Patterning of embryonic blood vessels - Wiley Online Library

Download PDF
18 downloads 159660 Views 511KB Size Report
Comment
tral side up for 7 hr in 1 ml of DMEM. (Gibco BRL/Life .... Adobe Photoshop 7.0 to measure the diameter of five ... size, NIH Image (version 1.62) was used to ...
DEVELOPMENTAL DYNAMICS 228:21–29, 2003

ARTICLE

Patterning of Embryonic Blood Vessels Amanda C. LaRue,1 Vladimir A. Mironov,1 W. Scott Argraves,1 Andra ´ s Cziro ´ k,2 Paul A. Fleming,1 and 1* Christopher J. Drake

Morphometric methods were developed to characterize the geometry of vascular patterns in avian and murine embryos. By using these methods, we found that networks of blood vessels formed during vasculogenesis share similar geometric properties (i.e., mean blood vessel diameters and avascular space diameters) regardless of developmental stage, location, or species in which they form. We also found that endothelial cell density within a unit area of an embryonic vasculature could be used to accurately distinguish between a small diameter, capillary-like vascular network (low endothelial cell density) and a large diameter, presinusoidal network (high endothelial cell density). Furthermore, we show that endothelial cell size remains constant in small and large diameter vessels, indicating that increased endothelial cell size is not the basis for diversity in vessel diameter. These observations serve as a foundation for future studies seeking to evaluate the effects of agents or genetic mutations on aspects of vasculogenesis. Developmental Dynamics 228:21–29, 2003. © 2003 Wiley-Liss, Inc. Key words: vascular patterning; vasculogenesis; morphogenesis; endothelial cell; coronary Received 25 February 2003; Accepted 8 May 2003

INTRODUCTION The morphogenesis of embryonic blood vessels has been studied since the beginning of the twentieth century. Sabin was one of the first to characterize the process, describing the formation of vascular plexi from isolated “angioblast clumps” within the area pellucida of the early chick embryo (Sabin, 1920). Later studies used scanning electron microscopy to examine blood vessel formation from cords of angioblasts in situ (Hirakow and Hiruma, 1981). A major advance was the development of antibodies to several angioblast/endothelial-specific molecules (QH1, Pardanaud et al., 1987; SCL/TAL-1, Kallianpur et al., 1994; Drake et al., 1997; and platelet endothelial cell

adhesion molecule-1 [PECAM], Newman et al., 1990; Newman and Albelda, 1992). Throughout this history, qualitative assessments of the dynamics of vascular development have predominated. Quantitative methods that can describe the spectrum of vascular patterns as they occur under both normal and experimental vasculogenesis have not been developed. We began this study by first establishing a method for quantitatively characterizing vascular patterns by using vessel and avascular space diameter metrics. We applied our method to the vascular patterns found at different stages in development, at various locations within developing embryos and in different

embryonic species. We also examined the number and size of constituent endothelial cells found in the various vascular pattern types in embryos. Our findings highlight both the high degree of conservation in the geometric configurations of embryonic blood vessels and the extent to which embryonic blood vessel size is a function of endothelial cell number as opposed to an increase in cell size or cell spreading.

RESULTS Vascular Pattern as Defined by Vascular and Avascular Diameter Measurements Figure 1A shows the developing vascular system of a representative 11-

1 Cardiovascular Developmental Biology Center, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 2 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas Grant sponsor: NIH; Grant number: HL57375; Grant number: HL52813; HL61873; Grant sponsor: DOD; Grant number: DAMD 17-00-1-0338. Drs. LaRue and Mironov contributed equally to this manuscript. *Correspondence to: Christopher J. Drake, Cardiovascular Developmental Biology Center, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. E-mail: [email protected]

DOI 10.1002/dvdy.10339

© 2003 Wiley-Liss, Inc.

22 LARUE ET AL.

the significance of two-dimensional distributions for paired vessel and avascular space diameter values from capillary-like networks and presinusoidal networks. The results of this test indicate that there is a significant difference (P ⬍ 10⫺6) between the paired measurements for capillary-like networks and presinusoidal networks in normal embryos. When the paired diameter values were plotted, the relationship between vessel diameter (DV) and avascular space diameter (DA) was determined to be an inverse relationship and also showed segregation (Fig. 2B). The significant statistical difference between measurements taken from normal capillary-like and presinusoidal networks suggests that DV and DA are sensitive parameters for characterizing and distinguishing vascular network pattern types.

Pattern Classification Using Vascular and Avascular Diameter Measurements

Fig. 1. Diversity in the normal pattern of embryonic blood vessels. A: A panoramic view of the vasculature of a normal 11-somite quail embryo immunolabeled with the quail endothelial cell-specific antibody QH1. Regulation of vascular pattern formation is evident by the diverse arrangements of blood vessels into distinctive vascular patterns, including the capillary-like networks (CLN), presinusoidal networks (PSN), and large vessels such as the dorsal aorta (da). B: Highlighted are the extremes of the early vascular networks (CLN, upper box; PSN, lower box) and the intermediate networks found between these extremes (asterisk) at higher magnification. Scale bars ⫽ 200 ␮m in A, 100 ␮m in B.

somite stage quail embryo immunolabeled with monoclonal antibodies to QH1, a marker of quail endothelial cells. Overall, the vascular pattern is made up of very small-caliber vessels that are separated by relatively large avascular spaces, progressively larger vessels surrounded by proportionally smaller avascular spaces, and finally very large vessels and sinuses that lack avascular spaces (i.e., dorsal aortae and sinus venosus). The extremes of vascular networks that compose an early embryonic vascular pattern are highlighted in Figure 1B. At one end of the spectrum are capillary-like networks (CLNs; Fig. 1B, upper box), which are composed of small caliber vessels and separated by relatively large avascular spaces. At the other extreme are the presinoidal networks (PSNs; Fig. 1B, lower box)

having larger caliber vessels and smaller avascular spaces. We first determined whether paired vessel diameter (DV) and avascular space diameter (DA) measurements compiled from the vascular pattern extremes (capillarylike and presinusoidal networks) of five 11-somite stage embryos could be used to quantitatively discriminate between various vascular network types (Fig. 2A). We found that mean DV for presinusoidal networks (MV,PSN) were 4.5-fold greater (P ⬍ 10⫺12) than for capillary-like networks (MV,CLN) (Table 1). Furthermore, we found that mean DA for capillary-like networks (MA,CLN) were 3.18-fold greater (P ⬍ 10⫺20) than for presinusoidal networks (MA,PSN; Table 1). A two-dimensional Kolmogorov-Smirnov test (Press et al., 1992) was also performed to determine

Based on the above data, we developed an algorithm that would allow for quantitative classification of an observed vascular network. For a given vascular pattern to be categorized, vascular (DV) and avascular space (DA) diameter values are measured from at least five vessel/ avascular space pairs within a 41,616 ␮m2 window of the vasculature and averaged to obtain the mean vascular (MV) and avascular area (MA) diameter values. Five paired measurements are used because, in many practical cases, it is not feasible to have a larger sample size. Measurements performed accordingly on the same vascular networks by independent observers (n ⫽ 3) resulted in consistent MV and MA values (all P values ⬎ 0.05 and the interobserver variation was 13.62%). The normalized difference value, x, between the obtained mean vascular and avascular space diameter values (MV and MA) and that of the reference (standard) vasculature (MV,CLN, MA,CLN, MV,PSN, or MA,PSN; indicated in Table 1) are calculated using the following: xV,CLN ⫽ |(MV ⫺ MV,CLN)/␴V,CLN|, xA,CLN ⫽ |(MA ⫺ MA,CLN)/␴A,CLN|, xV,PSN ⫽ |(MV ⫺

EMBRYONIC VASCULAR PATTERNING 23

or MA value approaches the standard values determined for normal embryos for that network type (Table 1), indicating little difference between the observed and standard values. By using the obtained normalized difference values (x values), we classify a network as capillarylike (CLN) if xV,CLN ⬍ xV,PSN and xA,CLN ⬍ xA,PSN (Condition I). A PSN is characterized by xV,CLN ⬎ xV,PSN and xA,CLN ⬎ xA,PSN (Condition II). When the normalized difference values do not satisfy either Condition I or II, then the network is defined as an intermediate network (IN). Such networks satisfy either xV,CLNⱖ xV,PSN and xA,CLN ⱕ xA,PSN (Condition III) or xV,CLN ⱕ xV,PSN and xA,CLN ⱖ xA,PSN (Condition IV). The graphic representation of these conditions is shown in Figure 3. Intermediate networks can be found in the region located between CLNs and PSNs in the embryonic vasculature shown in Figure 1B. Blood vessels in this area were determined to have the following vascular and avascular diameter values: MV ⫽ 38.36 ␮m (␴ ⫽ 17.61 ␮m, n ⫽ 5) and MA ⫽ 47.12 ␮m (␴ ⫽ 16.39 ␮m, n ⫽ 5), respectively. Given these measurements, the network is classified as intermediate based on the x values derived from the use of the above equations (xV,CLN ⫽ 1.76, xV,PSN ⫽ 0.70, xA,CLN ⫽ 1.24, xA,PSN ⫽ 1.53; Fig. 3, triangle in the Condition III quadrant).

Fig. 2. DV and DA are inversely related. A: The methods used to obtain five paired vessel diameter (DV) and avascular space diameter (DA) measurements within each 4,616 ␮m2 area window within the labeled vasculature. DV measurements, represented by solid black lines in A, were made from straight lines drawn from edge to edge of the vessel at a point located mid-distance between adjacent branches. A contiguous line through the avascular space, representing the DA measurement, was then made so as to bisect avascular spaces into approximately equal halves (A, solid white lines). The dashed lines in A demonstrate unpaired measurements that would not be used in these methods. B: The inverse relationship between vessel diameter (DV) and avascular space diameter (DA) in normal embryos. Gray circles represent data points taken from the capillary-like networks (CLNs) of 11-somite quail embryos, whereas black squares represent points taken from the presinusoidal networks (PSNs) of the same embryos.

MV,PSN)/␴V,PSN|, and xA,PSN ⫽ |(MA ⫺ MA,PSN)/␴A,PSN|, where ␴ represents the population standard

deviation values given in Table 1. Note, as an x value (normalized difference) approaches zero, the MV

Blood Vessels Found in Different Stage Embryos Retain Geometric Characteristics of Those Observed at the 11Somite Stage To test whether blood vessels observed in younger and older embryos have the same geometric properties and could be characterized by the mathematical methods developed herein, we analyzed the vasculature of 6- and 14-somite stage quail embryos. The geometry of the lateral network of a six-somite stage quail embryo (Fig. 4A) generated a mean vessel diameter (MV) of 6.67 ␮m (␴ ⫽ 2.45 ␮m, n ⫽ 10) and mean avascular space diameter (MA) of 54.63 ␮m (␴ ⫽ 15.64 ␮m, n ⫽

24 LARUE ET AL.

TABLE 1. Standard Mean Vascular or Avascular Space Diameter Measurements and Their Respective Population Standard Deviation (␴) Mean vascular diameter (␮m) Capillary-like network Presinusoidal network 1

16.42 (n⫽46) 65.64 (n⫽46)

␴V (␮m) 12.46 38.92

Mean avascular space diameter (␮m) 82.61 (n⫽46) 24.76 (n⫽46)

␴A (␮m) 28.73 14.58

Values indicated in the table were derived from measurements made on 41,616 ␮m2 windows in five 11-somite stage quail embryos. n ⫽ the number of measurements made within multiple standard areas (4,616 ␮m2) in multiple embryos.

ues from these measurements, regardless of developmental stage or bed location within the embryo.

Interspecies Conservation in Vessel and Avascular Space Diameter Values of Embryonic Vascular Patterns

Fig. 3. Pattern classification using MA and MV measurements and normalized difference values. Shown are plots of mean avascular space (MA) versus mean vascular (MV) diameter values from vascular networks shown in Figures 1, 4, and 5. Quadrants delineate the range of values that define a given vascular bed as capillary-like networks (CLN; Condition I quadrant), presinusoidal networks (PSN; Condition II quadrant), or intermediate networks (IN; Conditions III and/or IV quadrants). The position of the vertical dashed line was defined as the MV value for which the normalized difference values xV,CLN ⫽ xV,PSN. The position of the horizontal dashed line was defined as the MA value for which the xA,CLN ⫽ xA,PSN. dpc, days postcoitum.

10). Based on these values, the vessels were found to be capillary-like networks (xV,CLN ⫽ 0.78, xV,PSN ⫽ 1.52, xA,CLN ⫽ 0.97, xA,PSN ⫽ 2.05; Fig. 3, black circle in the Condition I quadrant). We next sought to determine whether blood vessels formed later in quail development (14-somite stage) also shared geometric properties with those formed at the 11somite stage. Yolk sac vessels of 14somite stage quail embryos (Fig. 4B) were determined to have mean vascular and avascular space mea-

surements of 29.19 ␮m (␴ ⫽ 11.55 ␮m, n ⫽ 11) and 49.65 ␮m (␴ ⫽ 7.25 ␮m, n ⫽ 11), respectively. Based on these MV and MA measurements, the vessels were defined as intermediate (xV,CLN ⫽ 1.02, xV,PSN ⫽ 0.94, xA,CLN ⫽ 1.18, xA,PSN ⫽ 1.64; Fig. 3, black square in the Condition III quadrant). These results indicate that this classification system can accurately define a vascular network based on measurements of vessel and avascular space diameter (DV and DA) and the mean val-

We next used the algorithm to determine whether pattern types within embryonic networks that form in species other than the quail have similar vessel diameter (DV) and avascular space diameter (DA) values. By using antibodies to PECAM (platelet endothelial cell adhesion molecule-1), DV and DA measurements were made on yolk sac blood vessels of early 8.5 days postcoitus (dpc) mouse embryos, vessels generated from the culture of 8.5 dpc mouse allantoides and on 12.5–13 dpc mouse coronary vessels (Fig. 5). In 8.5 dpc murine yolk sacs (Fig. 5A), the mean vessel diameter was determined to be 23.71 ␮m (␴ ⫽ 11.16 ␮m, n ⫽ 6), whereas the mean avascular space diameter was determined to be 29.65 ␮m (␴ ⫽ 10.32 ␮m, n ⫽ 6). By using these values, the yolk sac vascular networks of 8.5 dpc murine embryos were defined as intermediate (xV,CLN ⫽ 0.59, xV,PSN ⫽ 1.08, xA,CLN ⫽ 1.84, xA,PSN ⫽ 0.34; Fig. 3, gray circle in the Condition IV quadrant). When the vasculature forming within 8.5 dpc allantois explants cultured for 24 hr was evaluated (Fig. 5B), mean vessel and avascular space diameter values of 16.67 ␮m (␴ ⫽ 6.59 ␮m, n ⫽ 6) and 124.56 ␮m (␴ ⫽ 35.14 ␮m, n ⫽ 6), respectively, were obtained. By using these values, the networks were defined as capillary-like (xV,CLN ⫽ 0.02, xV,PSN ⫽ 1.26, xA,CLN ⫽ 1.46, xA,PSN ⫽ 6.85; Fig.

EMBRYONIC VASCULAR PATTERNING 25

proportional to vascular diameter (Fig. 6E).

Increased Endothelial Cell Size Does Not Contribute Vascular Sinus Formation

Fig. 4. Vascular networks from early and late stage quail embryos. A: A lateral network of a 6-somite stage quail embryo as revealed by QH1 immunolabeling. B: An image of a QH1 immunolabeled vascular network located at the boundary separating the intra- and extraembryonic vasculatures of a 14-somite quail embryo. Scale bars ⫽ 50 ␮m in A,B.

3, gray square in the Condition I quadrant). In addition to evaluating the extraembryonic vasculatures of mouse embryos, we also examined the developing coronary vasculature of 12.5–13 dpc mice (Fig. 5C). The mean vessel diameter value for these vessels was found to be 12.42 ␮m (␴ ⫽ 3.44 ␮m, n ⫽ 5), whereas the mean avascular space value was found to be 48.84 ␮m (␴ ⫽ 11.46 ␮m, n ⫽ 5). These values defined the coronary networks as capillary-like networks (xV,CLN ⫽ 0.32, xV,PSN ⫽ 1.37, xA,CLN ⫽ 1.18, xA,PSN ⫽ 1.65; Fig. 3, open circle in the Condition I quadrant). These findings indicate that the first vessels to form on the developing heart, the coronaries, conform to the defined morphometric characteristics of capillary-like networks.

Number of Constituent Endothelial Cells Is Proportional to the Size of a Vessel Both capillary-like and presinusoidal networks are components of the primitive embryonic circulatory system. How the diversity in networks, as represented by these two distinctive network types, is generated is unclear. To evaluate regulation of endothelial cell number in a given area (endothelial cell den-

sity) as a patterning mechanism, we evaluated the relationship between the pattern of a normal network, as represented by mean vascular diameter (MV), and the number of constituent endothelial cells forming that network. To accomplish this, we used the TAL1 antibody to label endothelial cell nuclei. TAL1 is a transcription factor that is expressed by cells of the endothelial lineage (Drake et al., 1997). Counting of TAL1-labeled nuclei can be used to determine the number of constituent endothelial cells within the vessels that compose a given network (e.g., capillary-like and presinusoidal networks in Fig. 6A and B, respectively). Measurement of the number of TAL1⫹ nuclei within a series of 41,616 ␮m2 fields (n) in capillary-like versus presinusoidal networks (Fig. 6C and D, respectively) of 11somite stage quail embryos shows that there are significantly (P ⬍ 0.0016) more endothelial cells in presinusoidal networks compared with capillary-like networks, 184.25 (␴ ⫽ 75.01, n ⫽ 4) vs. 67.75 (␴ ⫽ 27.92, n ⫽ 4), respectively. Comparing the constituent number of endothelial cells within a network with the mean vessel diameter values (MV) of that network showed that number of constituent cells is

Although the finding that there is a correlation between endothelial cell number and vascular sinus formation in normal embryos would suggest that endothelial cell density is a key regulator of patterning, it does not rule out other possible mechanisms. Alternative mechanisms that could modulate vascular patterns include vasodilation and changes in endothelial cell size. Given that the vessels that are the subject of this study are not yet invested with a smooth muscle layer, we have focused on the potential contribution of alterations in endothelial cell size. To determine whether the differences in mean vessel diameter values in capillary-like networks and presinusoidal networks could also be, in part, the result of increased endothelial cell size, we compared the sizes of endothelial cells in capillary-like networks with those in presinusoidal networks. To determine endothelial cell size in ␮m2, the following equation is used: [(c/ 100) ⫻ standard area]/n; where c represents the endothelial cell coverage index (percent of 41,616 ␮m2 standard area occupied by QH1⫹ cells). We found that the average size of endothelial cells in capillarylike networks was 142.62 ␮m2 (␴ ⫽ 18.35, n ⫽ 4) and the average cell size in presinusoidal networks was 177.24 ␮m2 (␴ ⫽ 46.41, n ⫽ 4). Based on a Student’s t-test, there was no statistical difference between these values (P ⬎ 0.21). Assuming relatively constant vessel wall thickness, this can be interpreted to mean that there is no difference in endothelial cells from the two networks types. Therefore, an increase in endothelial cell size does not seem to contribute to the change in mean vessel diameter between capillary-like and presinusoidal networks.

DISCUSSION In the present study, we describe morphometric methods that allow

26 LARUE ET AL.

Fig. 5. Geometric configurations of murine embryonic vascular networks. Shown are laser scanning confocal microscope images of 8.5 days postcoitus (dpc) murine yolk sac vessels (A), vessels generated from an 8.5 dpc cultured murine allantois (B), and 12.5–13 dpc murine coronary vessels (C). All networks are immunolabeled with antibodies to platelet endothelial cell adhesion molecule-1. Scale bars ⫽ 50 ␮m in A–C.

embryonic vascular patterns to be categorized quantitatively based on measurements of vessel and avascular space diameter and endothelial cell density. Whereas other morphometric methods exist for examining the pattern of blood vessels, they were developed to measure characteristics associated with angiogenesis (for review, see Vu et al., 1985; Auerbach et al., 2000, 2003; Ribatti et al., 2001). For example, these methods quantify the extent of sprouting or branching from an existing vascular bed. Importantly, many of the metrics used in the analysis of angiogenesis do not apply to networks that form during vasculogenesis. Through implementation of our methods described herein, we demonstrate that the initial patterns of blood vessels generated as part of in vivo vasculogenesis are similar, irrespective of the region within the embryo, the developmental stage, or the species in which vasculogenesis occurs. Specifically, we showed that capillary-like networks formed lateral to the axis of 11-somite quail embryos at either the level of the fourth or eighth somites are geometrically similar. In addition, we show that capillary-like networks found in quail embryos (i.e., lateral to axis at the 6-somite stage) exhibit similar geometrical properties as those that arise as part of vasculogenesis in cultured murine allantoides and those generated as part of murine coro-

nary vasculogenesis. The finding that capillary-like networks formed from mesoderm lateral to the axis and allantoic mesoderm are geometrically similar indicates that capillary-like network patterning is conserved in intraembryonic and extraembryonic compartments. This conservation also applies to the geometry of networks in transition from capillary-like networks to presinusoidal networks (i.e., intermediate sized networks in the extraembryonic region of 14somite quail embryos and those of 8.5 dpc mouse yolk sac). Taken together, these findings provide evidence that the rules governing embryonic blood vessel patterning are highly conserved. Perhaps the most dramatic example of the conservation of blood vessel patterning mediated by vasculogenesis is shown in the similarity between the geometry of the first blood vessels to form during vasculogenesis (i.e., those that form lateral to the axis at ⬃ 6-somite stage in the quail and 7.5– 8.0 dpc in the mouse) and the coronary vessels that form later in development. The earliest intraembryonic blood vessels form in the extracellular matrix (ECM) between the endoderm and splanchnic mesoderm (⬃7.5– 8.0 dpc), whereas the coronaries form in the ECM separating the myocardium and epicardium (⬃12 dpc). Our finding that the geometric configurations of nascent coronary vessels formed de novo and the first embry-

onic blood vessels formed in the embryo fell within the same category using our classification system suggests that the process of patterning capillary-like networks is endothelialspecific and proceeds independent of tissue-specific influences. Alternatively, there may be conservation in the composition of the ECMs in which the early embryonic and coronary blood vessel formation takes place. At least one common denominator to the environments in which these blood vessels form is that there is expression of vascular endothelial growth factor (VEGF) by adjacent epithelia (i.e., visceral endoderm and myocardium; Aitkenhead et al., 1998; Miquerol et al., 1999). Indeed, in the case of the coronaries, their formation coincides temporally with the expression of VEGF by the myocardium that lies subjacent to the epicardium (Tomanek et al., 1999). Our findings indicate that there is a correlation between embryonic vascular diameter and the number of constituent endothelial cells. They also indicate that endothelial cell size is similar between small diameter (capillary-like networks) and larger diameter (presinusoidal networks) vessels. These findings support the hypothesis that vessels increase their diameter during vasculogenesis by increasing endothelial cell numbers. This hypothesis is also supported by other findings showing that augmentation of VEGF signaling leads to in-

EMBRYONIC VASCULAR PATTERNING 27

creased endothelial cell numbers and the formation of large-caliber sinusoidal vessels (Fong et al., 1999; Kearney et al., 2002). Increases in endothelial cell numbers can be achieved by regulation of endothelial cell growth as well as through the recruitment of new endothelial cells by means of differentiation of mesoderm. Therefore, through regional regulation of endothelial cell numbers by factors such as VEGF, the spectrum of diversity in vascular patterning seen during vasculogenesis can be generated.

EXPERIMENTAL PROCEDURES Animals Fertile Japanese quail (Coturnix coturnix japonica; Manchester Farms, Dalzel, SC) were staged according to Hamburger and Hamilton (1951). Murine embryos (Mus musculus; Charles River Laboratories, Raleigh, NC) were staged by using somite number to accurately determine developmental stage (Drake and Fleming, 2000).

Avian Whole Embryo Culture Detailed methods for culturing quail embryos have been described previously (Packard and Jacobson, 1979; Drake et al., 1992). Briefly, injected embryos were cultured ventral side up for 7 hr in 1 ml of DMEM (Gibco BRL/Life Technologies, Baltimore, MD), 10% chicken serum (Gibco), 1% penicillin, streptomycin/ L-glutamine (Gibco) in a humidified CO2/air mixture (5%/100%) at 37°C.

Avian Embryo Fixation, Immunolabeling, and Laser Scanning Confocal Microscopy

Fig. 6. Mean endothelial cell density is proportional to mean vessel diameter (MV). A,B: Characteristic examples of normal capillary-like networks (CLNs) and presinusoidal networks (PSNs) labeled with antibodies to QH1, respectively. C,D: To correlate vascular patterns with endothelial cell density (number of endothelial cells per unit area), embryos were coimmunolabeled with TAL1, an angioblast/endothelial cell-specific marker. E: Analysis of the endothelial cell density of vascular pattern types depicted in A and B demonstrates that CLNs (gray circles) have smaller mean vessel diameter values and fewer mean constituent endothelial cells than PSNs (black squares). Scale bar ⫽ 50 ␮m in A (applies to A–D).

Embryos were rinsed in phosphate buffered saline (PBS) and fixed in 3% paraformaldehyde (45 min). After washing in Dulbecco’s PBS with 0.01% azide (DPBSA), the vitelline membrane was removed, embryos were permeabilized in absolute methanol (1 hr; ⫺20°C), rehydrated through a graded series of ethanol/ water solutions and then blocked in 3% bovine serum albumin/DPBSA (8 –12 hr; 4°C). The embryos were washed and double immunolabeled (8 –12 hr; 4°C) by using QH1

28 LARUE ET AL.

hybridoma supernatant (QH1 hybridoma supernatant developed by F. Dieterlen was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA) and rabbit anti-recombinant TAL1 IgG (obtained from Dr. Steven Brandt, Vanderbilt University, diluted to 10 ␮g/ml). After washing, secondary fluorochrome-conjugated antibodies (Jackson Immunological Research Labs, Inc., West Grove, PA) were added at 10 ␮g/ml and incubated (4 – 8 hr; 4°C). Labeled embryos were mounted under a #0 coverslip by using anti-photobleaching mounting medium (5% n-propyl gallate, 0.25% 1,4 diaza-bicyclo(2,2,2) octane, and 0.0025% phenylenediamine in glycerol; Giloh and Sedat, 1982). Embryos were analyzed by using a Bio-Rad MRC-1024 laser scanning confocal microscope (LSCM), NIH Image 1.62 and Adobe PhotoShop 7.0 image processing software. By using previously described procedures, the embryos were scanned in a plane parallel to the embryonic plate (Drake et al., 1997). Individual optical planes were sequentially analyzed for immunolabeling within the intraembryonic vasculature between the third and last formed somites, immediately lateral to the dorsal aorta extending to the boundary of the area pellucida/ opaca. The final images were projections of 20 optical planes collapsed by using Bio-Rad software to produce a single virtual image representing all blood vessels in the optical field.

Murine Embryo Dissection, Fixation, Immunolabeling, and Laser Scanning Confocal Microscopy Embryos were dissected into a planar format by cutting the yolk sac lateral to the embryonic axis and removing the amniotic sac (Drake and Fleming, 2000). Embryos were fixed by infusion of 3% paraformaldehyde into DPBS (5 min) followed by fixation in 3% paraformaldehyde (15–20 min). Embryos were permeabilized in DPBSA containing 0.02% Triton X-100

(40 min), exposed to a blocking solution of 3% bovine serum albumin/ DPBSA, incubated in anti-PECAM antibodies (BD Pharmingen, San Diego, CA) diluted to 10 ␮g/ml in DPBSA for 12–18 hr at 4°C, and washed with DPBSA. Fluorochromeconjugated secondary antibodies (Jackson ImmunoResearch Labs, Inc.) were added at 10 ␮g/ml and incubated for 12–18 hr, 4°C. Whole embryos were mounted and the vasculature of the extraembryonic yolk sac, or the heart was imaged and processed as described above for avian embryos.

Allantois Culture, Fixation, Immunolabeling, and Laser Scanning Confocal Microscopy Embryos were dissected from mice and placed into DPBS (4°C; Drake and Fleming, 2000). Allantoides were then removed from embryos, transferred to wells of a four-well chamber slide (Nalgene Nunc, Naperville, IL) containing 0.4 ml of DMEM, 10% fetal bovine serum (Gibco), 1% penicillin, and streptomycin/L-glutamine, and cultured for 24 hr (37°C, 5% CO2), during which time they attached and flattened onto the tissue culture plastic. For fixation, the culture medium was infused with 0.6 ml of 3% paraformaldehyde (20 min, 25°C). Fixative was removed, and the cultures were washed twice in DPBSA. Cultures were permeabilized with 0.02% Triton X-100/DPBSA (40 min), blocked with 3% bovine serum albumin/DPBSA (40 min), exposed to anti-PECAM antibodies, diluted to 10 ␮g/ml in DPBSA (12–18 hr; 4°C), and washed with DPBSA. Fluorochromeconjugated secondary antibodies (Jackson ImmunoResearch Labs, Inc.) were added at 10 ␮g/ml (12–18 hr; 4°C). After washing with DPBSA, mounted allantois cultures were imaged by using LSCM as described above.

Morphometric Analyses A magnification bar image was superimposed on LSCM images in Adobe Photoshop 7.0 to measure the diameter of five vessels (DV) and the diameter of the adjacent avascular space (DA) in a 204 ␮m ⫻ 204

␮m (41,616 ␮m2) area window within the labeled vasculature, resulting in five pairs of data from each window (Fig. 2A). The DV measurements were made from a straight line drawn from edge to edge of the vessel (diameter) at a point in the vessel located mid-distance between adjacent branches (Fig. 2A, solid black lines). A contiguous line through the avascular space was then made so as to bisect avascular spaces into approximately equal halves (Fig. 2A, solid white lines). Measurement of this line represents the DA value. The dashed lines in Figure 2A demonstrate unpaired measurements that would not be used in these methods. Mean diameter values (MV and MA) was then determined from paired data from each standard area evaluated. The Kolmogorov-Smirnov test (Press et al., 1992) was performed by using the Numerical Recipes in C software to determine the significance of twodimensional distributions of paired DV and DA data. Photoshop 7.0 was also used to determine the number of TAL1⫹ endothelial cell nuclei (n) per standard unit area (41,616 ␮m2). Microsoft Excel was used to perform two-way Student’s t-tests to determine the statistical significance of all data presented (significance level of P is 0.05). To quantify endothelial cell (EC) size, NIH Image (version 1.62) was used to determine the endothelial cell coverage index (c), which represents the percentage of a standard area (41,616 ␮m2) occupied by endothelial cells (QH1⫹ cells). To accomplish this analysis, LSCM grayscale image files were converted into binary images so that white pixels corresponded to the QH1-immunolabeled blood vessels and black pixels to the avascular regions. The NIH Image program was used to calculate the percentage of white pixels in a standard area ([number of white pixels/total number of pixels2] ⫻ 100%). To determine EC size in ␮m2, the following equation is used: [(c/100) ⫻ standard area]/n, where the standard area is in ␮m2. The value for n is the number of TAL1⫹ endothelial cell nuclei (n) per standard unit area (41,616 ␮m2). Microsoft Excel was used to perform

EMBRYONIC VASCULAR PATTERNING 29

two-way Student’s t-tests to determine the statistical significance of all data presented.

ACKNOWLEDGMENTS The authors thank Dr. Grier Page (University of Alabama at Birmingham, Birmingham, AL) for his statistical assistance. We also thank Dr. Stephen J. Brandt (Vanderbilt University and Veterans Affairs Medical Center, Nashville, TN) for providing us with TAL1 antibodies.

REFERENCES Aitkenhead M, Christ B, Eichmann A, Feucht M, Wilson DJ, Wilting J. 1998. Paracrine and autocrine regulation of vascular endothelial growth factor during tissue differentiation in the quail. Dev Dyn 212:1–13. Auerbach R, Akhtar N, Lewis RL, Shinners BL. 2000. Angiogenesis assays: problems and pitfalls. Cancer Metastasis Rev 19:167–172. Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. 2003. Angiogenesis assays: a critical overview. Clin Chem 49:32–40. Drake CJ, Fleming PA. 2000. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95:1671–1679. Drake CJ, Davis LA, Little CD. 1992. Antibodies to beta 1-integrins cause alterations of aortic vasculogenesis, in vivo. Dev Dyn 193:83–91. Drake CJ, Brandt SJ, Trusk TC, Little CD. 1997. TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and

defines a dorsal-to-ventral gradient of vasculogenesis. Dev Biol 192:17–30. Fong GH, Zhang L, Bryce DM, Peng J. 1999. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 126:3015–3025. Giloh H, Sedat JW. 1982. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217:1252–1255. Hamburger V, Hamilton HL. 1951. A series of normal stages in the development of the chick embryo. J Morphol 88:49 – 92. Hirakow R, Hiruma T. 1981. Scanning electron microscopic study on the development of primitive blood vessels in chick embryos at the early somitestage. Anat Embryol (Berl) 163:299 –306. Kallianpur AR, Jordan JE, Brandt SJ. 1994. The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis. Blood 83:1200 –1208. Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL. 2002. Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 99:2397–2407. Miquerol L, Gertsenstein M, Harpal K, Rossant J, Nagy A. 1999. Multiple developmental roles of VEGF suggested by a LacZ-tagged allele. Dev Biol 212:307– 322. Newman PJ, Albelda SM. 1992. Cellular and molecular aspects of PECAM-1. Nouv Rev Fr Hematol 34:S9 –S13. Newman PJ, Berndt MC, Gorski J, White GC Jr, Lyman S, Paddock C, Muller WA.

1990. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 247:1219 –1222. Packard DS Jr, Jacobson AG. 1979. Analysis of the physical forces that influence the shape of chick somites. J Exp Zool 207:81–91. Pardanaud L, Altmann C, Kitos P, Dieterlen-Lievre F, Buck CA. 1987. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 100:339 –349. Press WH, Teukolsky SA, Vetterling WT, Flannery BP. 1992. Numerical recipes. Cambridge: Cambridge University Press. Ribatti D, Nico B, Vacca A, Roncali L, Burri PH, Djonov V. 2001. Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and antiangiogenesis in vivo. Anat Rec 264:317– 324. Sabin RR. 1920. Studies on the origin of blood vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Carnegie Institute Washington Contrib. Embryology 9: 215–262. Tomanek RJ, Ratajska A, Kitten GT, Yue X, Sandra A. 1999. Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis. Dev Dyn 215:54 –61. Vu MT, Smith CF, Burger PC, Klintworth GK. 1985. An evaluation of methods to quantitate the chick chorioallantoic membrane assay in angiogenesis. Lab Invest 53:499 –508.