Limits...
Tracking and ablating subpopulations of epiblast cells in the chick embryo.

Gerhart J, Neely C, Pfautz J, George-Weinstein M - Biol Proced Online (2008)

Bottom Line: Embryos are removed from the shell and incubated on the yolk with an antibody.Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement.This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA. gerhartj@mlhs.org

ABSTRACT
The early chick embryo contains subpopulations of cells that express lineage-specific transcription factors. We have developed protocols to examine the role of these cells during development that involve labeling them for cell tracking purposes and ablating them within the epiblast. The procedures take advantage of the fact that subpopulations of epiblast cells differentially express cell surface antigens recognized by monoclonal antibodies. Embryos are removed from the shell and incubated on the yolk with an antibody. Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement. For long-term analyses, embryos are returned to a host shell and placed in an incubator. This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.

No MeSH data available.


Labeling Embryos Ex-Ovo with Fluorescent AntibodiesStage 2 embryos were labeled with the G8 and E12 MAbs and fluorescent secondary antibodies (red). Nuclei were stained with Hoechst dye (blue). The inset in panels A and B illustrates the location of cells labeled with the G8 (A) and E12 MAbs (B) in the posterior epiblast of unsectioned stage 2 embryos. Labeled embryos developed normally after returning them to the shell and incubating them for three (C), five (D), or 13 days (E). The region of the somite outlined in the hematoxylin and eosin stained section of the 5 day embryo (F) is shown at higher magnification in the fluorescence photomicrograph (G). Epiblast cells stained with the G8 MAb were incorporated into the dorsomedial region of the dermomyotome and myotome (G). The locations of epiblast cells labeled with the E12 MAb at stage 4 are outlined in the drawing of the unsectioned stage 14 embryo (H). Cells stained with E12 were visible in the brain (I) and neural tube (J). Bar = 9 µm in A, B and C and 54 µM in F-I.
© Copyright Policy - open acces
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2683548&req=5

Figure 01: Labeling Embryos Ex-Ovo with Fluorescent AntibodiesStage 2 embryos were labeled with the G8 and E12 MAbs and fluorescent secondary antibodies (red). Nuclei were stained with Hoechst dye (blue). The inset in panels A and B illustrates the location of cells labeled with the G8 (A) and E12 MAbs (B) in the posterior epiblast of unsectioned stage 2 embryos. Labeled embryos developed normally after returning them to the shell and incubating them for three (C), five (D), or 13 days (E). The region of the somite outlined in the hematoxylin and eosin stained section of the 5 day embryo (F) is shown at higher magnification in the fluorescence photomicrograph (G). Epiblast cells stained with the G8 MAb were incorporated into the dorsomedial region of the dermomyotome and myotome (G). The locations of epiblast cells labeled with the E12 MAb at stage 4 are outlined in the drawing of the unsectioned stage 14 embryo (H). Cells stained with E12 were visible in the brain (I) and neural tube (J). Bar = 9 µm in A, B and C and 54 µM in F-I.

Mentions: Our ex-ovo/in-ovo procedure is a precise and efficient method for labeling and ablating cells in blastula stage chick embryos that supports development into the fetal period. We have used this procedure to fluorescently label subpopulations of epiblast cells that express myogenic and neurogenic transcription factors with the G8 and E12 MAbs, respectively (4-6). In the stage 2 embryo, the G8 and E12 MAbs bind to separate populations of cells in the posterior epiblast (Fig. 1A and B) (4). A second and larger subpopulation of E12-positive cells is present in the anterior epiblast (4,5).


Tracking and ablating subpopulations of epiblast cells in the chick embryo.

Gerhart J, Neely C, Pfautz J, George-Weinstein M - Biol Proced Online (2008)

Labeling Embryos Ex-Ovo with Fluorescent AntibodiesStage 2 embryos were labeled with the G8 and E12 MAbs and fluorescent secondary antibodies (red). Nuclei were stained with Hoechst dye (blue). The inset in panels A and B illustrates the location of cells labeled with the G8 (A) and E12 MAbs (B) in the posterior epiblast of unsectioned stage 2 embryos. Labeled embryos developed normally after returning them to the shell and incubating them for three (C), five (D), or 13 days (E). The region of the somite outlined in the hematoxylin and eosin stained section of the 5 day embryo (F) is shown at higher magnification in the fluorescence photomicrograph (G). Epiblast cells stained with the G8 MAb were incorporated into the dorsomedial region of the dermomyotome and myotome (G). The locations of epiblast cells labeled with the E12 MAb at stage 4 are outlined in the drawing of the unsectioned stage 14 embryo (H). Cells stained with E12 were visible in the brain (I) and neural tube (J). Bar = 9 µm in A, B and C and 54 µM in F-I.
© Copyright Policy - open acces
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683548&req=5

Figure 01: Labeling Embryos Ex-Ovo with Fluorescent AntibodiesStage 2 embryos were labeled with the G8 and E12 MAbs and fluorescent secondary antibodies (red). Nuclei were stained with Hoechst dye (blue). The inset in panels A and B illustrates the location of cells labeled with the G8 (A) and E12 MAbs (B) in the posterior epiblast of unsectioned stage 2 embryos. Labeled embryos developed normally after returning them to the shell and incubating them for three (C), five (D), or 13 days (E). The region of the somite outlined in the hematoxylin and eosin stained section of the 5 day embryo (F) is shown at higher magnification in the fluorescence photomicrograph (G). Epiblast cells stained with the G8 MAb were incorporated into the dorsomedial region of the dermomyotome and myotome (G). The locations of epiblast cells labeled with the E12 MAb at stage 4 are outlined in the drawing of the unsectioned stage 14 embryo (H). Cells stained with E12 were visible in the brain (I) and neural tube (J). Bar = 9 µm in A, B and C and 54 µM in F-I.
Mentions: Our ex-ovo/in-ovo procedure is a precise and efficient method for labeling and ablating cells in blastula stage chick embryos that supports development into the fetal period. We have used this procedure to fluorescently label subpopulations of epiblast cells that express myogenic and neurogenic transcription factors with the G8 and E12 MAbs, respectively (4-6). In the stage 2 embryo, the G8 and E12 MAbs bind to separate populations of cells in the posterior epiblast (Fig. 1A and B) (4). A second and larger subpopulation of E12-positive cells is present in the anterior epiblast (4,5).

Bottom Line: Embryos are removed from the shell and incubated on the yolk with an antibody.Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement.This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA. gerhartj@mlhs.org

ABSTRACT
The early chick embryo contains subpopulations of cells that express lineage-specific transcription factors. We have developed protocols to examine the role of these cells during development that involve labeling them for cell tracking purposes and ablating them within the epiblast. The procedures take advantage of the fact that subpopulations of epiblast cells differentially express cell surface antigens recognized by monoclonal antibodies. Embryos are removed from the shell and incubated on the yolk with an antibody. Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement. For long-term analyses, embryos are returned to a host shell and placed in an incubator. This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.

No MeSH data available.