Antibody:	anti-Brachury (N-19; sc-17743)
Detection system:	Biotinylated secondary antibody/[Strept]avidin coupled to horseradish peroxidase

Structure	Level	Pattern	Image	Note
TS11: primitive streak	Strong		1B
TS11: embryo mesoderm	Strong	Regionally restricted	1B	Expression in posterior nascent mesoderm.
TS11: allantois	Present		1B

Structure	Level	Pattern	Image	Note
TS11: primitive streak	Strong		1C
TS11: embryo mesoderm	Strong	Regionally restricted	1C	Expression in posterior nascent mesoderm.
TS11: allantois	Present		1C

Structure	Level	Pattern	Image	Note
TS9: primitive streak	Present		2A	Expression within the nuclei of cells of the nascent primitive streak.
TS9: extraembryonic component	Absent		2A	Expression was not seen in cells of the extraembryonic mesoderm and visceral endoderm.

Structure	Level	Pattern	Image	Note
TS10: extraembryonic mesoderm	Present	Regionally restricted	2B	Expression in newly emerging extraembryonic mesoderm, not in cells that had emerged earlier and were further from the primitive streak.
TS10: primitive streak	Present	Widespread	2B	Expression was detected along the length of the streak.
TS10: extraembryonic visceral endoderm	Strong		2B

Structure	Level	Pattern	Image	Note
TS12: primitive streak	Present		2C	Expression within the nuclei.
TS12: allantois	Present		2C
TS12: node	Present		2C
TS12: notochord	Present		2C
TS12: embryo mesenchyme	Present	Regionally restricted	2C	Expression in nascent embryonic mesoderm, with staining forming an axial continuum between the allantois and the primitive streak.

Structure	Level	Pattern	Image	Note
TS13: neural ectoderm	Present	Regionally restricted	2D	Expression in nuclei of the posterior neurectoderm.
TS13: embryo mesenchyme	Present	Regionally restricted	2D	Expression in nuclei of the posterior embryonic mesoderm.
TS13: notochord	Present		2D
TS13: embryo endoderm	Present	Regionally restricted	2D	Expression in hindgut endoderm prior to delamination of the notochord.

Structure	Level	Pattern	Image	Note
TS10: primitive streak	Strong		3A
TS10: allantois	Absent		3A	Expression was negative in newly formed allantoic bud.
TS10: primitive endoderm	Absent		3A	By the neural plate stage (E7.0-7.5), no expression was observed in the posterior visceral endoderm overlying the primitive streak.

Structure	Level	Pattern	Image	Note
TS11: allantois	Strong	Regionally restricted	3B	Darkly stained nuclei were observed in the midline of the proximal allantoic core.
TS11: allantois mesoderm	Absent		3B	Expression was negative in the cells of the outer allantoic mesothelium.
TS11: primitive streak	Strong		3B

Structure	Level	Pattern	Image	Note
TS11: allantois	Present	Regionally restricted	3C	Expression in a densely packed region of cells extending from the proximal midline through the midline of the middle-third of the allantois.
TS11: allantois mesoderm	Absent		3C	Expression was negative in the cells of the outer allantoic mesothelium.

Structure	Level	Pattern	Image	Note
TS12: allantois	Strong	Regionally restricted	3D	Expression in a densely packed region of cells extending from the proximal midline through the midline of the middle-third of the allantois.
TS12: allantois mesenchyme	Absent		3D	Expression was negative in the cells of the outer allantoic mesothelium.
TS12: primitive streak	Strong		3D

Structure	Level	Pattern	Image	Note
TS12: primitive streak	Strong		3E
TS12: allantois	Present	Regionally restricted	3E	Expression in a densely packed region of cells extending from the proximal midline through the midline of the middle-third of the allantois.
TS12: allantois mesenchyme	Absent		3E	Expression was negative in the cells of the outer allantoic mesothelium.

Structure	Level	Pattern	Image	Note
TS12: primitive streak	Strong		3F
TS12: allantois	Weak	Regionally restricted	3F	Expression was only in a small population of nuclei in the base of the allantois.

Structure	Level	Pattern	Image	Note
TS12: cardiac muscle tissue	Present		4_inset in B , 4A , 4B	Expression within the cytoplasm of the myocardium and myocardial trabeculae.
TS12: endocardial tube	Strong		4B , 4A , 4_inset in B	Expression within the cytoplasm of the endocardium.

Structure	Level	Pattern	Image	Note
TS13: cardiac muscle tissue	Present		4C	Expression within the cytoplasm of the myocardium.
TS13: endocardial tube	Present		4C	Expression within the cytoplasm of the endocardial layers.

Structure	Level	Pattern	Image	Note
TS10: primitive endoderm	Present	Regionally restricted	5A	Expression is highly specific to transitional embryonic visceral endoderm residing at the extraembryonic-embryonic junction, and located in basally positioned cytoplasmic pits.

Structure	Level	Pattern	Image	Note
TS10: extraembryonic visceral endoderm	Strong	Regionally restricted	5_Upper inset in C , 5C	Expression within cytoplasmic pit-like structures, associated with the basally located nuclei in distal visceral endoderm.
TS10: extraembryonic mesoderm	Absent		5C	No expression in the extraembryonic mesoderm lining the fully formed exocoelomic cavity, which includes the mesodermal components of the yolk sac, chorion, amnion, and mesothelium of the allantois.
TS10: extraembryonic ectoderm	Present		5C
TS10: primitive streak	Present	Widespread	5C	Expression was detected along the length of the streak.

Structure	Level	Pattern	Image	Note
TS10: yolk sac endoderm	Present		5D
TS10: extraembryonic visceral endoderm	Strong	Non-Uniform	5D	Robust expression remained in the distal extraembryonic visceral endoderm. Once the chorion formed, low-level expression was seen in the proximal extraembryonic visceral endoderm near a developing yolk sac blood island.
TS10: extraembryonic mesoderm	Absent		5D	No expression in the extraembryonic mesoderm of the lining the fully formed exocoelomic cavity and chorion.

Structure	Level	Pattern	Image	Note
TS11: yolk sac endoderm	Present	Regionally restricted	5E	As yolk sac vascularization spread toward the embryonic region, expression within proximal extraembryonic visceral endoderm coincided with vascular morphogenesis.
TS11: extraembryonic mesenchyme	Absent		5E	No expression in the extraembryonic mesoderm lining the fully formed exocoelomic cavity.

Structure	Level	Pattern	Image	Note
TS12: yolk sac endoderm	Present		5F
TS12: extraembryonic mesenchyme	Absent		5F	No expression in the extraembryonic mesoderm lining the fully formed exocoelomic cavity.

Structure	Level	Pattern	Image	Note
TS9: extraembryonic ectoderm	Present		6A
TS9: extraembryonic visceral endoderm	Strong	Non-Uniform	6A	Robust expression in the extraembryonic visceral endoderm, particularly strong in distal regions.
TS9: primitive streak	Present	Widespread	6A	Expression was detected along the length of the streak.

Structure	Level	Pattern	Image	Note
TS11: chorion ectoderm	Strong	Punctate	6B , 6C	Most signal was punctuate cytoplasmic staining in the unfused chorionic ectoderm, but nuclear staining was present.
TS11: chorion mesoderm	Absent		6C , 6B

Structure	Level	Pattern	Image	Note
TS12: chorion ectoderm	Strong	Punctate	6D	Punctuate staining in the chorionic ectoderm, which has not fused with the ectoplacental cone.
TS12: chorion mesenchyme	Absent		6D

Structure	Level	Pattern	Image	Note
TS12: chorion ectoderm	Weak		6E	Diminished expression in the chorionic ectoderm coincides with the time of contact with the allantois.

Structure	Level	Pattern	Image	Note
TS7: embryo	Absent

Structure	Level	Pattern	Image	Note
TS10: posterior visceral endoderm	Strong	Regionally restricted		Strong expression was seen within posterior visceral endoderm overlying the primitive streak.
TS10: anterior visceral endoderm	Weak	Regionally restricted		Expression was less abundant in anterior visceral endoderm overlying the prospective heart field.

Structure	Level	Pattern	Image	Note
TS12: primitive heart tube	Trace	Single cells		Occasional positive cell was observed in the cardiogenic field at 4-somite embryos.

Structure	Level	Pattern	Image	Note
TS12: extraembryonic visceral endoderm	Present	Regionally restricted		No expression in the distal extraembryonic visceral endoderm overlaying the nascent chorionic disk.
TS12: amnion mesenchyme	Absent
TS12: chorion mesenchyme	Absent

Structure	Level	Pattern	Image	Note
TS13: embryo endoderm	Present	Regionally restricted		Expression in a few isolated cells in the hindgut not associated with the notochord.
TS13: allantois	Absent			Expression was no longer detectable by 8-somite stage.

Structure	Level	Pattern	Image	Note
TS13: yolk sac endoderm	Present			Expression diminished between 8- and 10-s.
TS13: extraembryonic mesenchyme	Absent			No expression in the extraembryonic mesoderm lining the fully formed exocoelomic cavity, which includes the mesodermal components of the yolk sac, chorion, amnion, and mesothelium of the allantois.

Structure	Level	Pattern	Image	Note
TS13: primitive ventricle cardiac muscle	Present	Regionally restricted		Between 8- and 12-somite stage, expression became restricted in the myocardium of the developing ventricles.

Structure	Level	Pattern	Image	Note
TS13: embryo mesenchyme	Present	Regionally restricted		Expression in the posterior embryonic mesoderm.
TS13: notochord	Present
TS13: yolk sac endoderm	Absent			Expression was absent by 12-somite stage.
TS13: extraembryonic mesenchyme	Absent			No expression in the extraembryonic mesoderm lining the fully formed exocoelomic cavity, which includes the mesodermal components of the yolk sac, chorion, amnion, and mesothelium of the allantois.
TS13: chorion	Absent			No expression in the chorionic ectoderm by 12-somite.