Arterial-Venous Differentiation in the Yolk Sac of Chick Embryos

Introduction

Vascular formation is a fascinating progression within chicken embryonic development. Its characteristic extrauterine (occurring outside the uterus) development enables an individual to visualize progression of the embryo. Storing the egg in low temperature environments allows one to temporarily freeze the embryo and resume experimental observations at a later date. Along with a regular laying cycle, these characteristics make the chicken a model organism for studying the formation of the circulatory system within the yolk sac and embryo of the egg. Such studies are crucial for us to gain understanding of the development of our own vascular system. The purpose of this page is to categorize the way in which arterial-venous differentiation occurs within the chick embryo yolk sac as described by Ferdinand Noble et al.

Vascular Development of a Chicken Embryo

Yolk Sac

The yolk sac is incredibly important in the development of the embryo. Through proper formation of the yolk sac vascular system vital nutrients such as, vitamins, ions and fatty acids can be transported to the developing embryo. This occurs because mesodermal cells migrate, along with the endodermal epithelial cell layer, into non-vascular spaces of the yolk sac ultimately forming the capillary plexus for initial nutrient transport [1,2]. The endodermal cells play a vital role in breaking down proteins into soluble amino acids to be transported within the vascular tissue [3].

More about the Yolk Sac:

Interesting fact – Human embryos develop a yolk sac providing nourishment before the onset of internal circulation.

The Onset of Blood Flow

With the onset of circulation, the capillary vascular network in the yolk sac begins forming the vitelline artery — artery associated with the yolk of the egg (Fig I A,B). Further expansion/remodeling results in the formation of a closed loop of veins and arteries within the yolk sac. This process of angiogenesis (formation of blood vessels) comprises the disconnection, splitting and remodeling of the capillary network within the yolk sac to form larger vessels [4]. The authors, Ferdinand Noble et al., wanted to know what regulates the formation of arterial-venous system in vitelline circulation. They show that it is achieved through the onset of blood flow, requiring endothelial plasticity.

Key Markers

ephrinB2 & NRP1: Receptor proteins involved in arterial differentiation

NRP2 & Tie2: Receptor proteins involved in venous differentiation

The authors, Ferdinand Noble et al., suggest a four step method

  1. Endothelial cells expressing genes specific for arterial or venous formation are localized to the posterior arterial pole and anterior venous pole, respectively. The border between  each vessel type is located in the same vascular tube [5].
  2. After the onset of circulation the vitelline artery begins to form near the posterior pole.
  3. Through disconnection of small side branches, arterial tube formation progresses further into the yolk sac.
  4. Because blood flow ceases in these disconnected segments, arterial markers (ephrinB2 and NRP1) become significantly downregulated. Subsequently, venous markers (NRP2 and TIE2) become upregulated to form veins from the disconnected segments. Once formed, capillary sprouts project outward from the vein towards the artery for reconnection to the arterial system.

Formation of the Vitelline Artery

The formation of the vitelline artery from the primary capillary plexus can easily be seen in Fig 2 A-H, beginning within the posterior regions of the embryo. Blood is brought back to the embryo in the anterior regions by veins. This vessel forms shortly after the onset of circulation. Blood flows from the heart through the aorta and into the arterial plexus indicated by the red arrows(Fig 2 A,B). This plexus becomes remodeled into the vitelline artery as indicated by the black arrows(Fig 2 C). This occurs through fusion of some capillary segments and the disconnection of others.

Expressions of Vascular Markers

Expression of arterial markers EphrinB2 and NRP1 in the early formation of the vitelline artery can be seen in Fig 3 A,B. As an advanced vascular system is yet to be established, most of the blood flows from the arteries to veins without passing through capillary vessels (Fig 3C). At the border between arteries and veins these markers become significantly downregulated (Fig 3 D,E). Arterial markers in the anterior (top) region in become significantly downregulated ultimately forming veins (Fig 3A).

Formation of Venous System from Disconnected Arterial Capillaries

The first vitelline veins that become connected to the arterial system are those that run parallel  to the vitelline artery. These were capillary vessels that were disconnected from the artery (Fig 5A). These disconnected vessels form the dark blood spots seen in Fig 4 A-H. Once blood was unable to perfuse through these vessels, arterial genes were significantly downregulated and venous markers become upregulated. These disconnected segments form veins, and capillary sprouts project outward for reattachment to the arterial system (Fig 5 B,D). As a result, the blood spots get flushed out of the veins (Fig 4 K,L). Enlargement of veins is achieved through the joining of other disconnected segments.

Role of Perfusion in the Formation of the Yolk Sac Vascular Plexus

The authors then asked what role does perfusion play in the formation of the yolk sac vascular plexus. It has been shown that the expression patterns of arterial and venous markers are established in the avian embryo before the onset of flow [6]. The authors have found similar results, however, vascular differentiation and patterning of the vascular plexus are entirely due to onset and continued progression of perfusion within the yolk sac. They performed several experiments effectively eliminating flow within the yolk sac of the chicken egg. In Fig 6 A, the yolk sac capillary plexus resembles that of an early stage embryo. The authors state that there is no arterial differentiation despite the presence of an arterial marker, ephrinB2, in some remote areas (Fig 6B). One of the experiments involved an incision that effectively separated the left side of the yolk sac from the embryo before the onset of perfusion. This resulted in the formation of a vitelline vein and an absence of arteries in the left side of the yolk sac (Fig. 6D). This does suggest that formation of the vitelline  artery does depend upon perfusion of the yolk sac. It is interesting to note that the right vitelline artery developed to twice its normal size and extended across the middle axis of the yolk sac.

Blood Flow within the Yolk Sac Controls Expression of Genes

In determining to what extent blood flow affects the expression of arterial and venous markers, the authors performed whole-mount in situ hybridization with ligated embryos (Fig. 7). They observed that expression of the arterial markers, NRP1 and ephrinB2,  were significantly downregulated as time progressed (Fig. 7 A-F). Examination of the expression of venous markers, Tie 2 and NRP2, one hour after ligation showed no apparent upregulation (Fig. 7 G,H). However, significant upregulation of these markers is apparent after several hours(Fig. 7 I,J). This suggests that the expression of both arterial and venous markers is regulated by blood flow, with regulation of venous markers less rapid in comparison. The authors allowed reperfusion of the vitelline artery at different times. This resulted in the restoration of arterial marker expression in a time dependent manner and often at reduced area (data not shown).

Fig 7: Arrows (A-H) indicate the site of ligation. A-F are arterial markers. G-J Indicate expression of venous markers. Notice G,H show no upregulation of venous markers. Source: Ferdinand Noble et al.

Strengths and Weaknesses

The authors did a great job at analyzing how blood flow affects the expression of arterial and venous markers. They also thoroughly studied the effects of perfusion on the formation of the initial vitelline artery. However, genetic studies could have been performed to study the effects of the expression of arterial and venous markers, particularly, on differentiation of arteries and veins in “no-flow” embryos. This may allude to a more definitive correlation between the expression of specific genes and perfusion in the differentiation of the vascular plexus in the yolk sac. However, genetics studies within chicken embryos is known to be rather limited and could be a reason why this was not properly addressed. Furthermore, in the experiments shown in Fig 6, studying the expression of more than one marker may yield stronger results. Other interesting questions that will need to be addressed include: how long after blood flow is ceased does the expression of arterial and venous markers change and what is the reason one is much more rapid than the other?

Summary

Prior to the onset of the perfusion, the yolk sac vascular plexus consists of capillaries. Once the heart of the embryo begins to beat and the yolk sac is perfused the vitelline artery begins to form and arterial markers, NRP1 and ephrinB2, become upregulated as blood flow persists. Progression is allowed through fusion of some capillary segments and disconnection of others. In disconnected segments, venous markers become upregulated because blood flow ceases in these areas. These disconnected segments form veins and reconnect to the arterial system.

Interesting Articles

Arterial Venous Specification During Development

Effect of Vascular Cadherin Knockdown on Zebrafish Vasculature during Development

Abnormal Vascular Development in Zebrafish Model for Fukutin and FKRP Deficiency

Resources

  1. Ferdinand Noble, Delphine Moyon, Luc Pardanaud et al. “Flow    Regualtes Arterial-Venous Differentiation in Chick Embryo.” Department of  Physiology, Cardiovascular Research Institute Maastricht (CARIM). (2004)
    PMID14681188
    http://www.ncbi.nlm.nih.gov/pubmed/?term=14681188
  2. Raimund Bauer, Julia A. Plieschnig, Thomas Finkes et al . “The  Developing Chicken Yolk Sac Acquires Nutrient Transport Competence by an  Orchestrated Differentiation Process of its Endodermal Epithelial  Cells.” American Society for Biochemistry and Molecular Biology.  (2012) PMID: 23209291                                          http://www.ncbi.nlm.nih.gov/pubmed/?term=23209291
  3. National Center for Biotechnology Information(NCBI)Endoderm.    Sinauer Associates. (2000) Book ID: NBK10107  http://www.ncbi.nlm.nih.gov/books/NBK9983/
  4. Risau, W. Mechanism of Angiogenesis. Nature 386, 671-674 1997.  PMID: 9109485      http://www.ncbi.nlm.nih.gov/pubmed/?term=9109485
  5. Yancopoulos, G. D., Suri, C., Jones, P. F. et al. (1996). Requisite role of  angiopoietin-1, a ligand for the TIE2 receptor, during embryonic  angiogenesis. Cell 87,1171 -1180.                                                                                      http://www.cell.com/abstract/S0092-8674(00)81813-9
  6. Moyon, D., Pardanaud, L., Yuan, L., Breant, C. and Eichmann, A. (2001b). Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo. Mech. Dev. 106,133 -136.  http://www.ncbi.nlm.nih.gov/pubmed/11472842

Last Updated: 23 April 2013 by Tyler Brown

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