Orbital dissections of E13.5 to E15.5
Myf5WT/WT:IslMN:GFP and
Myf5Cre:IslMN:GFP embryos. Nerves and muscles are labeled and columns arranged as in
Figure 1. Orbits are imaged from a superior (
A,
A′,
B,
E,
E′,
F,
I,
I′,
J) or inferior (
C,
C′,
D,
G,
G′,
H,
K,
K′,
L) view, as per
Figures 2B through
2D schematics. (
A–
D) At E13.5,
Myf5WT/WT EOMs share a common origin at the annulus of Zinn (
A,
asterisk;
C). From the superior view (
A,
A′) the SO muscle begins to curve at its insertion onto the globe, and the rectus muscles have further differentiated from each other. CN3 superior division has multiple branches extending to the SR muscle (
A′,
triple-headed arrow). CN6 has crossed inferiorly to CN3 and extends terminal branches into the LR muscle. From the inferior view (
C,
C′), CN3 inferior division sends terminal branches to the IR and MR muscles (
C′,
long arrow), and a well-formed branch to the IO muscle (
C′,
caret). At E13.5, the
Myf5Cre orbit is devoid of EOMs (
B,
D). From the superior view (
B), CN4 has fasciculated but lacks an extensive distal decision region and terminal branches (
arrowhead), and CN3 superior division is not visible. From the inferior view (
D), CN3 distal decision region (
D,
long arrow) and the branch to the IO muscle (
D,
caret) are hypoplastic and misoriented, and CN6 is stunted and barely crosses over CN3 (
D,
double-headed arrow). (
E–
H) At E14.5, the
Myf5WT/WT EOMs continue to grow and change in orientation (
E,
G): the SR muscle is now a broad band inserting on the top of the globe, the LPS muscle has appeared between the SR and SO muscles, and the SO muscle has obtained its stereotypic curved shape. There is extensive terminal branching of CN3 superior division into the SR muscle (
E,
E′), and CN4 has developed a blunt-ended branch that is not in association with an EOM (
E,
E′). CN3 inferior division branch has continued extending terminal branches to the MR and IR muscles, and the branch to the IO muscle extends into the muscle (
G,
G′). In the absence of EOM, the
Myf5Cre CN4 appears thin and has developed multiple aberrant branches (
F), CN3 main trunk widens (
H) where the absent superior branch should have formed (
H,
triple arrowhead), the inferior division decision region is small, with no visible branches to the IR and MR muscles, and the branch to the IO muscle is now hypoplastic and aberrant (
H). CN6 trajectory is aberrantly parallel to CN3 and its growth cones pathologically wander (
H,
double arrowhead). (
I–
L) At E15.5,
Myf5WT/WT orbits reveal the near-final ocular motor innervation patterns, and each nerve sends terminal and intramuscular branches into the appropriate EOM. CN3 superior division branches have coalesced into a single trunk, and the blunted branch of CN4 is retracting (
I,
I′,
K,
K′). By contrast, in
Myf5Cre orbits the nerves have failed to develop further since E14.5 and appear to be regressing, while the abducens nerve wanders (
double arrowhead). Although more of the oculomotor nerve appears visible in
Figure 2J than in
Figure 2F, this is a staining and imaging artifact. In both images, the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery; the embryo in (
J) was imaged slightly deeper.
Arrows in the
lower left corner indicate direction relative to the eye.
Scale bars: 100 μm.