventricle trabecula appears; after 100 h, atrioventricular septum forms basically, thus the typical heart with four distinct characteristic chambers comes into being (Martinsen, 2005).
The new method that we developed offers a work platform for the proteomics research on chick cardiac development. It remedies the insufficient of traditional standards, such as HH and Witschi, about the division of chick embryo development, confirms and subdivides the relation between the subtle events during chick heart development and relevant morphology characteristics, such as the folding and shifting of amnion and solves the problem that the external development is too inconsistent to be used to confirm the developmental stage. This standard can ensure a definite developmental stage accurately and quickly, offer a more scientific and more reliable basis for the collection of specific tissue at certain stage and assure the accuracy and repeatability of the process.
We are grateful to all members of the Center for Heart Development, College of Life Sciences in Hunan Normal University for their excellent technical assistance and encouragement. This study was supported in part by the National Natural Science Foundation of China (No. 90508004, 30470867, 30270722, 30570934, 30571048, 30570265), PCSIRT of Education Ministry of China (IRT0445), National Basic Research Program of China (2005CB522505) and the Foundation of Hunan Province (No. 04FJ2006).
- Garcia-Martinez, V. and G.C. Schoenwolf, 1993. Primitive-streak origin of the cardiovascular system in avian embryos. Dev. Biol., 159: 706-719.
- Harh, J.Y. and M.H. Paul, 1975. Experimental cardiac morphogenesis. I. Development of the ventricular septum in the chick. J. Embryol. Exp. Morphol., 33: 13-28.
- Manning, A. and J.C. McLachlan, 1990. Looping of chick embryo hearts in vitro. J. Anat., 168: 257-263.
- Martinsen, B.J., 2005. Reference guide to the stages of chick heart embryology. Dev. Dyn., 233: 1217-1237.