Antibody:	anti-En1 (C-19)
Detection system:	Biotinylated secondary antibody/[Strept]avidin coupled to horseradish peroxidase
Assay notes:	Fixative was 4% paraformaldehyde, 20% picric acid.

Structure	Level	Pattern	Image	Note
TS20: hindbrain	Present		2D' , 2A , 2D
TS20: midbrain	Present		2D' , 2A , 2D
TS20: midbrain-hindbrain junction	Present		2D' , 2A , 2D
TS20: diencephalon	Present	Regionally restricted	2G' , 2G , 2A	Expression is around Rathke's pouch.

Structure	Level	Pattern	Image	Note
TS17: midbrain-hindbrain junction	Present	Regionally restricted	3A , 3B	Expression is in the ventricular and intermediate zones of the junction.
TS17: hindbrain lateral wall	Present	Regionally restricted	3A , 3C	Expression is in a subset of cells of the intermediate zone.

Structure	Level	Pattern	Image	Note
TS19: midbrain-hindbrain junction	Present		3D
TS19: midbrain lateral wall	Present	Regionally restricted	3D	Expression is ventral and is mostly restricted to the intermediate zone.
TS19: midbrain	Present	Regionally restricted	3D	Expression is in a group of positive cells in the mesencephalic flexure.

Structure	Level	Pattern	Image	Note
TS20: midbrain-hindbrain junction	Present	Regionally restricted	3E , 3F	Expression is in the outer layers of the intermediate zone of the midbrain/hindbrain junction.
TS20: hindbrain	Present	Regionally restricted	3G , 3E , 3F	In the floor of the hindbrain expression is in the intermediate zone and is broken at several regions.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present	Regionally restricted	3H	Expression is ventral in a subset of cells of the mesencephalic flexure.

Structure	Level	Pattern	Image	Note
TS22: inferior colliculus	Present		3I
TS22: cerebellum	Present	Regionally restricted	3I
TS22: hindbrain	Weak	Regionally restricted	3I	Expression is in the intermediate zone in the hindbrain floor.

Structure	Level	Pattern	Image	Note
TS22: midbrain	Present	Regionally restricted	3J , 3J'	Expression is ventral in the mesencephalic flexure.

Structure	Level	Pattern	Image	Note
TS27: periaqueductal grey	Present	Scattered	3K
TS27: cerebellum	Present	Regionally restricted	3K
TS27: inferior colliculus	Present	Regionally restricted	3K

Structure	Level	Pattern	Image	Note
TS27: midbrain	Present	Regionally restricted	3L , 3L'	Expression is ventral.
TS27: substantia nigra	Present		3L' , 3L

Structure	Level	Pattern	Image	Note
TS17: head mesenchyme	Strong	Regionally restricted	4A	Expression is in a stream extending dorsoventrally in the mesenchyme adjacent to the midbrain/hindbrain junction.

Structure	Level	Pattern	Image	Note
TS19: head mesenchyme	Strong	Regionally restricted	4B' , 4B	Expression is in a stream extending dorsoventrally in the mesenchyme adjacent to the midbrain/hindbrain junction.

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Present	Single cells	4C' , 4C	Expression is in a few positive cells scattered in the dorsal surface.

Structure	Level	Pattern	Image	Note
TS21: embryo	Present	Regionally restricted	4D' , 4D	Expression is in the mandibular arch.

Structure	Level	Pattern	Image	Note
TS17: head mesenchyme	Present	Regionally restricted	4F	Expression is in the primodium of craniofacial bones.
TS17: Rathke's pouch	Absent		4F

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Present	Regionally restricted	4G	Expression is in the craniofacial mesenchyme.

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Present	Regionally restricted	4H	Expression is in the craniofacial mesenchyme.

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Present	Regionally restricted	4I	Expression is in the craniofacial mesenchyme.
TS20: face	Present		4I

Structure	Level	Pattern	Image	Note
TS20: dermomyotome	Present	Regionally restricted	5A' , 5A	Expression is in two lateral stripes.
TS20: sclerotome	Present	Regionally restricted	5A , 5A'	Expression is in the rostral halves.

Structure	Level	Pattern	Image	Note
TS20: dermomyotome	Present		5B' , 5B
TS20: sclerotome	Present	Regionally restricted	5B , 5B'	Expression is segmental in the rostral halves.

Structure	Level	Pattern	Image	Note
TS22: tail dermomyotome	Present		5C
TS22: tail sclerotome	Present	Regionally restricted	5C	Expression is in the rostral halves.

Structure	Level	Pattern	Image	Note
TS22: dermomyotome	Present	Single cells	5D

Structure	Level	Pattern	Image	Note
TS20: dermomyotome	Present		5E' , 5E
TS20: dorsal root ganglion	Present	Non-Uniform	5E' , 5E	Expression is predominantly ventral.

Structure	Level	Pattern	Image	Note
TS20: sympathetic ganglion	Strong		5F

Structure	Level	Pattern	Image	Note
TS22: sympathetic ganglion	Present		5G

Structure	Level	Pattern	Image	Note
TS24: sympathetic ganglion	Present	Single cells	5H , 5H'

Structure	Level	Pattern	Image	Note
TS20: vagus nerve	Present		5I

Structure	Level	Pattern	Image	Note
TS24: vagus nerve	Present	Scattered	5J' , 5J

Structure	Level	Pattern	Image	Note
TS23: heart ventricle wall	Present		5K' , 5K

Structure	Level	Pattern	Image	Note
TS17: cloaca	Present	Regionally restricted	5L , 5L'	Expression is in the wall of the cloaca, mostly restricted to the ventral parts.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present		S2A	Expression is in the nuclei.

Structure	Level	Pattern	Image	Note
TS27: midbrain	Present		S2B	Expression is in the nuclei.

Structure	Level	Pattern	Image	Note
TS20: face	Present		S2C	Expression is non-nuclear.

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Present		S2D

Structure	Level	Pattern	Image	Note
TS20: vertebral cartilage condensation	Present		S2E

Structure	Level	Pattern	Image	Note
TS17: cloaca	Present		S2F	Expression is non-nuclear.

Structure	Level	Pattern	Image	Note
TS24: cerebellum	Present		S3B , S3A
TS24: inferior colliculus	Present		S3B , S3A
TS24: midbrain	Present	Regionally restricted	S3A , S3C	Expression is ventral.

Structure	Level	Pattern	Image	Note
TS27: cerebellum	Present		S3E , S3D
TS27: inferior colliculus	Present		S3E , S3D
TS27: midbrain	Present	Regionally restricted	S3F , S3D	Expression is ventral.

Structure	Level	Pattern	Image	Note
TS28: midbrain	Present	Regionally restricted	S3G , S3H	Expression is ventral.

Structure	Level	Pattern	Image	Note
TS28: cerebellum	Absent		S3I

Structure	Level	Pattern	Image	Note
TS20: spinal cord	Weak	Regionally restricted		Expression is in the ventral lateral portion from E12.5 to neonatal ages.

Structure	Level	Pattern	Image	Note
TS27: spinal cord	Weak	Regionally restricted		Expression is in the ventral lateral portion from E12.5 to neonatal ages.