Probe:	clone PRK
Probe preparation:	Antisense RNA
Visualized with:	Alkaline phosphatase
Assay notes:	One- and two-color in situ hybridization was performed using digoxygenin- and/or fluorescein-labeled probes, detected with alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies. Color reactions were carried out using different alkaline phosphatase substrates, resulting in different colored products as indicated in some of the figure legends.

Structure	Level	Pattern	Image	Note
TS17: telencephalon	Absent		4C	No expression in the medial wall of the telencephalon.
TS17: diencephalon	Present	Regionally restricted	4C	Expression at the dorsal midline of the diencephalon and midbrain.
TS17: midbrain	Present	Regionally restricted	4C	Expression at the dorsal midline of the diencephalon and midbrain.

Structure	Level	Pattern	Image	Note
TS17: brain	Strong	Regionally restricted	4H	Sites of strong expression include the dorsal midline of neural tube (does not continue caudal to isthmus), optic cup, and otic vesicle (pigmented epithelium of the retina).
TS17: optic cup	Strong	Regionally restricted	4H	Sites of strong expression include the dorsal midline of neural tube (does not continue caudal to isthmus), optic cup, and otic vesicle (pigmented epithelium of the retina).
TS17: otocyst	Strong	Regionally restricted	4H	Sites of strong expression include the dorsal midline of neural tube (does not continue caudal to isthmus), optic cup, and otic vesicle (pigmented epithelium of the retina).

Structure	Level	Pattern	Image	Note
TS17: nasal epithelium	Present

Structure	Level	Pattern	Image	Note
TS19: telencephalon	Present	Regionally restricted	4F	The cortical hem (see below) is delineated by the expression of Wnt2b, Wnt3a and Wnt5a.

Structure	Level	Pattern	Image	Note
TS19: hindbrain	Weak	Regionally restricted		Weak expression next to the hindbrain site of choroid plexus generation.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	1A	Strong expression in a band along the caudal 2/3rds of the medial wall, adjacent to the lateral ventricle, defines the cortical hem. At rostral levels, band is dorsal to CPe; at caudal levels, band curves ventrally to surround caudal end of CPe.
TS20: diencephalon	Present	Regionally restricted	1A	Expression marks the dorsal midline and two patches on either side of the midline.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	1B	Strong expression in a band along the caudal 2/3rds of the medial wall, adjacent to the lateral ventricle, defines the cortical hem. At rostral levels, band is dorsal to CPe; at caudal levels, band curves ventrally to surround caudal end of CPe.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	2A	Strong expression in a band along the caudal 2/3rds of the medial wall, adjacent to the lateral ventricle, defines the cortical hem. At rostral levels, band is dorsal to CPe; at caudal levels, band curves ventrally to surround caudal end of CPe.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	1D	Expression marks the cortical hem; abuts the CPe dorsally.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	1E	Expression marks the cortical hem; no expression in the junctional epithelium between the hem and CPe; abuts the CPe dorsally and caudoventrally.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	1F	Expression marks the cortical hem; abuts the CPe caudally.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	2C	Expression marks the cortical hem; no expression in the junctional epithelium between the hem and CPe; abuts the CPe.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Strong	Regionally restricted	7I	Expression marks the cortical hem; no expression in the junctional epithelium between the hem and CPe; abuts the CPe.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Present	Regionally restricted	2E	Expression at the ventricular side of the hem, but not in cells close to the pial surface.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Present	Regionally restricted	2F	Expression at the ventricular side of the hem, but not in cells close to the pial surface.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Present	Regionally restricted	2G	Expression at the ventricular side of the hem, but not in cells close to the pial surface.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Absent		7A	In brains of non-exencephalic homozygous mutant mice, no expression was detectable in the telencephalon.
TS20: diencephalon	Present	Regionally restricted	7A	Expression in some mutant mice at the dorsal midline of the diencephalon.
TS20: hindbrain	Present	Regionally restricted	7A	Expression next to the fourth ventricle where Ttr-expressing choroid plexus formed.

Structure	Level	Pattern	Image	Note
TS20: telencephalon	Absent		7G	In brains of non-exencephalic homozygous mutant mice, no expression detected at the margin of the medial telencephalic wall.

Structure	Level	Pattern	Image	Note
TS20: inner ear	Strong	Regionally restricted	7H	Strong expression at the otic vesicle (inner ear).

Structure	Level	Pattern	Image	Note
TS20: nasal cavity epithelium	Strong

Structure	Level	Pattern	Image	Note
TS22: telencephalon	Present	Regionally restricted	5A	By the age at which a hippocampal anlage is identifiable by morphology, expression marks the shrinking cortical hem, which is clearly separate from regions of neuroepithelium though to generate hippocampal neurons.

Structure	Level	Pattern	Image	Note
TS23: telencephalon	Present	Regionally restricted	5B	Overlapping expression of Wnt2b, Wnt3a and Wnt5a marks a thin layer of cells along the ventricular side of the hippocampal fimbria-fornix.

Structure	Level	Pattern	Image	Note
TS24: telencephalon	Absent			Five non-exencephalic homozygous mutants showed no expression in the medial telencephalon.