Low cost labeling with highlighter ink efficiently visualizes developing ...

The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 792–801

doi: 10.1111/dgd.12106

Original Article

Low cost labeling with highlighter ink efficiently visualizes developing blood vessels in avian and mouse embryos Yuta Takase, 1,2 Ryosuke Tadokoro 1 and Yoshiko Takahashi 1,3 * 1

Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, 630-0192, and 3Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, 102-0076, Japan

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To understand how blood vessels form to establish the intricate network during vertebrate development, it is helpful if one can visualize the vasculature in embryos. We here describe a novel labeling method using highlighter ink, easily obtained in stationery stores with a low cost, to visualize embryo-wide vasculatures in avian and mice. We tested 50 different highlighters for fluorescent microscopy with filter sets equipped in a standard fluorescent microscope. The yellow and violet inks yielded fluorescent signals specifically detected by the filters used for green fluorescent protein (GFP) and red fluorescent protein (RFP) detections, respectively. When the ink solution was infused into chicken/quail and mouse embryos, vasculatures including large vessels and capillaries were labeled both in living and fixed embryos. Ink-infused embryos were further subjected to histological sections, and double stained with antibodies including QH-1 (quail), a smooth muscle actin (aSMA), and PECAM-1 (mouse), revealing that the endothelial cells were specifically labeled by the infused highlighter ink. Highlighter-labeled signals were detected with a resolution comparable to or higher than signals of fluorescein isothiocyanate (FITC)-lectin and Rhodamine-dextran, conventionally used for angiography. Furthermore, macroconfocal microscopic analyses with ink-infused embryos visualized fine vascular structures of both embryo proper and extra-embryonic plexus in a Z-stack image of 2400 lm thick with a markedly high resolution. Together, the low cost highlighter ink serves as an alternative reagent useful for visualization of blood vessels in developing avian and mouse embryos and possibly in other animals. Key words: endothelium, fluorescent labeling, macroconfocal microscopy, vascular formation.

Introduction The vascular network of blood vessels is widely distributed in the body, and conveys substances including oxygen, nutrients, and hormones to peripheral tissues. Recent studies show that blood vessels also play an important role in their adjacent morphogenesis during vertebrate development (Lammert et al. 2001; Matsumoto et al. 2001; Honma et al. 2002; Makita et al. 2008; Saito et al. 2012). However, because of intricate structures, it remains incompletely understood how the vasculature forms, in particular, how the body-wide *Author to whom all correspondence should be addressed. Email: [email protected] Received 4 October 2013; revised 20 October 2013; accepted 20 October 2013. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists

patterns of the vascular network are established. To address these questions, direct visualization of forming vessels in vivo must be useful. In the past decade, blood vessel-visualizing techniques have advanced mostly by using transgenic lines of zebrafish and mice, in which an endothelialspecific promoter drives reporter/enhanced green fluorescent protein (EGFP) (Constien et al. 2001; Lawson & Weinstein 2002; Ishitobi et al. 2010). However, such techniques are not applicable to animals unavailable for genetics, such as chickens (except for a few cases (Sato et al. 2010)). Alternatively, blood vessels are visualized by perfusion (intra-vascular injection) with fluorescent reagents such as fluorescein isothiocyanate (FITC)-dextran and Q-dot (Bates et al. 2002; Mayes et al. 2008). However, as an embryo grows, an increasing amount of reagent is required with a high demand of cost. Furthermore, since these reagents primarily label the blood flow,

Blood vessel labeling by highlighter

endothelial cells hardly retain the signal, particularly, in fixed specimens, that is, cryo-sections, hampering detailed assessments by double-staining with antibodies. Another reagent FITC-lectin, although retained in the endothelium after fixation, binds to animal species-specific substance (different types of lectin need to be used for mice and chickens) (Laitinen 1987; Jilani et al. 2003). Thus, alternative techniques/ reagents have been awaited that can widely be used for vascular visualization both in vivo and in histological sections. We here demonstrate a novel technique that fulfilled such requirements using the ink of highlighter pens that can easily be obtained in an ordinary stationery store (Fig. 1A). Three advantages are highlighted: First, among a wide range of color collections, one can select a type of fluorescence suitable for one’s experiments. Second, the highlighter ink costs considerably lower than FITC-Dextran and Q-dot. Third, when the ink is infused into chicken embryos, the fluorescent signal is detected in all types of blood vessels including body-wide capillaries, and the endothelial signal is retained even after cryo-sectioning of fixed specimens, thus enabling subsequent immunohistochemistry with various antibodies. The method we describe in this study might also be applicable to a wide range of animal species in which blood vessels are amenable to infusion.

Materials and methods Embryological manipulations Fertilized chicken and quail eggs were commercially obtained from the poultry farm Shiroyama Farm (Kanagawa, Japan) and Motoki Corporation (Saitama, Japan), respectively. Embryos were staged according to Hamburger & Hamilton (1992). C57BL/6 mouse embryos were obtained from CLEA Japan (Japan). Detection of fluorescent signals Ink was taken from a highlighter pen (Table 1), and diluted with phosphate-buffered saline (PBS) if necessary. Fluorescent signals of rubbed ink on a white paper were obtained using an Axioplan 2 microscope with the Apotome system (Carl Zeiss). Highlighter inks were compared with 1 mg/mL LCA-Lectin-FITC/PBS (J-oil, J507) or 1 mg/mL TRITC-conjugated high MW dextran/PBS (Molecular Probes, D-7139). For stereo microscopy, Leica MZ10 F (Leica) was used equipped with AxioCam HRc CCD camera (Carl Zeiss). Fluorescent intensity of obtained images was measured by Image J software.

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Visualization of blood vessels by infusion Chicken embryos at E3.5 or E7.5 were infused with ink or fluorescent reagents (1–3 lL) into the vitelline artery using a micropipette pulled from a glass capillary of 1 mm diameter (Narishige, GD-1) with a vertical micropipette puller (Narishige, PC-10). Mouse embryos were infused into the umbilical cord at E12.5 or E16.5 with 1–3 lL of ink. Infused embryos were incubated at 38°C for an additional period of 10 min. The fluorescent images were obtained using a Leica MZ10 F (Leica) with AxioCam HRc CCD camera (Carl Zeiss). Immunohistochemistry Chicken and mouse embryos were fixed overnight in PBS containing 4% paraformaldehyde (PFA) at 4°C. Frozen sections (10 lm thick) of fixed embryos were prepared with a cryostat (MICROM, HM500 OM). The sections were washed in PBS three times (each 5 min). After blocking with 1% blocking reagent (Roche, 1096176)/PBS for 1 h at room temperature (RT), the sections were incubated overnight at 4°C with a 1:2 dilution of culture supernatant of QH-1 mouse monoclonal antibody (Developmental Studies Hybridoma Bank), a 1:400 dilution of anti-a smooth muscle actin (aSMA) mouse monoclonal antibody (Sigma, 1A4), a 1:50 dilution of anti-Integrin aVb3 mouse monoclonal antibody (Chemicon, LM609), or a 1:500 dilution of anti-fibronectin mouse monoclonal antibody (Sigma, F614) in 1% blocking reagent/PBS. After washing three times in PBS (each 5 min), the specimens were reacted with a 1:500 dilution of Alexa 488 donkey antimouse IgG (Invitrogen, A-21202) or Alexa 568 goat anti-mouse IgG (Invitrogen, A-11004) in 1% blocking reagent/PBS for 1 h at RT. The reaction was terminated by washing three times in PBS (each 5 min), and the sections were sealed by FluorSave reagent (Calbiochem, 345789). Fluorescent images were obtained using an Axioplan 2 microscope with Apotome system (Carl Zeiss). For immuno-staining of abdominal skin in mice, a piece of skin was peeled from fixed embryos and washed in 1% Tween 20/PBS (PBST) three times (each 10 min). After blocking with 2% skim milk (BD, 232100)/PBST for 1 h at RT, the specimens were incubated overnight at 4°C with a 1:100 dilution of anti-mouse CD31 (PECAM-1) rat monoclonal antibody (BD Pharmingen, 550274) in 2% skim milk/PBST. After washing three times in PBST (each 1 h), the skin pieces were reacted with a 1:500 dilution of Alexa 568 goat anti-rat IgG (Invitrogen, A-11077) in 2% skim milk/PBST for overnight at 4°C. After washing three

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Fig. 1. Fluorescent microscopy with highlighter ink. (A) Representative highlighters used in this study, which were easily obtained in a stationery store. (B) Ten highlighter inks of PILOT spotliter were rubbed on a piece of white paper, and tested with four different excitation/emission filters for fluorescent microscopy (see also Table 1). Signals obtained by the green fluorescent protein (GFP), red fluorescent protein (RFP), and Cy 5 filters were digitally processed by the Apotome system (Carl Zeiss) so that they appeared in green, red, and blue, respectively. Scale bar: 200 lm. See text for details.

times in PBST (each 1 h), the skin was cleared in 60% glycerol/PBS as shown below. Fluorescent images were obtained using AZ-C1 macroconfocal microscope system (Nikon).

Embryo clearing Fixed embryos were washed in PBS three times (each 5 min). They were incubated in 20% (w/v) glycerol/

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Table 1. Ink of different highlighter products were tested with fluorescent microscopy Manufacturer products PILOT spotliter

Ink type Aqueous pigment

DAPI filter GFP filter RFP filter Cy 5 filter Ex 300–390 nm Ex 450–490 nm Ex 550–580 nm Ex 625–655 nm Endothelium Em 420–470 nm Em 500–550 nm Em 590–650 nm Em 665–715 nm labeling

Color

Green Yellow Cream yellow Orange Red Pink Purple Violet Blue Emerald green PILOT frixion Unknown Green Soft green Yellow Orange Red Baby pink Pink Violet Light blue Blue Brown Black MITSUBISHI Aqueous Green propus2 pigment Yellow Orange Red Pink Red purple Purple Sky blue Blue Brown MITSUBISHI Aqueous Yellow pure color dye Violet ZEBRA mildsign Aqueous Blue pigment Pink Yellow Orange Green ZEBRA Aqueous Blue sparky-2 pigment Yellow Red ZEBRA optex Aqueous Yellow care pigment Blue ZEBRA check Aqueous Green pen dye Red Pentel art brush Aqueous Lemon dye yellow Purple Teranishi chemical Oily pigment Blue magic ink Red

9 9 9

s

M 9

M 9 9

+ + nt

9 9 9 9 9 9 9

M M M M 9 9 9

⌾ ⌾ ⌾ ⌾

9 9 9 9 9 s M

nt + nt + + + nt

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

M s s M 9 s M 9 9 9 9 9

9 M 9 M s s s s 9

s s s M 9 9 9 s 9 9 9 s

⌾ ⌾ ⌾ ⌾ ⌾

M M 9 9 9 9 9 M s s M s s 9 9 9 9 M s

9 9 9

9 9 9

⌾ ⌾

⌾ ⌾

⌾

s s 9

⌾

s

⌾

9 M 9 9

⌾

s s M

⌾

s s M 9

s

⌾ ⌾

9 M s

⌾

9 s s s 9

⌾

9 s 9

⌾

9

s 9

⌾

⌾ ⌾

s 9 M s 9 9 9 s s 9 M 9 s M M 9 M 9 s

nt nt nt nt nt nt nt nt nt + + nt nt nt nt + nt nt nt

nt nt + nt nt + + + + +

nt nt

9, signal undetected; M, weak signal; s, fair signal; ⌾, intense signal; nt, not tested. DAPI, 4´6´-diamidino-2-phenylindole dihydrochloride; GFP, green fluorescent protein; RFP, red fluorescent protein.

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PBS for 2–3 h at RT, and serially replaced by 40% and 60% glycerol/PBS.

Results Fluorescent microscopy to detect highlighter signals with different color filters We tested 50 different highlighter pens produced by five different Japanese major manufacturers (Table 1). Out of 50, representative results obtained by 10 of the PILOT spotliter series are shown in Figure 1B. The ink of each highlighter was rubbed on a white paper, and subjected to fluorescent microscopy. Four different filters were tested whose excitation/emission wavelengths were: 300–390 nm/420–470 nm (often used for detection of DAPI [4´6´-diamidino-2-phenylindole dihydrochloride] signals, and hereafter called “DAPI filter”), 450–490 nm/500–550 nm (“GFP filter”), 550– 580 nm/590–650 nm (“RFP filter”), and 625–655 nm/ 665–715 nm (“Cy 5 filter”) (Fig. 1B, Table 1). Microscopy with DAPI filter did not yield signals for any highlighters tested. The GFP filter permitted a detection of seven different inks (two with high signal, five with low signal). The RFP filter gave five intense signals and four weak signals. The Cy 5 filter yielded three moderate signals (Fig. 1B). Eight ink types out of the 10 exhibited signals detected by more than one filter, which is an unfavorable situation if one combines the ink-perfusion of embryos with subsequent immunohistochemistry. In this context, yellow and violet inks were preferable; the yellow was detected only by the GFP filter, and the violet by the RFP filter. Nevertheless, if one merely visualizes blood vessels, all 10 inks can be used, and in particular, the green, blue, and emerald green inks would be good for detection by the Cy 5 filter. The results for all 50 inks are summarized in Table 1, which shows more cases of specific detection by a single filter set: yellow of PILOT frixion by GFP filter, light blue of PILOT frixion by Cy 5 filter, yellow of ZEBRA mildsign by GFP filter, yellow of ZEBRA sparky-2 by GFP filter, and yellow of ZEBRA optex care by GFP filter. In the following experiments, we used the yellow and violet inks of PILOT spotliter. Infused highlighter ink labeled blood vessels Prior to the infusion into embryos, we optimized the dilution condition of the yellow and violet inks (PILOT spotliter). Serial dilutions with PBS followed by fluorescent microscopy revealed that 1:200 dilution gave a sufficient intensity of fluorescence for the yellow ink (Fig. 2A), which was higher than that of 1 mg/mL

FITC-lectin, the concentration commonly used for infusion. In the following studies, we used 1/200 and 1/50 dilutions for the yellow and violet inks, respectively, unless otherwise indicated. We injected the ink into an embryonic day 3.5 (E3.5) embryo through the vitelline artery using a glass capillary. Infused ink underwent embryo-wide circulation within a few minutes, and fluorescently labeled vasculature emerged as shown in Figure 2E. The perfused embryos were subsequently fixed in PFA, cryo-sectioned, and stained for several antibodies. Figure 2G,H showed a dorsal aorta of quail embryo, which was costained for QH-1 to identify endothelial cells (Pardanaud et al. 1987). QH-1-positive endothelial cells were overlapped with ink-labeled cells. These ink-positive cells were located inside the smooth muscle cells (aSMA-positive), indicating that perfused ink used in this study labeled specifically the endothelial component of developing blood vessels. The perfused ink not only labeled large vessels like dorsal aorta, but also capillaries as evidenced by intraneural vascular plexus growing in the early spinal cord of E4 embryos. Yellow ink signals coincided with immunohistochemically stained signals for integrin aVb3 (Fig. 2I; Alexa 568) and fibronectin (Fig. 2J; Alexa 568). It should be noted that such endothelial labeling was enabled solely by the type of pigment ink, in which fluorescent pigment particles are suspended in aqueous medium, but not by the type of water-soluble dye ink or PILOT frixion, whose ink type is unknown (Table 1). Indeed, we sometimes observed punctated precipitates attached to the endothelium of spotliterinfused embryos, and they are likely aggregates of pigment particles (not shown for chicken, but see Fig. 4 for mice). In frixion ink-infused embryos, although pigment particles were observed in blood vessels, they were not attached to the endothelium. Water-soluble dye ink, which did not label the endothelium, underwent diffusion into stroma. Comparison of the highlighter ink with Rhodaminedextran and FITC-lectin We next compared the highlighter ink with conventionally used vessel-labeling reagents. An E3.5 chicken embryo was infused with a mixture of yellow ink (1/ 200) and Rhodamine-dextran (1 mg/mL), and examined under fluorescent stereomicroscope. In live embryos, yellow ink (by GFP filter) yielded fine structures of blood vessels, whereas such clarity was not obtained by Rhodamine-dextran (by RFP filter). Following fixation, the difference in signal clarity between the two reagents was more pronounced (Fig. 3C). As for lectin, since only the FITC-conjugated version (by GFP

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Fig. 2. Highlighter ink visualized embryonic vasculatures both in live and in fixed specimens. (A, B) Fluorescent stereomicroscopy with a solution placed in a 200 lL test tube, which is usually used for polymerase chain reaction (PCR) reaction. Highlighter yellow ink was serially diluted in phosphate-buffered saline (PSB) (A), and their fluorescence intensity was measured (B). (C) Schematic drawing of infusion into an embryonic day 3.5 (E3.5) quail embryo though the vitelline artery. (D) Infused yellow ink was circulated, and an embryo-wide distribution of vasculature became visible within 10 min. (E) Ink-infused quail embryos were fixed and subjected to histological cryo-sectioning. (F) Dorsal aorta double-stained with QH-1 (endothelial marker) showed that the infused ink specifically marked the endothelium (arrows). (G) Dorsal aorta was double-stained with aSMA (a smooth muscle actin). Infused violet ink-positive cells were located inside aSMA-stained cells. (H, I) Neural tube was stained for Integrin aVb3 (H) and Fibronectin (I), visualizing co-stained intraneural vascular plexus (arrow). Scale bars: 1 mm for (D), 50 lm for (F–I).

filter) was available for chickens, we used violet highlighter (by RFP filter) for comparison. Vascular signals visualized by these reagents were of comparable clarity in both live and fixed embryos (Fig. 3D,E). We further compared these reagents in older embryos. For these experiments, we used the yellow ink yielding more intense signals than the violet (Fig. 1B). Highlighter yellow ink, FITC-lectin, and Rhodamine-dextran were separately infused into E7.5 embryos through the umbilical cord (Fig. 3F). Again, fluorescent signals were quickly circulated to label the embryo-wide vasculature. Despite the growing size

and thickness of tissues, superficial vasculatures were recognized by the yellow ink (Fig. 3G), but not by FITC-lectin or Rhodamine-dextran (Fig. 3H,I). In magnified views of mesencephalon, known as a highly vascularized tissue, the yellow ink visualized the fine structure of blood vessels (Fig. 3J), which were hardly seen in the mesencephalon infused with FITC-lectin or Rhodamine-dextran (Fig. 3K,L). Likewise, in the tip of forelimbs wherein digital patterning of vasculature began to form, yellow ink-infused tissues exhibited digitally associated blood vessels (Fig. 3M). Again, FITC-lectin or Rhodamine-dextran hardly yielded such

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Fig. 3. Comparison of the highlighter ink with conventional vessel-labeling reagents. (A) An embryonic day 3.5 (E3.5) chicken embryo co-infused with highlighter ink and fluorescent reagent/dye. (B, C) Comparison between yellow ink and Rhodamine-dextran either in ovo (B) or in paraformaldehyde (PFA)-fixed embryos (C). Rhodamine signal was detected by the red fluorescent protein (RFP) filter (see Fig. 1). (D, E) Comparison between violet ink and fluorescein isothiocyanate (FITC)-lectin either in ovo (D) or in PFA-fixed embryos (E). FITC signals were detected by the green fluorescent protein (GFP) filter (see Fig. 1). (F) An E7.5 chicken embryo infused with either yellow ink, FITC-lectin, or Rhodamine-dextran. (G–I) Whole embryos fixed in PFA. (J–L) Mesencephalon. Hyper-vascularization was recognized in the yellow ink-infused specimen (J), but not in the Rhodamine-dextran- or FITC-lectin infused (K, L). (M–O) A tip of fore-limb bud, which was fixed and cleared in 60% glycerol/PBS. Digital patterns of vasculature were recognized by the yellow ink (M), but not by Rhodamine-dextran or FITC-lectin (N–O). Scale bars: 1 mm.

signals (Fig. 3N,O). The limbs shown in Figure 3M–O were the specimens cleared in glycerol (see Materials and Methods). In conclusion, the highlighter ink successfully labeled vascular structures of chicken live embryos, and labeled endothelial signals were retained even after fixation. Whereas the signal clarity was either comparable to or higher than FITC-lectin or Rhodamine-dextran at

earlier stages, the usefulness of the highlighter ink was more pronounced as embryos grew in size at later stages. Application of the highlighter ink to mouse embryos We examined whether the highlighter ink could also label blood vessels in mouse embryos. We infused the

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yellow ink of 1/5 solution into an E16.5 embryo through the umbilical cord (Fig. 4A). Superficial blood vessels were seen in a way similar to chicken embryos (Fig. 4B). To further scrutinize blood vessels, a piece of skin was peeled off from the abdomen, and subjected to double staining with rat anti-PECAM-1, the most commonly used antibody to detect endothelium in mice, followed by a final detection with Alexa 568conjugated anti-rat antibody. Confocal microscopy with a Z-stack of 40 slices of 8 lm each (=320 lm thick) revealed that a majority of vascular plexus was co-stained for yellow ink and PECAM-1, although some cells were stained only by PECAM-1 (Fig. 4C). Given that embryo-wide capillaries were ink-labeled in chickens (see above), it is likely that the PECAM-1positive and ink-negative cells reflect endothelial precursors prior to participating to lumenized blood

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vessels. Punctated precipitations of ink were sporadically seen (see also Discussion). Infused mouse specimens were also examined in transverse sections after fixation in PFA. Figure 4D shows a dorsal aorta double-stained with aSMA (detected by RFP filter). As seen for chicken embryos, the yellow ink-labeled endothelial cells of a single layer were located interior to the aSMA-positive perivascular component. Vascular images with higher resolution obtained by macroconfocal microscopy Cleared violet-embryos in glycerol, which provided a higher resolution of microscopic images than the noncleared (Fig. 5A,B), were examined using the macroconfocal microscope AZ-C1 (Nikon). A processed image with a Z-stack of 240 slices of 10 lm each (=2400 lm thick) highlighted an intricate vascular network governing both the embryo proper and extra embryonic membranes (Fig. 5C). The image resolution obtained by the macroconfocal microscopy was markedly higher than that by standard fluorescent stereomicroscope.

Discussion

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Fig. 4. Highlighter ink visualized vasculature in mouse embryos. (A) An embryonic day 16.5 (E16.5) mouse embryo was infused with yellow ink into the umbilical cord. (B) Blood vessels in superficial tissues were visible in a whole embryo. (C) A piece of abdominal skin was co-stained with anti PECAM-1 antibody (detected by Alexa 568 with red fluorescent protein [RFP] filter). Confocal microscopy processed a Z-stack of 40 slices of 8 lm each (=320 lm thick). A majority of vascular plexus was costained for yellow ink and PECAM-1 (white arrows), although some cells were stained only by PECAM-1 (black arrowheads). (D) Dorsal aorta was double-stained with a smooth muscle actin (aSMA). Yellow ink-positive cells were located inside aSMAstained cells. Scale bars: 1 mm for (B, C), 50 lm for (D).

We have demonstrated that the highlighter ink serves as a convenient reagent to visualize vascular structures of chicken and mouse embryos both in live and in fixed specimens. We found that the highlighter ink of pigment type can be used for vascular visualization either by the GFP, RFP, or Cy 5 filters. And if one demands a high specificity in color detection, the yellow ink is recommended since this color is specifically detected by the GFP filter with no leak through RFP and Cy 5 filters. We have also succeeded in obtaining images of macroconfocal microscopy of E3.5 infused chicken embryos with a markedly higher resolution compared to conventional fluorescent stereomicroscopy. Historically, avian embryos have made a considerable contribution to vascular developmental biology, where the QH-1 antibody has widely been used to identify endothelial cells (Pardanaud et al. 1987) mostly in histological sections. In sections, however, vascular plexus of mesh-like structure is often recognized as fractured pieces, making it difficult to obtain a 3D image of vasculature. Thus, reagents that can conveniently be used for angiography have been awaited. The method described in this study provides a new way to advance our understanding of how the patterning of blood vessels is regulated in developing embryos. In addition, this method might be applicable to other animals available for vascular infusion.

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for regular observation under non-fluorescent microscope, this is not amenable to fluorescent microscopy including confocal analyses. Disadvantages using highlighter ink

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Infusion with highlighter ink sometimes causes small precipitates or punctated signals in blood vessels probably due to an aggregate of pigment particles. Thus, the observed fluorescent intensity of the ink does not necessarily reflect the density of endothelial cells. If one needs to assess the endothelial density, FITC-lectin is a suitable reagent. Another caution is that highlighter inks tested in this study were the commercial products available in Japan. The same product name might sometimes indicate different products in other countries (i.e. PILOT spotliter). Nevertheless, it is highly expected that yellow color of pigment type of ink would work well for endothelial labeling detected by the GFP filter regardless of manufacturers’ products.

Acknowledgments Fig. 5. Macroconfocal microscopy visualized embryo wide vasculature. (A, B) A comparison between non-cleared (A) and cleared (in 60% glycerol/PBS) embryonic day 3.5 (E3.5) chicken embryos (B), which were infused with violet ink. (C) A cleared embryo as shown in (B) was further subjected to macroconfocal microscopy using AZ-C1 (Nikon). Z-stack image was obtained with 240 slices of 10 lm each (=2400 lm thick) (2048 bit 9 2048 bit). The resolution of visualized vasculature obtained by the macroconfocal microscopy was markedly higher than that by standard fluorescent stereomicroscope (B). Scale bars: 1 mm

Advantages using highlighter ink over conventional reagents for angiography Cost. Highlighter ink of $US1-worth covers approximately 200 000 E5 chicken embryos, whereas it costs $US4200–10 000 if Rhodamine-dextran (Molecular Probes) or FITC-Lectin (J-oil) are used for the same number of embryos. The extremely low cost of highlighter ink is particularly appreciated when progressively growing embryos are used, which demand an increasing amount of reagents. Signal clarity. The highlighter ink yields at least comparable or higher clarity in endothelial signals compared to FITC-lectin or Rhodamine-dextran in early living embryos. Such differences are more pronounced in PFA-fixed specimens and also in the late stage. embryos. Although classically used Indian ink is useful

We thank Dr Y. Atsuta for helpful discussion. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, MEXT, Japan, CREST (JST), The Mitsubishi Foundation, and Takeda Science Foundation.

References Constien, R., Forde, A., Liliensiek, B., Grone, H. J., Nawroth, P., Hammerling, G. & Arnold, B. 2001. Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line. Genesis 30, 36–44. Hamburger, V. & Hamilton, H. L. 1992. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272. Honma, Y., Araki, T., Gianino, S., Bruce, A., Heuckeroth, R. O., Johnson, E. M. & Milbrandt, J. 2002. Artemin is a vascularderived neurotropic factor for developing sympathetic neurons. Neuron 35, 267–282. Ishitobi, H., Matsumoto, K., Azami, T., Itoh, F., Itoh, S., Takahashi, S. & Ema, M. 2010. Flk1-GFP BAC Tg mice: an animal model for the study of blood vessel development. Exp. Anim. 59, 615–622. Jilani, S. M., Murphy, T. J., Thai, S. N., Eichmann, A., Alva, J. A. & Iruela-Arispe, M. L. 2003. Selective binding of lectins to embryonic chicken vasculature. J. Histochem. Cytochem. 51, 597–604. Laitinen, L. 1987. Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues. Histochem. J. 19, 225–234. Lammert, E., Cleaver, O. & Melton, D. 2001. Induction of pancreatic differentiation by signals from blood vessels. Science 294, 564–567.

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Lawson, N. D. & Weinstein, B. M. 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318. Makita, T., Sucov, H. M., Gariepy, C. E., Yanagisawa, M. & Ginty, D. D. 2008. Endothelins are vascular-derived axonal guidance cues for developing sympathetic neurons. Nature 452, 759–763. Matsumoto, K., Yoshitomi, H., Rossant, J. & Zaret, K. S. 2001. Liver organogenesis promoted by endothelial cells prior to vascular function. Science 294, 559–563. Mayes, P., Dicker, D., Liu, Y. & El-Deiry, W. 2008. Noninvasive vascular imaging in fluorescent tumors using multispectral unmixing. Biotechniques 45, 459–460, 461–464.

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Pardanaud, L., Altmann, C., Kitos, P., Dieterlen-Lievre, F. & Buck, C. A. 1987. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 100, 339–349. Saito, D., Takase, Y., Murai, H. & Takahashi, Y. 2012. The dorsal aorta initiates a molecular cascade that instructs sympathoadrenal specification. Science 336, 1578–1581. Sato, Y., Poynter, G., Huss, D., Filla, M. B., Czirok, A., Rongish, B. J., Little, C. D., Fraser, S. E. & Lansford, R. 2010. Dynamic analysis of vascular morphogenesis using transgenic quail embryos. PLoS ONE 5, e12674.

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