Antibody:	anti-rabbit type II collagen
Detection system:	Secondary antibody coupled to fluorescein
Assay notes:	For most of the work the anti-rabbit type II collagen antibody was used but a second antibody (anti-chicken type II collagen, MGI:2148747) was also used. Identical results were obtained with both. Embryos were fixed in 95% ethanol.

Structure	Level	Pattern	Image	Note
TS15: head mesenchyme	Strong	Regionally restricted	1B , 1A	Strong staining around the notochord which extended a short distance into the adjacent mesenchyme.
TS15: notochord	Strong	Regionally restricted	1B , 1A	Strong staining around the notochord which extended a short distance into the adjacent mesenchyme.

Structure	Level	Pattern	Image	Note
TS15: optic vesicle	Strong	Regionally restricted	1C , 1D	Staining at the interface between the basal surfaces of the lateral aspect of the optic vesicle and adjacent ectoderm, but did not extend around the remainder of the vesicle.

Structure	Level	Pattern	Image	Note
TS16: future hindbrain	Present	Regionally restricted	2A , 2B	Patchy staining along the basal surface of the neural tube. Staining around the otocyst and on the ventrolateral aspect of the adjacent (rhombencephalic) neural tube increased in intensity. The strongest reaction was found between the two structures.
TS16: otocyst	Present	Regionally restricted	2A , 2B	Patchy staining along the basal surface of the neural tube. Staining around the otocyst and on the ventrolateral aspect of the adjacent (rhombencephalic) neural tube increased in intensity. The strongest reaction was found between the two structures.
TS16: future midbrain	Present	Patchy	2A , 2B	Patchy staining along the basal surface of the neural tube.

Structure	Level	Pattern	Image	Note
TS16: future hindbrain	Present	Regionally restricted	3B , 3A	Staining around the otocyst and on the ventrolateral aspect of the adjacent (rhombencephalic) neural tube increased in intensity. The strongest reaction was found between the two structures.
TS16: otocyst	Present	Regionally restricted	3B , 3A	Staining around the otocyst and on the ventrolateral aspect of the adjacent (rhombencephalic) neural tube increased in intensity. The strongest reaction was found between the two structures.

Structure	Level	Pattern	Image	Note
TS16: otocyst	Present	Regionally restricted	3C , 3D	Staining around the remaining aspects of the otic vesicle and absence of staining under the ectoderm.

Structure	Level	Pattern	Image	Note
TS14: heart	Strong	Regionally restricted	4A	Strong staining of the interface between the endocardial and mesocardial layers of the heart wall and also deep within the mesocardium itself.

Structure	Level	Pattern	Image	Note
TS14: heart	Strong	Regionally restricted	4C , 4B	Strong staining of the interface between the endocardial and mesocardial layers of the heart wall and also deep within the mesocardium itself.

Structure	Level	Pattern	Image	Note
TS19: lens vesicle	Present	Regionally restricted	5D , 5A , 5B , 5C	Staining in the forming posterior chamber of the eye between the medial surface of the lens and the neural retina; immunoreactivity appeared to be associated almost exclusively with the basal surface of the retina.
TS19: retina	Present	Regionally restricted	5D , 5A , 5B , 5C	Staining in the forming posterior chamber of the eye between the medial surface of the lens and the neural retina; immunoreactivity appeared to be associated almost exclusively with the basal surface of the retina.

Structure	Level	Pattern	Image	Note
TS18: Rathke's pouch	Very strong	Regionally restricted	6A , 6B	Staining between the basal surfaces of the infundibulum and the hypophyseal pouch.
TS18: infundibular recess of 3rd ventricle	Very strong	Regionally restricted	6A , 6B	Staining between the basal surfaces of the infundibulum and the hypophyseal pouch.

Structure	Level	Pattern	Image	Note
TS21: inner ear	Very strong	Regionally restricted	7	Staining within the periotic mesenchyme, wich was starting to display a morphology characteristic of mature cartilage. Staining in all cartilages of the inner ear, in particular the tympanic ring surrounding the cochleal and saccular epithelia.
TS21: otic capsule	Strong		7	Staining within the periotic mesenchyme, wich was starting to display a morphology characteristic of mature cartilage. Staining in all cartilages of the inner ear, in particular the tympanic ring surrounding the cochleal and saccular epithelia.
TS21: eye	Absent		7
TS21: axial skeleton	Present		7	Staining in the facial, branchial arch, vertebral and limb cartilages.
TS21: chondrocranium	Present		7	Staining in the facial, branchial arch, vertebral and limb cartilages.

Structure	Level	Pattern	Image	Note
TS21: inner ear	Present	Regionally restricted	8B , 8A	All rudiments of the inner ear appear to express type II collagen.
TS21: otic capsule	Present		8B , 8A

Structure	Level	Pattern	Image	Note
TS21: inner ear	Present	Regionally restricted	8C , 8D	All rudiments of the inner ear appear to express type II collagen.
TS21: otic capsule	Present		8C , 8D

Structure	Level	Pattern	Image	Note
TS21: basioccipital pre-cartilage condensation	Strong		8E , 8F

Structure	Level	Pattern	Image	Note
TS20: hindbrain	Present	Regionally restricted	9B	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)
TS20: inner ear	Present	Regionally restricted	9B	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)

Structure	Level	Pattern	Image	Note
TS20: hindbrain	Present	Regionally restricted	9C	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)
TS20: inner ear	Present	Regionally restricted	9C	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)

Structure	Level	Pattern	Image	Note
TS20: hindbrain	Present	Regionally restricted	9D	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)
TS20: inner ear	Present	Regionally restricted	9D	Immunopositive interface between the basal surfaces of the rhombencephalon and the otocyst in the intervening reticulate lamina and on the outer surface of the lamina densa. Gold particles were located exclusively extracellularily. (TEM)

Structure	Level	Pattern	Image	Note
TS17: nasal pit	Present	Regionally restricted	10B , 10A	Staining surrounding the olfactory vesicles subjacent to the epithelium.
TS17: hindbrain	Present	Patchy	10A , 10B	Patchy staining along the basal surface of the neural tube throughout the mesencephalic and rhombencephalic regions
TS17: midbrain	Present	Patchy	10A , 10B	Patchy staining along the basal surface of the neural tube throughout the mesencephalic and rhombencephalic regions

Structure	Level	Pattern	Image	Note
TS20: nose	Present	Regionally restricted	10D , 10C	Expression in the optic cup localized to its interior, confined to the forming posterior chamber; expression apparent at the margin of the optic cup.
TS20: eye	Present	Regionally restricted	10D , 10C	Authors report expression in the olfactory primordia.
TS20: ear	Present	Regionally restricted	10D , 10C	Expression in the otocyst.
TS20: notochord	Present		10D , 10C

Structure	Level	Pattern	Image	Note
TS11: embryo	Absent

Structure	Level	Pattern	Image	Note
TS13: embryo	Absent

Structure	Level	Pattern	Image	Note
TS14: notochord	Weak	Regionally restricted		Expression was as a faint halo surrounding the notochord.
TS14: otic pit	Weak	Regionally restricted		Expression was between the basal surfaces of the invaginating otic vesicle and the neural epithelium.
TS14: forelimb bud mesenchyme	Present	Regionally restricted		Central and apically located limb mesenchyme (beneath the apical ectodermal ridge).
TS14: branchial arch endoderm	Present	Regionally restricted		Staining in the lateral and basal surfaces of the pharyngeal endoderm.

Structure	Level	Pattern	Image	Note
TS18: otocyst	Present	Regionally restricted		Staining in the otic region was adjacent and between the basal surfaces of the vestibular and cochleal epithelia, but also extended into the periotic mesenchyme.

Structure	Level	Pattern	Image	Note
TS20: neurohypophysis infundibulum	Very strong	Regionally restricted		Staining between the basal surfaces of the infundibulum and the hypophyseal pouch.
TS20: remnant of Rathke's pouch	Very strong	Regionally restricted		Staining between the basal surfaces of the infundibulum and the hypophyseal pouch.

Structure	Level	Pattern	Image	Note
TS21: remnant of Rathke's pouch	Absent			Absence of staining between the basal surfaces of the infundibulum and the hypophyseal pouch.

Structure	Level	Pattern	Image	Note
TS20: head mesenchyme	Strong	Regionally restricted		Staining around the notochord which extended well into the perinotochordal mesenchyme.
TS20: notochord	Strong	Regionally restricted		Staining around the notochord which extended well into the perinotochordal mesenchyme.
TS20: mandibular process	Weak	Regionally restricted		Faint staining in the central region of the mandibular processes.
TS20: inner ear	Strong	Regionally restricted		Staining around the otic epithelia, for instance basal and apical surfaces of the sacculus, utriculus, ampulus, and cochlea.
TS20: saccule	Strong	Regionally restricted		Staining around the otic epithelia, for instance basal and apical surfaces of the sacculus, utriculus, ampulus, and cochlea.
TS20: utricle epithelium	Strong	Regionally restricted		Staining around the otic epithelia, for instance basal and apical surfaces of the sacculus, utriculus, ampulus, and cochlea.

Structure	Level	Pattern	Image	Note
TS22: inner ear	Present	Regionally restricted		Staining in all rudiments of the inner ear which has now become restricted to the capsular cartilages.
TS22: otic capsule	Present	Regionally restricted		Staining in all rudiments of the inner ear which has now become restricted to the capsular cartilages.