Antibody:	anti-Gata4
Detection system:	Secondary antibody coupled to Alexa Fluor
Assay notes:	Heterozygous and homozygous embryos were indistinguishable.

Structure	Level	Pattern	Image	Note
TS5: inner cell mass	Present	Regionally restricted	1A GATA4 , 1A merge	Expression is localized to the primitive endoderm layer of the blastocyst; it co-localized with another expression marker for this layer.

Structure	Level	Pattern	Image	Note
TS6: primitive endoderm	Present		1D GATA4 , 1D merge	Expression is localized to the primitive endoderm layer of the blastocyst; it co-localized with a reporter for another gene that is a marker for this layer.

Structure	Level	Pattern	Image	Note
TS4: blastocyst	Absent		4A merge , 4A Gata4

Structure	Level	Pattern	Image	Note
TS5: inner cell mass	Present	Single cells	4B merge , 4B Gata4	Of the embryos examined, 60% (n=15) showed expression at this stage. The number of expressing cells observed ranges from 2 to 13. They are distributed heterogeneously.
TS5: trophectoderm	Absent		4B merge , 4B Gata4

Structure	Level	Pattern	Image	Note
TS5: blastocyst	Absent			40% of embryos examined at this stage did not show expression.

Structure	Level	Pattern	Image	Note
TS6: primitive endoderm	Present		4C merge , 4C Gata4
TS6: trophectoderm	Absent		4C Gata4 , 4C merge

Structure	Level	Pattern	Image	Note
TS6: primitive endoderm	Present		4D Gata4 , 4D merge	Of the 96 embryos of >100 cells examined, 80.2% of them had expression restricted to the primitive endoderm layer.
TS6: epiblast	Absent		4D merge , 4D Gata4
TS6: trophectoderm	Absent		4D merge , 4D Gata4

Structure	Level	Pattern	Image	Note
TS5: inner cell mass	Present	Single cells	5A	There is a salt and pepper distribution of expression.
TS5: trophectoderm	Absent		5A

Structure	Level	Pattern	Image	Note
TS5: inner cell mass	Present	Single cells	5B	There is a salt and pepper distribution of expression.
TS5: trophectoderm	Absent		5B

Structure	Level	Pattern	Image	Note
TS6: primitive endoderm	Present		5C	At this stage approximately half the embryos exhibit partial or complete segregation of expression to this layer; the other embryos exhibit the salt and pepper distribution pattern of expression seen at earlier stages. One or two expressing cells may persist in the deeper layers of the inner cell mass.
TS6: trophectoderm	Absent		5C

Structure	Level	Pattern	Image	Note
TS6: primitive endoderm	Present		5D
TS6: trophectoderm	Absent		5D