Antibody:	anti-OTX2
Detection system:	Biotinylated secondary antibody/[Strept]avidin coupled to horseradish peroxidase
Assay notes:	Tissue was frozen in cooled isopentane and then immersed in methanol.

Structure	Level	Pattern	Image	Note
TS9: primitive endoderm	Present	Non-Uniform	2c	In the hypoblast, there is higher expression in rostral regions as compared to more caudal ones.
TS9: embryo ectoderm	Present	Widespread	2c	Expression is present in the entire epiblast.

Structure	Level	Pattern	Image	Note
TS10: primitive endoderm	Present	Non-Uniform	2d	In the hypoblast, there is higher expression in rostral regions as compared to more caudal ones.
TS10: embryo ectoderm	Present	Widespread	2d	Expression is present in the entire epiblast.
TS10: mesendoderm	Weak	Single cells	2d	There is expression in a few cells ingressing through the primitive streak.

Structure	Level	Pattern	Image	Note
TS11: embryo ectoderm	Strong	Non-Uniform	2e	There is strong expression in the anterior neuroectoderm of the headfold; there is a progressive decrease in more posterior ectoderm.
TS11: neural ectoderm	Strong	Non-Uniform	2e	There is strong expression in the anterior neuroectoderm of the headfold; there is a progressive decrease in more posterior ectoderm.
TS11: mesendoderm	Present	Regionally restricted	2e	There is anterior expression.

Structure	Level	Pattern	Image	Note
TS12: embryo ectoderm	Strong	Non-Uniform	2f	There is strong expression in the anterior neuroectoderm of the headfold; there is a progressive decrease in more posterior ectoderm.
TS12: neural ectoderm	Strong	Non-Uniform	2f	There is strong expression in the anterior neuroectoderm of the headfold; there is a progressive decrease in more posterior ectoderm.

Structure	Level	Pattern	Image	Note
TS16: future forebrain	Present	Widespread	3a	Expression is not present in the preoptic recess.
TS16: telencephalon	Weak	Regionally restricted	3a	Expression levels are lower in the presumptive cerebral cortex.

Structure	Level	Pattern	Image	Note
TS18: forebrain	Present	Regionally restricted	3b	Expression has almost totally receeded from the dorsal telencephalon.
TS18: diencephalon	Present	Regionally restricted	3b	Expression is present in the mammillary area.

Structure	Level	Pattern	Image	Note
TS21: diencephalon	Present	Regionally restricted	3c	Areas of expression include the zona limitans intrathalamica.
TS21: hypothalamus	Present	Regionally restricted	3c	Expression is present in the mammillary area.

Structure	Level	Pattern	Image	Note
TS21: diencephalon	Moderate	Regionally restricted	3d	Areas of expression include the zona limitans intrathalamica.
TS21: epithalamus	Present		3d
TS21: thalamus ventricular layer	Present	Regionally restricted	3d	Expression is present in the dorsal thalamus.
TS21: hypothalamus	Present	Regionally restricted	3d	Expression is present in the mammillary area.

Structure	Level	Pattern	Image	Note
TS24: epithalamus	Present		3e
TS24: hypothalamus	Present	Regionally restricted	3e	Expression is present in the preoptic area.

Structure	Level	Pattern	Image	Note
TS24: hypothalamus	Present	Regionally restricted	3f	Expression is present in the mammillary area and around the infundibular cavity.

Structure	Level	Pattern	Image	Note
TS15: future midbrain	Present	Regionally restricted	4a	Expression is distributed homogeneously across the whole width of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS15: future midbrain	Present	Regionally restricted	4a'	Expression is distributed homogeneously across the whole width of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS15: future midbrain	Present	Regionally restricted	4a''	Expression is distributed homogeneously across the whole width of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS19: midbrain	Present	Regionally restricted		Expression is distributed homogeneously across the whole width of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present	Regionally restricted	4b	Expression is almost completely restricted to the marginal portion of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present	Regionally restricted	4b'	Expression is almost completely restricted to the marginal portion of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS20: midbrain	Present	Regionally restricted	4b''	Expression is almost completely restricted to the marginal portion of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS21: midbrain	Present	Regionally restricted		Expression is almost completely restricted to the marginal portion of the tectum anlage.

Structure	Level	Pattern	Image	Note
TS22: tectum	Present

Structure	Level	Pattern	Image	Note
TS24: inferior colliculus	Present	Regionally restricted	4c	Expression is ventricular.
TS24: superior colliculus	Present	Regionally restricted	4c	Expression is in the marginal layers.

Structure	Level	Pattern	Image	Note
TS24: inferior colliculus	Present	Regionally restricted	4c'	Expression is ventricular.
TS24: superior colliculus	Present	Regionally restricted	4c'	Expression is in the marginal layers.

Structure	Level	Pattern	Image	Note
TS24: inferior colliculus	Present	Regionally restricted	4c''	Expression is ventricular.
TS24: superior colliculus	Present	Regionally restricted	4c''	Expression is in the marginal layers.

Structure	Level	Pattern	Image	Note
TS17: rhombomere	Present	Regionally restricted	5a	Expression is restricted to a few cells in a rostral-alar portion.
TS17: midbrain	Present		5a

Structure	Level	Pattern	Image	Note
TS19: midbrain	Present		5b

Structure	Level	Pattern	Image	Note
TS22: cerebellum	Present	Regionally restricted	5c	Expression is restricted to the germinal trigone, immediately overlying the choroid plexus of the fourth ventricle.

Structure	Level	Pattern	Image	Note
TS23: cerebellum	Present	Regionally restricted	5d	Expression is present in the germinal trigone, immediately overlying the choroid plexus of the fourth ventricle. It also extends rostrally along the marginal surface that is the external granular layer, with maximum expression caudually.

Structure	Level	Pattern	Image	Note
TS24: cerebellum	Present	Regionally restricted	5e	Expression extends rostrally along the marginal surface that is the external granular layer, with maximum expression caudually and minimum rostrally.

Structure	Level	Pattern	Image	Note
TS25: cerebellum	Present	Regionally restricted	5f	Expression extends rostrally along the marginal surface that is the external granular layer, with maximum expression caudually and minimum rostrally.

Structure	Level	Pattern	Image	Note
TS27: cerebellum external granule cell layer	Present		5h
TS27: cerebellum internal granule cell layer	Present		5h

Structure	Level	Pattern	Image	Note
TS28: cerebellum external granule cell layer	Strong	Widespread	5i
TS28: cerebellum internal granule cell layer	Strong	Widespread	5i

Structure	Level	Pattern	Image	Note
TS28: cerebellum internal granule cell layer	Weak	Regionally restricted	5j	Expression is detectable only in the most caudal IGL.

Structure	Level	Pattern	Image	Note
TS21: hindbrain lateral wall	Present	Regionally restricted	6a	There is a cluster of cells located slightly dorsal to the sulcus limitans and midway between the ventricular and marginal surface. Additional scattered cells are located in the basal plate.

Structure	Level	Pattern	Image	Note
TS21: hindbrain lateral wall	Present	Regionally restricted	6b	There is a cluster of cells located slightly dorsal to the sulcus limitans and midway between the ventricular and marginal surface. Additional scattered cells are located in the basal plate.

Structure	Level	Pattern	Image	Note
TS21: hindbrain lateral wall	Present	Regionally restricted	6c	There is a cluster of cells located slightly dorsal to the sulcus limitans and midway between the ventricular and marginal surface. Additional scattered cells are located in the basal plate.

Structure	Level	Pattern	Image	Note
TS21: spinal cord lateral wall	Present	Regionally restricted	6d	There is a cluster of cells located slightly dorsal to the sulcus limitans and midway between the ventricular and marginal surface. Additional scattered cells are located in the basal plate.

Structure	Level	Pattern	Image	Note
TS16: nasal pit	Present		7a	Expression is present in the olfactory placode.
TS16: telencephalon	Present		7a

Structure	Level	Pattern	Image	Note
TS18: olfactory epithelium	Present		7b

Structure	Level	Pattern	Image	Note
TS20: olfactory epithelium	Present		7c	Expression is mainly restricted to nuclei lying in luminal position, suggesting expression in post-mitotic and differentiated cells.

Structure	Level	Pattern	Image	Note
TS22: olfactory epithelium	Present		7d	Expression is mainly restricted to nuclei lying in luminal position, suggesting expression in post-mitotic and differentiated cells.
TS22: nasal mesenchyme	Present	Regionally restricted	7d	Expression is present in two parallel rows of small cell clusters that lie between the nasal pits. The form longitudinal columns, extending from the olfactory epithelium to the olfactory bulb region in the anterior medio-basal forebrain.

Structure	Level	Pattern	Image	Note
TS22: vomeronasal organ	Present	Single cells	7e	Expression is present in only a very few nuclei.

Structure	Level	Pattern	Image	Note
TS19: nose	Present	Single cells	7f	Expression is present in scattered cells localized just ventral to the anlage of the olfactory bulb.
TS19: olfactory epithelium	Present		7f
TS19: telencephalon	Present		7f

Structure	Level	Pattern	Image	Note
TS21: olfactory epithelium	Present		7g	Expression is mainly restricted to nuclei lying in luminal position, suggesting expression in post-mitotic and differentiated cells.
TS21: nasal mesenchyme	Present	Regionally restricted	7g	Expression is present in two parallel rows of small cell clusters that lie between the nasal pits. The form longitudinal columns, extending from the olfactory epithelium to the olfactory bulb region in the anterior medio-basal forebrain.