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Keywords:

  • chick embryos;
  • gastrulation;
  • immunolocalization;
  • microinjection;
  • tissue transplantation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three-dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor-intensive fate mapping studies. Developmental Dynamics 230:309–315, 2004. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Fate mapping studies play an important role in understanding the organization of the early embryo. Classic studies in chick embryos at gastrula and neurula stages have involved labeling the cell surface with chalk, vital dyes, iron oxide particles, or carbon particles; or alternatively labeling cells internally with tritiated thymidine (summarized in Schoenwolf et al., 1992). Modern studies use two main approaches to follow the movement of groups of cells: construction of quail–chick transplantation chimeras, or labeling groups of cells with fluorescent dyes either by injection or transplantation of labeled grafts (summarized in Darnell et al., 2000). These two approaches offer their own advantages and disadvantages. Constructing chimeras, for example, allows for fate mapping of a discrete population of grafted cells. The quail marker is detected by using antibodies and is not diluted (as is tritiated thymidine labeling) by cell proliferation. However, close matching of donor and host embryos for homotopic and isochronic transplantation is required; damage during excision and time for healing of the graft must be taken into consideration, as well as possible regeneration of host (i.e., extirpated) tissue. In contrast to constructing chimeras, microinjection establishes a pool of dye that maps a site over time, rather than a discrete population of cells. A small injection reduces the size of the pool of dye available for transfer to cells as they pass through the original injection site, and it limits the amount of diffusion that can occur to adjacent sites. However, the dye is diluted, with reduction or loss of the signal, as cell proliferation and lysosomal degradation of the dye occurs. Recently, the use of two “colors” to map relative cell movements has become particularly attractive. Cytoplasmic dyes are either injected directly into the site of interest, or they are used to label donor embryos from which groups of cells are then transplanted into host embryos. The dyes are detected and amplified by using antibodies to produce permanent markers, although the technique requires extensive tissue processing over several days (e.g., Garton and Schoenwolf, 1996; Lopez-Sanchez et al., 2001).

The improved method described here, combining in situ hybridization and double immunoctyochemistry in the same embryo, enables the investigator to rapidly extract substantially more information from individual embryos, maximizing the results obtained from limited sample sizes produced in labor-intensive fate mapping studies. With this approach double labeling facilitates the tracking of relative movements of fluorescently labeled cells in the living embryo; the movements of two groups of cells can be mapped simultaneously over a period of time by fluorescent microscopy and digital image capture. The ability to detect the two fluorescent labels, by simultaneously using distinct antibodies during immunocytochemistry, significantly shortens the time required to obtain and analyze the data. When combined with in situ hybridization to ascertain cell identity by using specific riboprobes, fate mapping can be conducted using molecular criteria rather than position and morphology only, allowing for detailed three-dimensional analysis in histological serial sections.

RESULTS AND EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Six different experimental procedures were used in this study: whole embryo culture, microinjection of fluorescent dyes, transplantation of labeled groups of cells, double immunocytochemistry, in situ hybridization, and paraffin histology.

Whole Embryo Culture

Fertile chicken eggs were incubated at 38°C in forced-draft, humidified incubators, until embryos reached gastrulation stages (stages 3 and 4; Hamburger and Hamilton, 1951; with substaging of stage 3 embryos as described by Chapman et al., 2003). Culture dishes (35 × 10 mm) containing an agar–albumen substrate were prepared by mixing together equal parts of thin albumen from unincubated fertile chicken eggs and a 0.6% solution of agar (Bacto-Agar, Difco, Detroit, MI) in 123 mM NaCl. Culture dishes were prepared in advance and stored in humidified containers at 4°C until needed. On the day of the experiment, culture dishes were equilibrated at 38°C in a humidified incubator before use. Embryos were prepared for either modified New (1955) culture (Darnell and Schoenwolf, 2000) or EC culture (Chapman et al., 2001).

Microinjection of Fluorescent Dyes

To track the movement of various populations of cells within the blastoderm, two neighboring areas were sequentially microinjected with different fluorescent dyes (Fig. 1). In the embryo shown in Figure 1A, a small bolus of a solution of DiI/CRSE (Molecular Probes, Inc., Eugene, OR) was first microinjected into the rostral primitive streak (red fluorescent spot; DiI = 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, catalog no. D-282, CRSE = 5-[and 6-] carboxytetramethylrhodamine, succinimidyl ester, catalog no. C-1171). DiI intercalates into cell membranes and is included in the solution because it is intensely fluorescent, allowing living cells to be easily followed by fluorescence microscopy. To make the DiI solution, dissolve 2.5 mg of DiI in 50 μl of dimethylsulfoxide (DMSO), then add 950 μl of ethanol, for a concentration of 2.5 mg/ml. CRSE passes through cell membranes and becomes trapped in the cytoplasm; it is included in the solution because its fluorescent label is rhodamine, against which commercial antibodies exist allowing for processing of a permanent marker (see Double Immunocytochemistry section, below). To make the CRSE solution, dissolve 2.5 mg of CRSE in 1 ml of DMSO, for a concentration of 2.5 mg/ml. DiI/CRSE solution consists of a 1:1 mixture of the two dyes; thus, the final working concentration of each dye in DiI/CRSE is 1.25 mg/ml.

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Figure 1. Whole-mount chick blastoderm (A–D) and a transverse section (E; level indicated on D) showing the results of DiI/CRSE (red fluorescence) and DiO/CFSE (green fluorescence) dye microinjections at stage 3b. A: Immediately after injection of two levels of the rostral primitive streak (ps). B: Same embryo 16 hours after injection (n, node). C: Immunocytochemistry with anti-rhodamine (blue reaction product; n, node). D: Double immunocytochemistry with anti-rhodamine (blue reaction product) and anti-fluorescein (red reaction product). E: Transverse section of the embryo shown in D. Arrows, CRSE-labeled cells; arrowheads, CFSE-labeled cells. nt, neural tube. DiI = 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; CRSE = 5-[and 6-] carboxytetramethylrhodamine, succinimidyl ester; DiO = 3,3′-dioctadecyloxacarbocyanine perchlorate; CFSE = 5-[and 6-] carboxyfluorescein diacetate, succinimidyl ester.

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Next, as shown in Figure 1A, a small bolus of a solution of DiO/CFSE (green fluorescent spot; Molecular Probes) was microinjected immediately caudal to the first injection (DiO = 3,3′-dioctadecyloxacarbocyanine perchlorate, catalog no. D-275; CFSE = 5-[and -6] carboxyfluorescein diacetate, succinimidyl ester, catalog no. C-1157); DiO, like DiI, intercalates into cell membranes and is included in the solution because it is intensely fluorescent, allowing living cells to be easily followed by fluorescence microscopy. To make the DiO solution, dissolve 2.5 mg of DiO in 50 μl of DMSO then mix with 950 μl of ethanol, for a concentration of 2.5 mg/ml. CFSE, like CRSE, passes through cell membranes and becomes trapped in the cytoplasm; it is included in the solution because its fluorescent label is fluorescein, again allowing for processing of a permanent stain using commercially available antibodies (see Double Immunocytochemistry section, below). To make the CFSE solution, dissolve 2.5 mg of CFSE in 1 ml of DMSO, for a concentration of 2.5 mg/ml. DiO/CFSE solution consists of a 1:1 mixture of the two dyes; thus, the final working concentration of each dye in DiO/CFSE is 1.25 mg/ml. CFSE is colorless in solution (unlike CRSE) and including DiO, therefore, is very useful for visualizing the injection. However, DiO seems to increase the viscosity of the solution. If preferred, it can be omitted and a 0.25 ethanol:1 CFSE mixture is used. This change increases the final concentration of CFSE/ethanol to 2 mg/ml rather than 1.25 mg/ml for DiO/CFSE, and it is sufficient for following the fluorescence in living embryos.

Dye solutions were microinjected by using a Picospritzer II (General Valve Corp., Fairfield, NJ) and a micropipette attached to a micromanipulator. Images of labeled embryos were taken after microinjection and subsequently at regular intervals during incubation using an epifluorescence microscope equipped with a digital camera.

By using a green excitation filter, cells labeled with DiI/CRSE fluoresce red, and by using a blue excitation filter, cells labeled with DiO/CFSE fluoresce green (Fig. 1A). After 8–16 hr of incubation, final images of labeled embryos at neurulation stages were obtained (Fig. 1B); embryos were then dissected from the vitelline membranes and fixed overnight with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS; subsequently called 4% paraformaldehyde; to make 1× PBS, add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4 to 1 liter of distilled water and adjust pH to 7.4).

Transplantation of Labeled Groups of Cells

Grafts from donor embryos were transplanted homotopically and isochronically to host embryos (Fig. 2). Two groups of donor embryos were labeled with fluorescent markers: one group was labeled with a solution of CRSE; the other group was labeled with a solution of CFSE. A working solution of each label was made by adding 30 μl of a stock solution (10 mg dye/1 ml DMSO) to 1 ml of saline. Donor embryos, placed in modified New culture, were incubated for 60–90 min in the dye solution. Grafts were obtained from donor embryos as small 125-μm square fragments, cut from desired areas using a cactus needle. Subsequently, grafts were transferred with a micropipette to a depression slide containing saline, washed, and transferred to a host embryo (in New or EC culture) in which a similar fragment had been previously removed. As shown in Figure 2A,B, two grafts were transplanted, one labeled with CRSE (red fluorescent spot; placed more caudally within the primitive streak; Fig. 2A), and one labeled with CFSE (green fluorescent spot; placed immediately rostral to the CRSE-labeled graft; Fig. 2B). After 8–16 hr of incubation, embryos were dissected from the vitelline membranes and fixed overnight in 4% PFA.

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Figure 2. A,B: Whole-mount chick host blastoderm at stage 3b after homotopic and isochronic grafting of segments of the chick primitive streak (ps) from donor blastoderms, one labeled with CRSE (A, red fluorescent spot) and another labeled with CFSE (B green fluorescent spot). C,D,F,G: Two whole-mount embryos, 16 hr after transplantation at the levels shown in A,B (n, node). C,F: Immunocytochemistry with anti-rhodamine (C, blue reaction product; F, red reaction product; n, node). D,G: Double immunocytochemistry with anti-rhodamine (D, blue reaction product; G, red reaction product) and anti-fluorescein (brown reaction product). E: Transverse section of the embryo shown in D (level indicated on D). Arrows, CRSE-labeled cells; arrowheads, CFSE-labeled cells. nt, neural tube. CRSE = 5-[and 6-] carboxytetramethylrhodamine, succinimidyl ester; CFSE = 5-[and -6] carboxyfluorescein diacetate, succinimidyl ester.

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Double Immunocytochemistry

Embryos subjected to microinjection or transplantation were processed for double whole-mount immunocytochemistry to identify the fluorescently (i.e., CRSE- and CFSE-) labeled cells at the end of the incubation period (Figs. 1, 2). Immunocytochemistry was used to provide a permanent label that could be detected in whole-mounts or serial sections after processing for paraffin histology.

After fixation in 4% PFA, embryos were washed in PBS, dehydrated in a methanol series, and stored in methanol (for at least 12 hr at −20°C). Embryos were then rehydrated to PBS, washed in PBT (0.25% Triton X-100, 0.2% bovine serum albumin in PBS), and blocked in PBT and N (5% normal goat serum). Primary antibodies conjugated with fluorescein or rhodamine against both CRSE and CFSE, diluted in PBT and N, were applied together, and embryos were incubated overnight at 4°C. As primary antibodies were labeled with fluorescent markers, the need for secondary antibodies was eliminated, greatly reducing the amount of time required to generate results. The following specific protocols were used.

To detect cells labeled with CRSE, we used an alkaline phosphatase (AP) -conjugated goat anti-rhodamine (Vector Laboratories, Burlingame, CA, catalog no. MB-1920), diluted 1:500 to detect cells labeled by microinjection, or 1:100 to detect transplanted labeled cells. We processed this antibody in two different ways to obtain different color reactions. To obtain a blue reaction product, we used a solution of 20–200 μl of NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Roche, Indianapolis, IN, catalog no. 1681451) in 10 ml of NTMT (100 mM NaCl, 100 mM Tris-HCl, 50 mM MgCl2, 0.25% Tween-20), pH 9.5 (Figs. 1C, 2C). To obtain a red reaction product, we used, according to the manufacturer's instructions, the Vector red substrate kit to detect alkaline phosphatase (Vector Laboratories, catalog no. SK-5100), pH 8.2–8.5 (Fig. 2F).

To detect cells labeled with CFSE, we used a horseradish peroxidase (POD) -conjugated sheep anti-fluorescein (Roche, catalog no. 1426346), diluted 1:200 (regardless of whether cells to be detected were labeled by microinjection or were transplanted). We also processed this antibody in two different ways to obtain different color reactions. To obtain a red reaction product, we used, according to the manufacturer's instructions, the Vector VIP substrate kit (to detect peroxidase; Vector Laboratories, catalog no. SK-4600; Fig. 1D,E). To obtain a brown reaction product, we used a standard peroxidase reaction according to the manufacturer's instructions (solution obtained from diaminobenzidine [DAB] tablets, Sigma, St. Louis, MO, catalog no. D-5905) (Fig. 2D,E,G). For the best contrast between the different colors, in both whole-mounts and paraffin sections, we recommend one of the following three color combinations: blue and red (Fig. 1D,E), blue and brown (Fig. 2D,E), or red and brown (Fig. 2G).

The CRSE (AP) antibody should be developed before CFSE (POD) antibody to avoid background staining by a nonspecific reaction due to the different pHs used (i.e., develop the more basic pH reaction first to avoid cross-reaction). After developing the CRSE antibody, it is necessary to wash embryos several times in PBT over 2 hr before developing the CFSE antibody. The embryo should not be post-fixed before developing the second color. All steps for the double immunocytochemical procedure require less than 2 working days to obtain the final photographed embryos.

In Situ Hybridization and Double Immunocytochemistry

To achieve triple staining in a single embryo, we used a combination of in situ hybridization and double immunocytochemistry (Figs. 3, 4). Embryos were first microinjected in adjacent areas with CRSE and CFSE or transplanted with grafts labeled with CRSE and CFSE (Fig. 3A,B). After incubation of the embryo to the desired stage (Fig. 3C,D), the embryos were fixed and processed first for in situ hybridization using a chick riboprobe for Nkx2.5 (marks the early heart rudiments) and then for double immunocytochemistry to detect CRSE and CFSE (Fig. 3E,F). In situ hybridization was performed as described by Nieto et al. (1996).

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Figure 3. A,B: Whole-mount chick blastoderm at stage 3d, 6 hr after homotopic and isochronic grafting of chick segments of the chick primitive streak (ps) from donor blastoderms, one labeled with CRSE (A, red fluorescence) and another labeled with CFSE (B, green fluorescence). C,D: Same embryo 12 hr after grafting (n, node). E: Same embryo after in situ hybridization (blue reaction product) with a chick Nkx2.5 riboprobe (marks the heart rudiments, hr) and immunocytochemistry with anti-rhodamine (red reaction product; n, node). F: Same embryo after in situ hybridization with a chick Nkx2.5 riboprobe (blue reaction product) and double immunocytochemistry with anti-rhodamine (red reaction product) and anti-fluorescein (brown reaction product). CRSE = 5-[and 6-] carboxytetramethylrhodamine, succinimidyl ester; CFSE = 5-[and 6-] carboxyfluorescein diacetate, succinimidyl ester.

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Figure 4. A,B: Whole-mount chick blastoderm (A) and an oblique section (B; level and orientation indicated on A). Two segments of the primitive streak were grafted 16 hr earlier from donor embryos, one labeled with CRSE and the other labeled with CFSE. In situ hybridization (blue reaction product) was performed using a chick Hex riboprobe (marks the heart rudiments, hr, and the extraembryonic vasculature, v), followed by immunocytochemistry with anti-rhodamine (red reaction product) and anti-fluorescein (brown reaction product). Arrow, CRSE-labeled cells; arrowheads, CFSE-labeled cells; asterisks, Hex-labeled tissue; ps, primitive streak. CRSE = 5-[and 6-] carboxytetramethylrhodamine, succinimidyl ester; CFSE = 5-[and 6-] carboxyfluorescein diacetate, succinimidyl ester.

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As digoxigenin labeled probes are typically developed to yield a blue reaction product to detect alkaline phosphatase, the best color combination for subsequent double immunocytochemistry is the red and brown described above (Fig. 2G) and shown in combination with in situ hybridization in Figure 3E,F. However, it is also possible to develop the digoxigenin-labeled probe to yield a red reaction product by using the Vector red substrate kit to detect AP (catalog no. SK-5100), pH 8.2–8.5 and then to use the blue and brown combination described above (Fig. 2D,E). However, the red color will change to blue if NBT/BCIP in NTMT at pH 9.5 is used to develop one of the immunocytochemistry colors. This undesirable characteristic means that in situ hybridization using NBT/BCIP at pH 9.5 to produce a blue reaction product is preferable, followed by immunocytochemistry using red and brown colors at lower pH.

Combining in situ hybridization and double immunocytochemistry provides permanent labels that can be detected in whole-mounts and histological sections. For example, an embryo processed for in situ hybridization using the Hex riboprobe, developed in blue, followed by double immunocytochemistry for CRSE and CFSE, developed in red and brown, respectively, can be embedded in paraffin and serially sectioned (see below) without obvious loss of any of the three permanent labels (Fig. 4A,B).

Paraffin Histology

The blue reaction product generated by detecting alkaline phosphate with NBT/BCIP, in either immunocytochemistry or in situ hybridization, is often substantially faded by conventional paraffin histological procedures. To preserve the reaction product better, embryos for paraffin histology were dehydrated with an ethanol series and then cleared in isopropanol (2 changes of 100% for 30 min each at room temperature) rather than with a conventional clearing agent such as Histosol. Thereafter, embryos were transferred to a 1:1 solution of isopropanol:Paraplast at 60°C for 30 min, infiltrated with 100% Paraplast at 60°C for 30 min, and embedded in fresh Paraplast. Blocks were sectioned at 10–20 μm, and the sections were mounted onto glass slides, cover-slipped using Aqua Poly/Mount (Polysciences, Warrington, PA, catalog no. 18606; note: most mounting media containing organic solvents cause the NBT/BCIP reaction product to fade after cover-slipping), and examined with Hoffman modulation contrast optics.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We have developed a new, rapid method combining in situ hybridization and double immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. Recent fate mapping studies in chick have focused on defining regional domains of the early blastoderm, including the primitive streak, epiblast, and endoderm (e.g., Garcia-Martinez et al., 1993; Lopez-Sanchez et al., 2001; Lawson and Schoenwolf, 2003). Moreover, these regional domains are characterized by the expression of several specific marker genes (e.g., Lawson et al., 2001; Chapman et al., 2002).

Construction of quail–chick transplantation chimeras remains a good, although technically difficult, method for fate mapping studies, in which quail cells are identified in chimeras by the presence of a nucleolar marker after Feulgen staining (Le Douarin, 1973). This technique continues to be widely used, especially since pan-quail antibodies and antibodies against quail endothelial cells became commercially available, which allow the permanent labeling of cells in whole-mounts and sections (Garcia-Martinez and Schoenwolf, 1993).

The development of various fluorescent dyes (DiI, DiO [lipophilic], CFSE, CRSE [cytoplasmic]) has provided a recent alternative to the construction of quail-chick transplantation chimeras, allowing the observation of marked cells by fluorescence microscopy in living embryos. A major advantage of using dyes has been the production of antibodies against the cytoplasmic ones; using such antibodies allows cells to be permanently marked in whole-mounts and sections (Darnell et al., 2000). Unfortunately, use of fluorescent dyes and antibodies requires extensive tissue processing over several days, especially when multiple dyes are used.

In the present work, we have developed a rapid method for conducting fate mapping studies, using microinjections of two different fluorescent dyes, or transplantation of groups of cells labeled with such dyes. This allows whole-mount observation of living embryos with fluorescence microscopy, followed by in situ hybridization and double immunocytochemistry to produce permanent reaction products that are retained through processing for paraffin histology. Moreover, samples can be stored indefinitely after immunocytochemistry without loss of label.

Microinjection Vs. Transplantation

Currently, there is no perfect technique to label groups of cells in developing chick embryos to track their morphogenetic movements, as both microinjection and transplantation have their own strengths and weaknesses and potential artifacts. Hence, the safest approach in any fate mapping study is to use a combination of microinjection and transplantation. Many fate maps have been constructed in chick by microinjecting lipophilic membrane dyes (such as DiI, DiA, DiO). However, using these dyes has a major weakness. Because antibodies for these labels do not exist, to visualize labeled cells within the depth of the embryo and to know for sure exactly what cells are labeled by the injection requires that the dye be photoconverted to a permanent reaction product before serial sectioning. In our experience, photoconversion seems very unreliable, in some cases resulting in deposition of label in areas of the embryo where fluorescence was not present and labeling should not occur, and in other cases failing to deposit label, despite the presence of robust fluorescence. Moreover, DAB is a carcinogen, and photoconversion requires that embryos be submersed in a DAB solution and kept for several hours on the stage of a fluorescence microscope. Using CRSE and CFSE and antibodies against rhodamine and fluorescein eliminates these problems. Nonetheless, using these dyes still does not make microinjection a perfect technique. The resolution of labeling is only as good as the quality of the original injection (i.e., its size and precision of placement) and variation can occur from injection to injection. Thus, although easier than transplantation with appropriate equipment, microinjection still requires considerable micromanipulatory skill. Moreover, microinjection indirectly labels cells within a site in an embryo (i.e., the injection is not an intracellular one), and as cells move into the site (and the pool of extracellularly injected dye), they can become labeled over time. This can be an advantage or a disadvantage, depending on experimental design and the question one is asking. However, that a site is labeled by microinjection rather than a finite number of cells needs to be kept in mind during data interpretation.

Transplantation of groups of cells from donor embryos (either quail embryos or fluorescently labeled chick embryos) to host embryos (unlabeled chick embryos), allows one to track a finite number of cells, rather than those cells that pass through a site over a period of time. Transplantation requires more technical skill than does microinjection and, consequently, it is more time consuming. Moreover, transplantation involves tissue extirpation (which can result in cell death in both the host and donor tissue, as well as regeneration of host tissue) and subsequent healing of the graft into host tissue. For example, when epiblast grafts were studied over a time course, it was found that by 4–6 hr, approximately half the grafts had healed into place, and by 8 hr, virtually all had healed (Schoenwolf and Alvarez, 1989). Delayed or faulty healing can give misleading and erroneous results. For example, if the graft fails to heal into place and to undergo normal morphogenetic movements, the results of fate mapping can be grossly misinterpreted (i.e., concluding that contributions occur to more rostral or lateral regions; or to deeper tissues). Finally, transplantation requires that embryos be carefully staged (to ensure that grafting is done isochronically) and the graft position be accurately determined (to ensure that grafting is done homotopically).

Strengths of the New Method

Our results show that whole-mount images obtained by fluorescence microscopy of living embryos, after microinjection or transplantation, and after immunocytochemistry show strikingly similar patterns of labeling. Thus, the movements of cells can be tracked in living embryos and their final positions can be reliably determined in detail in conventional serial sections. By using two fluorescent markers and two reaction products during immunocytochemistry, the relative movements of cells can be followed and subsequently analyzed in three dimensions in individual embryos, providing better insight into how cells migrate to reach their final destinations. By applying both primary antibodies together (and by using conjugated primary antibodies rather than conjugated secondary antibodies) processing can be done rapidly, obtaining final results considerably quicker than with previous methods. By conducting in situ hybridization, populations of cells can be defined by their molecular characteristics, rather than just their morphological or positional characteristics, further increasing the value of fate mapping studies. Finally, for lineage studies it is possible to identify single cells doubled labeled with, say, an anti-quail antibody (which provides nuclear labeling) and an in situ probe (which provides cytoplasmic labeling; e.g., Garcia-Martinez et al., 1997) or a cell labeled with two in situ probes when combined with confocal microscopy (e.g., Denkers et al., 2004).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. A. Sawitzke for comments on the manuscript. C.L.S., V.G.M., and G.C.S. were funded by de Consejería de Educación, Ciencia y Tecnología of the Junta de Extremadura; S.C.C. received a Wellcome Trust Prize Fellowship; and G.C.S. was funded by the National Institutes of Health.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND EXPERIMENTAL PROCEDURES
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES