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Keywords:

  • mouse;
  • parietal yolk sac;
  • Reichert's membrane;
  • collagenase type IV;
  • dispase

Abstract

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

Crucial aspects of axial development in mice occur at early postimplantation stages from the time of implantation to the appearance of the primitive streak. However, this period of development is notoriously refractory to experimental approaches due to the small size of the conceptus and to the presence of the parietal yolk sac, a protective tripartite membrane that surrounds the developing egg cylinder. Here, we describe a method that combines enzymatic digestion and mechanical manipulation to remove the parietal yolk sac of conceptuses at stages between 5.5 and 6.5 days post coitum. This method, which is compatible with whole-mount in situ hybridization and immunostaining techniques, offers a significant improvement over conventional dissection techniques, and it will greatly facilitate research in early mammalian development. Developmental Dynamics 236:489–493, 2007. © 2006 Wiley-Liss, Inc.


INTRODUCTION

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

Recent experiments in mice show that the basic body plan is molecularly and morphologically evident at early postimplantation stages between 4.5 and 5.5 days post coitum (dpc) (Rossant and Tam,2004; Rivera-Pérez and Magnuson,2005; Takaoka et al.,2006). These experiments suggest that the process of axial specification commences soon after implantation of the blastocyst. As a consequence, research into the mechanisms that determine the main body axes of the embryo has focused on early postimplantation stages.

One of the main obstacles in experimental manipulation of early postimplantation stages is the presence of the parietal yolk sac (PYS), an extraembryonic membrane that envelops the egg cylinder. The parietal yolk sac is composed of trophoblast cells on the uterine side and parietal endoderm cells on the conceptus side. Between these two cellular layers lies Reichert's membrane, an acellular proteinaceous basement membrane composed mainly of type IV procollagen, laminin, entactin, and heparan sulfate proteoglycan (Hogan,1980; Smith and Strickland,1981; Semoff et al.,1982; Hogan et al.,1984). Removal of the parietal yolk sac is a prerequisite in the majority of experimental manipulations of egg cylinder conceptuses.

The conventional method for removal of the parietal yolk sac is to physically tear it away from the egg cylinder using forceps. This method is routinely used for dissection of embryos at 6.5 dpc and later. However, the task becomes increasingly difficult in younger conceptuses. For example, at 5.5 dpc, the egg cylinder measures a fifth of a millimeter in length (Rivera-Pérez et al.,2003) and exceptional manual dexterity is required to isolate the PYS without damaging the egg cylinder. An alternative method is to handle the conceptus using glass needles and micromanipulators (Miura and Mishina,2003). Both of these methods require skillful manipulations and/or mastering the use of micromanipulators.

Here, we report that a mixture of three enzymes, collagenase type IV, dispase, and hyaluronidase, in combination with pipetting, can be used to gently remove the parietal yolk sac without apparent damage to the conceptus. Moreover, we show that conceptuses dissected using this method show normal whole-mount in situ hybridization and immunostaining patterns.

RESULTS

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

Collagenase type IV and dispase are two enzymes commonly used to gently dissociate human stem cells (Shamblott et al.,2001; Carpenter et al.,2003; Amit et al.,2004; Ware et al.,2005). Collagenase type IV is a protease with high specificity for collagen, whereas dispase is a neutral metalloprotease that cleaves fibronectin and collagen type IV (Stenn et al.,1989). Because collagen type IV is a major component of Reichert's membrane (Hogan,1980; Smith and Strickland,1981; Hogan et al.,1984) and fibronectin is localized immediately adjacent to the trophoblast cells of the PYS (Semoff et al.,1982), we reasoned that a solution containing these enzymes would facilitate the removal of the PYS without affecting the egg cylinder-shaped conceptus. To determine whether a solution containing collagenase type IV and dispase facilitated removal of the PYS, we initially used a phosphate buffered saline (PBS) solution containing collagenase type IV and dispase at a concentration of 10 mg/ml. This concentration is approximately 10 times higher than that used in the dissociation of human embryonic stem cells. Using this solution, we were able to remove the PYS in conceptuses dissected at 6.5 dpc after incubation at 37°C for a period of 10 min and pipetting them through the bore of a pulled Pasteur pipette. However, this procedure made the PYS viscous and sticky, and the conceptuses tended to attach to each other, the pipette, or the tissue culture dish. To minimize this problem, we added hyaluronidase at a concentration of 0.3 mg/ml to the enzyme mixture. Hyaluronidase has been used to reduce the viscosity of amnion preparations (Nagy et al.,2003). Conceptuses treated with a solution composed of collagenase type IV, dispase, and hyaluronidase were less sticky and easier to manipulate.

Once we demonstrated the feasibility of removing the PYS with an enzymatic treatment and mechanical manipulation, we determined the minimal amount of collagenase type IV and dispase as well as the incubation time required to remove the PYS but minimize the potential damage to the egg cylinder. By using serial dilutions, we found that a solution containing 1.0 mg/ml of collagenase type IV, 1.0 mg/ml of dispase, and 0.3 mg/ml of hyaluronidase was sufficient for the removal of the parietal yolk sac. An incubation time of 3 or 5 min at 37°C was required for embryos at 5.5 and 6.5 dpc, respectively. A schematic representation of the procedure and the removal of the PYS in 5.5 dpc conceptuses is shown in Figure 1.

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Figure 1. Removal of the parietal yolk sac (PYS) in 5.5 days post coitum (dpc) embryos. A: Schematic representation of the method used for removal of the parietal yolk sac. The egg cylinder conceptus is isolated from the decidual tissue and incubated at 37°C in a mixture of three enzymes: collagenase type IV, dispase, and hyaluronidase in phosphate buffered saline. After incubation, the conceptus is drawn through a narrow pipette ectoplacental cone side first and pipetted in and out several times. The conceptus is then reversed and pulled into the pipette with the distal tip first. This procedure shears the PYS, which usually remains attached to the ectoplacental cone. Furthermore, pipetting removes the PYS completely. B: Conceptuses dissected at 5.5 dpc. The embryo to the left has the parietal yolk sac intact. In the embryos to the right, the parietal yolk sac has been removed but it is still attached to the ectoplacental cone (arrows). Scale bar = 80 μm.

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The majority of the embryos dissected enzymatically did not show obvious mechanical damage. However, approximately 10% of the embryos tended to lose the ectoplacental cone or were bent and misshapen. These damages happened when the egg cylinder entered the pipette sideways rather than through the ectoplacental cone or the distal epiblast end of the egg cylinder. Enzymatic removal of the PYS required approximately 45 min for a litter of 10 embryos as opposed to approximately 1 hr for manual dissection. Groups of four to six embryos were incubated simultaneously in the enzymatic solution to reduce the dissection time.

To determine the feasibility of using enzymatically dissected embryos in whole-mount in situ hybridization experiments, we conducted a side-by-side comparison of manually and enzymatically dissected embryos. We hybridized 5.5 dpc egg cylinders with Hhex. Hhex labels visceral endoderm cells located at the distal tip of the egg cylinder at approximately 5.5 dpc and later the anterior visceral endoderm (or AVE) located on one side of the epiblast (Thomas et al.,1998; Rodriguez et al.,2001). Both manually and enzymatically dissected embryos showed similar results (Fig. 2A,B). Embryos at 6.5 dpc were compared using a Brachyury probe. Brachyury labels the posterior epiblast/primitive streak and distal extraembryonic ectoderm (Perea-Gomez et al.,2004; Rivera-Pérez and Magnuson,2005). Embryos hybridization with Brachyury showed similar results independently of the dissection method used (Fig. 2C). Embryos at 6.5 dpc were also hybridized with a Wnt3 probe. Wnt3 labels the posterior epiblast and overlying visceral endoderm and tapers anteriorly on the visceral endoderm layer (Rivera-Pérez and Magnuson,2005). No obvious differences were observed between manually and dissected embryos using a Wnt3 probe (data not shown).

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Figure 2. Comparative analysis of the expression of Hhex and Brachyury in embryos dissected manually or enzymatically. A,B:Hhex expression in manually (A) and enzymatically (B) dissected 5.5 days post coitum (dpc) embryos. Hhex expression is visible on the anterior visceral endoderm located on one side of the distal epiblast (arrow). C: Early streak conceptuses dissected at 6.5 dpc and hybridized with Brachyury (T). The manually dissected embryo is shown on the left. Brachyury staining is observed in the primitive streak (arrowheads) and in the extraembryonic ectoderm (arrows). Scale bar = 30 μm in A,B, 100 μm in C.

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To determine whether enzymatically treated embryos were useful in immunostaining studies, we subjected 5.5 and 6.5 dpc conceptuses to whole-mount immunofluorescence using an anti–E-Cadherin antibody (Rivera-Pérez et al.,2003). E-Cadherin is a cell adhesion protein and, therefore, a good candidate to determine whether the enzymatic treatment of the egg cylinders had affected proteins located at the cell–cell interphase. We found comparable staining between manually and enzymatically dissected embryos (Fig. 3).

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Figure 3. Comparative analysis of E-cadherin immunostaining in manually and enzymatically dissected embryos. A,B: Embryos dissected manually (A) or enzymatically (B) at 5.5 days post coitum (dpc). C–F: Manually (C,D) or enzymatically (E,F) dissected 6.5 dpc embryos at early streak stage. D and F show a higher magnification of the anterior visceral endoderm (arrow) at the epiblast/extraembryonic ectoderm boundary of embryos in C and E, respectively. E-Cadherin is present at the cell–cell boundaries (red). The nuclei are stained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI, blue). Each panel is a confocal section through the middle of the proamniotic cavity of the embryo. Scale bars = 40 μm in A–C,E, 20 μm in D,F.

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In summary, we show that an enzymatic solution composed of collagenase type IV, dispase, and hyaluronidase along with pipetting is sufficient to gently remove the PYS in early postimplantation mouse conceptuses and that this treatment does not interfere with whole-mount in situ hybridization or immunostaining protocols.

DISCUSSION

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

The traditional method for removal of the parietal yolk sac in early postimplantation embryos requires skillful dissection using forceps. This method is hard to master and becomes more challenging the smaller the embryo. At 5.5 dpc for example, the dissection of intact egg cylinder conceptuses is a difficult task and some researchers have resorted to using glass needles and micromanipulators to remove the PYS (Miura and Mishina,2003). To facilitate the removal of the parietal yolk sac without damaging the conceptus, we devised a method that combines the use of a mild enzymatic solution composed of collagenase type IV, dispase, and hyaluronidase and pipetting.

We show that enzymatically treated embryos can be used in whole-mount in situ hybridization experiments in genes expressed in both the visceral endoderm, the external layer of the egg cylinder, and in the internal epiblast and extraembryonic ectoderm. In addition, we show that this method does not interfere with the detection of the cell surface protein E-Cadherin.

The methodology described here offers three major advantages over manual dissection, especially for embryos dissected at preprimitive streak stages. First, the percentage of embryos dissected without mechanical damage is improved. At 5.5 dpc, for example, approximately 90% of the enzymatically dissected embryos had no obvious mechanical damage. This finding is a significant improvement over the 50% success rate obtained in our previous manual dissection experiments (Rivera-Pérez et al.,2003). Second, using enzymatic dissection, we shortened the time of dissection approximately 25% over manual dissection; and third, this technique can lead to more reproducible results, because it relies less on the manual skills of the individual conducting the dissection.

We have shown that embryos dissected using the enzymatic method can be used in whole-mount in situ hybridization and immunostaining. Further studies will test the utility of this method for embryo culture. As more researchers focus their studies in early postimplantation stages, the need for simple ways to manipulate these embryos increases. Our method offers a simple and significant improvement over the traditional or micromanipulation-driven methods used to date.

EXPERIMENTAL PROCEDURES

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

Mouse Strains and Staging

Embryos were obtained from crosses between CD-1 mice (Charles River Laboratories). Embryos dissected at 5.5 dpc were staged according to the position of the visceral endoderm thickening (Rivera-Pérez et al.,2003). Embryos dissected at 6.5 dpc were staged as described (Downs and Davies,1993). The middle of the dark cycle (midnight) that preceded the morning in which the copulation plug was observed was considered the beginning of gestation.

Removal of the Parietal Yolk Sac

To remove the parietal yolk sac, a mixture of three enzymes was used, collagenase type IV (1.0 mg/ml; Gibco, catalog no. 17104-019), dispase (1.0 mg/ml; Gibco, catalog no. 17105-041), and hyaluronidase (0.3 mg/ml; Sigma H-4272) in Ca++/Mg++ free PBS. The solution was prepared fresh before every use and filtered through a 0.22-μm filter. Aliquots kept at −20°C for 1 month also worked well. Embryos were dissected from the deciduum and incubated in 500 μl of prewarmed enzymatic solution in four-well plates (Nunc, catalog no. 176740) at 37°C in a conventional CO2 incubator (Forma Scientific). Embryos at 5.5 and 6.5 dpc were incubated for 3 and 5 min, respectively. After the incubation period, they were transferred to medium consisting of MEMα (Gibco) containing 20 mM Hepes, 10% fetal calf serum, penicillin (100 U/ml), and Streptomycin (100 mg/ml). In this medium, they were gently pipetted up and down with a pulled Pasteur pipette. The bore of the pipette was of the same diameter as the egg cylinder or slightly narrower to allow friction between the pipette wall and the PYS during pipetting (the bore of the pipettes can be gauged using affi-gel blue beads from Bio-Rad, catalog no. 153-7301 or 153-7302). Embryos were passaged through the pipette with the ectoplacental cone side first several times and then inverted such that the epiblast tip passed first through the pipette bore (Fig. 1A). This treatment usually led to breakage of the PYS, which remained attached to the ectoplancental cone region. Further pipetting eliminated the PYS completely. To avoid clumping of the conceptuses, we pipetted them individually.

Whole-Mount In Situ Hybridization

For whole-mount in situ hybridization, Hhex (Thomas et al.,1998) and Brachyury (pSK75; Herrmann et al.,1990) probes were used as described previously (Rivera-Pérez and Magnuson,2005).

Immunofluorescence

Immunostaining was done as described previously (Rivera-Pérez et al.,2003). A rat anti–E-Cadherin primary antibody (Zymed, catalog no. 13-1900) and an Alexa Fluor 594 goat anti-rat secondary (Molecular Probes, catalog no. A-11007) were used. Confocal sections were done using a Leica SP2 AOBS microscope using ×10 or ×20 objectives.

Acknowledgements

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

We thank Stormy Chamberlain for helpful suggestions and Sundeep Kalantry for critical reading of the manuscript and help with confocal microscopy. T.M. was funded by a grant from the NIH.

REFERENCES

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