Antibody:	Anti-HEF1 (2G9)
Detection system:	Secondary antibody coupled to Cy3

Structure	Level	Pattern	Image	Note
TS17: neural tube	Present	Regionally restricted	2C	Expression was exclusively restricted to Sox2+ progenitors of the neuroepithelium.

Structure	Level	Pattern	Image	Note
TS17: brain	Present	Regionally restricted	3A	Expression was observed at posterior regions of the brain including diencephalon.
TS17: diencephalon	Present		3A
TS17: midbrain	Present	Regionally restricted	3A	Expression was detected in ventral midbrain.

Structure	Level	Pattern	Image	Note
TS17: midbrain	Present	Regionally restricted	3B	Expression was detected in the ventral midbrain in Sox2+/Nurr1(Nr4a2)- progenitors. Low expression levels were noted in the prospective occulomotor nuclei.

Structure	Level	Pattern	Image	Note
TS17: midbrain	Present	Regionally restricted	3C	Expression was detected in the ventral midbrain in Sox2+/Nurr1(Nr4a2)- progenitors. Low expression levels were noted in the prospective occulomotor nuclei.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present	Regionally restricted	3I	Expression was detected in the ventricular zone of the ventral midbrain. Expression was downregulated in cells with Nurr1 (Nr4a2) positive staining.

Structure	Level	Pattern	Image	Note
TS20: diencephalon	Present	Regionally restricted		Expression was restricted to the Sox2+/Nurr1+/Th+ neuroepithelial cells.

Structure	Level	Pattern	Image	Note
TS20: pons ventricular layer	Present		4A	There was some overlap with Sox2 expression.
TS20: pons	Present	Regionally restricted	4A	Expression was downregulated in Nurr1+(Nr4a2) serotoninergic nuclear complex.

Structure	Level	Pattern	Image	Note
TS20: pons ventricular layer	Present		4B
TS20: pons	Present	Regionally restricted	4B	Expression was decreased in Nurr1+(Nr4a2) serotoninergic nuclear complex.

Structure	Level	Pattern	Image	Note
TS20: cerebellum primordium	Strong	Regionally restricted	4C	Expression was detected in the ventricular zone of the cerebellum.

Structure	Level	Pattern	Image	Note
TS17: hindbrain	Present	Regionally restricted	4E	Expression was detected in the dorsal motor nucleus of the vagus nerve. Expression was seen in Sox2 positive cells.

Structure	Level	Pattern	Image	Note
TS17: hindbrain	Present	Regionally restricted	4F	Expression was detected in the hypoglossal nucleus but not in cells strongly expressing Neurog2 or Nr4a2 (Nurr1).

Structure	Level	Pattern	Image	Note
TS17: trigeminal ganglion	Present	Regionally restricted	4H	Expression was detected in the otic branch of the trigeminal ganglion.

Structure	Level	Pattern	Image	Note
TS17: primitive ventricle cardiac muscle	Present	Regionally restricted	5C	Expression was detected in cardiomyocyte progenitors of the heart ventricle.

Structure	Level	Pattern	Image	Note
TS17: optic cup inner layer	Present	Regionally restricted	5F	Expression was detected in Sox2+ cells in the prospective retina in the area corresponding to the ventricular zone of the neuroepithelium.

Structure	Level	Pattern	Image	Note
TS17: nasal pit epithelium	Present		5G	Expression was detected in some Sox2 positive cells in the olfactory pit epithelium.

Structure	Level	Pattern	Image	Note
TS20: gonad germinal epithelium	Present		6B	Expression was detected in the gonad somatic epithelium where it was co-expressed with low levels of Nurr1 (Nr4a2).
TS20: mesonephros	Present		6B

Structure	Level	Pattern	Image	Note
TS20: cartilage	Present	Regionally restricted	6C	Expression was detected in progenitor cells of the prospective cartilage at trunk level.

Structure	Level	Pattern	Image	Note
TS20: cartilage	Present	Regionally restricted	6D	Expression was detected in progenitor cells of the prospective cartilage at trunk level where it was co-expressed with low levels of Nurr1 (Nr4a2).
TS20: dorsal root ganglion	Absent		6D

Structure	Level	Pattern	Image	Note
TS20: cartilage	Present	Regionally restricted	6F	Expression was detected in progenitor cells of the prospective cartilage at trunk level.

Structure	Level	Pattern	Image	Note
TS20: cartilage	Present	Regionally restricted	6H	Expression was detected in progenitor cells of the prospective cartilage at cranial level where it was co-expressed with low levels of Nurr1 (Nr4a2).

Structure	Level	Pattern	Image	Note
TS20: gut epithelium	Present		7C	Expression was seen in Sox2 positive progenitors of the gut epithelium.

Structure	Level	Pattern	Image	Note
TS20: gut epithelium	Present		7D	Expression was seen in Rxra positive progenitors of the gut epithelium.

Structure	Level	Pattern	Image	Note
TS20: gut epithelium	Present		7E	Expression was seen at moderate levels in Nurr1 (Nr4a2) positive progenitors of the gut epithelium.

Structure	Level	Pattern	Image	Note
TS17: Rathke's pouch	Present	Scattered	7F	Expression was detected in Rathke's pouch in scattered Sox2 positive cells.