Fig 1.
Lineage contributions to the EOM functional unit.
(A-B”) Neural crest (Tg:Wnt1Cre;R26Tom) and (C-D”) mesodermal (Mesp1Cre;R26Tom) lineage contributions to the periocular region of E13.5 embryos, combined with immunostaining for tdTomato (Tom), GFP (Tg:Scx-GFP reporter), and muscle (PAX7/MYOD/MYOG, myogenic markers). Coronal sections at ventral (A-A”, C-C”) and dorsal (B-B”, D-D”) levels. Asterisks in B’,B” denote Tom negativity at the tendon origin. Asterisks in C’,C” denote Tom negativity at the tendon insertion site. (A1, D1) High-magnification views of muscle areas in panels A and D. (n = 3 per condition). a, anterior; c, choroid; cs, corneal stroma; d, dorsal; E, embryonic day; eom, extraocular muscle; l, lateral; m, medial; MCT, muscle connective tissue; p, posterior; r, retina; ti, tendon insertion; to, tendon origin; v, ventral.
Fig 2.
Developmental time course of EOM development.
(A-C) WMIF for MYOD/MYOG/Desmin (myogenic differentiation markers) (A, B) and MyHC (myofibers) (C) at the indicated embryonic stages. EOMs were segmented from adjacent head structures and 3D-reconstructed in Imaris (Bitplane). (A’-C’) EOMs are shown as isosurfaces for clarity of visualization. Medial views as schemes (left eye). (D) WMIF for MYOD/Desmin (labeling the EOM anlage, isosurface) and PITX2 (labeling the POM and EOM anlage) on E11.75 embryos (left eye). Lateral and medial views as schemes. (E) Whole-mount LysoTracker Red (LysoR) staining of the periocular region at the indicated stages (left eye). The POM is delimited with dashed lines. Asterisks indicate apoptotic foci with reduced intensity from E12.5 onwards. anl, anlage; a, anterior; d, dorsal; 3D-rec, 3D reconstruction; E, embryonic day; i, inferior EOM anlage projection; io, inferior oblique; ir, inferior rectus; l, lateral; lg, lacrimal gland sulcus; lr, lateral rectus; m, medial; m-pom, medial periocular mesenchyme; mr, medial rectus; p, posterior; pom, periocular mesenchyme; pom-r, periocular mesenchyme ring; rb, retractor bulbi; s, superior EOM anlage projection; so, superior oblique; sr, superior rectus; v, ventral; WMIF, whole-mount immunoflurorescence.
Fig 3.
Developmental time course of EOM insertions in the POM.
(A-D) Immunostaining of lateral POM in coronal sections of E11.75 Tg:Wnt1Cre;R26Tom;Scx-GFP embryos for the indicated markers. B, Immunostaining of section adjacent to the one shown in (A, C, D; channels split for clarity). (E-H) Immunostaining of lateral POM in coronal sections of E13.5 Tg:Wnt1Cre;R26Tom;Scx-GFP embryos for the indicated markers. G, Immunostaining of section adjacent to the one shown in (E, F, H; channels split for clarity). Bracket show overlap between Scx-GFP and SOX9 expression domains (high-magnification views in S3C” Fig). The asterisk in G points to Scx-GFP-negative SOX9+ lateral-most POM at the insertion site. (I,J) Immunostaining of lateral POM in coronal sections of E12.5 Tg:Scx-GFP embryos pretreated with LysoR. Arrowheads in J’ point to LysoR+ SOX9+ cells. (J”) TUNEL and LysoR staining of a section adjacent to the one shown in (I,J). (K) Quantification of the total number of SOX9+ cells per square-micrometer in LysoR+ regions (red square, J) and more medial LysoR-negative regions (yellow square, J). Mann–Whitney test. Cell density was 33% higher in the LysoR+ area compared with the more medial POM. See S1 Data for individual values. (L-N’) Immunostaining on coronal sections of E14.5 Tg:Scx-GFP embryos. (L,L’) Tnc and Scx-GFP co-localize in tendons at level of insertion (arrowhead). (M,M’) SOX9 expression in the POM is greatly reduced at the insertion site and no longer overlaps with Scx-GFP (asterisk, cyan). Low levels of SOX9 expression in the RPE and sclera. (N,N’) PITX2 remains expressed in the POM at the insertion site overlaping with Scx-GFP (asterisk) and in the sclera. MyHC (L’) and SMA (M-N’) were used to label EOM muscle. Dashes in L-N were drawn according to GFP labeling in L’-N’. Images in A-N’ correspond to insertion site of superior rectus muscle in the POM as shown in the scheme. (n = 3 per condition). a, anterior; apop, apoptosis spots; d, dorsal; E, embryonic day; eom, extraocular muscle; l, lateral; LysoR, LysoTracker Red; m, medial; p, posterior; POM, periocular mesenchyme; rpe, retinal pigmented epithelium; s, sclera; t, tendon; ti, tendon insertion, v, ventral.
Fig 4.
Extraocular muscle morphogenesis is dependent on ATRA.
(A) Immunostaining for ALDH1A3 on coronal sections of E10.5, E11.5, and E12.5 control embryos (n = 3). (B) Scheme of retinoic acid signaling pathway with key enzymes for oxidation of retinol (Vitamin A) and retinaldehyde (pink) and mutants/inhibitors used in this study (blue). (C-D”’) Micro-CT-based 3D-reconstruction of chondrogenic mesenchymal condensations of nasal capsule, trabecular cartilage, EOM, eyeball, optic nerve, and lens in E13.5 control, Aldh1a3KO and BMS493-treated embryos. EOM visualization in context of whole head (D), nasal capsule (D’-D”), trabecular cartilage (D”’). (E) Raw micro-CT data (n = 2). (F-H) 3D-reconstructions of WMIF for MyHC of E13.5 control (F), Aldh1a3KO (G) and BMS493-treated embryos (H) (n > 9). EOMs were segmented from adjacent head structures and 3D-reconstructed in Imaris (Bitplane). EOMs are shown as isosurfaces for clarity of visualization. (1–7) denote non-segregated muscle masses with differential fiber orientation (see also S5D Fig). Raw immunostaining data for MyHC (myofibers) and Tnc (tendon) are shown in panels F’-H’. Double asterisk in G,H indicate fused muscle mases. Asterisk in G indicate misoriented medial rectus. (I) Relative EOM volume (compared with control) of WMIF in F-H. Each dot represents an individual embryo (n > 9). Mann–Whitney test. See S2 Data for individual values. a, anterior; ATRA, all-trans retinoic acid; ce, presumptive corneal epithelium; ctrl, control; d, dorsal; dr, dorsal retina; 3D-rec, 3D-reconstruction; e, eyelid groove; E, embryonic day; EOM, extraocular muscle; hc, hypochiasmatic cartilage; io, inferior oblique; ir, inferior rectus; l, lateral; le, lens; lr, lateral rectus; m, medial; mr, medial rectus; micro-CT, micro computed tomography; os, optic stalk; p, posterior; rb, retractor bulbi; rpe, retinal pigmented epithelium; RAR, retinoic acid receptor; RXR, retinoid X receptor; se, surface ectoderm; so, superior oblique; sr, superior rectus; v, ventral; vr, ventral retina.
Fig 5.
Periocular connective tissues are responsive to retinoic acid signaling.
(A) Scheme of mouse alleles used. (B) Strategy used to determine cell types reponsive to retinoic acid signaling in Tg:RARE-CreERT2;R26mTmG embryos. Tamoxifen was injected to pregnant females at E9.75 or E10.5 and analysis performed (bulk, sections or WMIF) at E11.75 or E12.5. (C) The percentage of recombined (GFP+) cells within the non-myogenic or myogenic populations (PAX7+, MYOD+, MYOG+) was assessed by immunostaining on bulk cell preparations of the periocular region of Tg:RARE-CreERT2;R26mTmG embryos following different tamoxifen treatments. Each dot represents an individual embryo (n > 4 embryos/condition). See S3 Data for individual values. (D-F’) WMIF of Tg:RARE-CreERT2;R26mTmG embryos for SMA (differentiated muscle) and GFP (ATRA-responsive cells) at the indicated embryonic stages. The number of reporter positive cells at the place where the developing medial rectus muscle will project increases from E11.75 (blue arrowhead) to E12 (white arrowheads). Asterisks mark the optic nerve (n = 3). (G-I) Coronal sections of E12.5 Tg:RARE-CreERT2;R26Tom;Scx-GFP embryos immunostained for GFP, Tom (ATRA-responsive cells), MYOD/MYOG (muscle), and CD31 (endothelial cells). Higher-magnification views as insets. Asterisks in G1 indicate sporadic labeling in myogenic cells (n = 3). (J-K’) WMIF for MyHC (myofibers) of E12.5 control (Tg:Wnt1Cre;Rarbfl/+;Rargfl/+) (J,J’) and mutant (Tg:Wnt1Cre;Rarbfl/fl;Rargfl/fl) (K,K’) EOM. Medial and lateral views are shown. Note absence of splitting in mutant EOM. Arrowheads indicate split EOM in controls (n = 2 per genotype). In D-F’ and J-K’ EOMs were segmented from adjacent head structures and 3D-reconstructed in Imaris (Bitplane). EOMs are shown as isosurfaces for clarity of visualization. a, anterior; ATRA or RA, all trans retinoic acid; ctrl, control; d, dorsal; 3D-rec, 3D-reconstruction; E, embryonic day; EOM, extraocular muscle; l, lateral; m, medial; mGFP, membrane-tagged GFP; p, posterior; RAR, retinoic acid receptor; RXR, retinoid X receptor; SMA, alpha smooth muscle actin; Tam, Tamoxifen; v, ventral; WMIF, whole-mount immunofluorescence.
Fig 6.
Altered EOM insertions in the POM upon ATRA deficiency.
(A-C’) Immunostaining for ColXII on E12 coronal sections. ColXII expression is lost in the medial POM (future sclera) but not in the corneal stroma of Aldh1a3KO (B,B’) and BMS493-treated embryos (C,C’). Higher magnification of ColXII staining as insets in (A’-C’). (D-G) Immunostaining for PITX2 (muscle progenitors, POM) and MYOD/MYOG (myogenic cells) on E12 coronal sections. Asterisks indicate superior oblique muscle. Red dashed lines delimitate the POM in controls (D) and Myf5nlacZ (G) embryos. Higher magnification of PITX2 staining as insets in (D’-G’). Note absence of PITX2 expression in medial-POM of Aldh1a3KO (E,E’) and BMS493-treated embryos (F,F’). (H-P) WMIF for SOX9 and PITX2 of E12 control (H-J), Aldh1a3KO (K-M) and BMS493-treated (N-P) embryos preincubated with LysoR (left eyes). Aldh1a3KO and BMS493 embryos do not show a complete POM-ring as controls (dashed lines in H,I). Arrowheads mark remaining expression of SOX9/PITX2 in the POM of Aldh1a3KO (K,L) or inhibitor-treated (O) embryos. Asterisks in (J, M, P) mark apoptosis spots in POM. (Q-R) WMIF for MyHC (myofibers) and GFP on E12 control (Q) and BMS493-treated Tg:Scx-GFP embryos (R). WMIF for β-gal (myogenic progenitors) and GFP on E12 Tg:Scx-GFP;Myf5nlacZ/nlacZ E12 embryos (S) (left eyes). The periocular area was segmented from adjacent structures for ease on visualization (dashed lines). White arrowheads in Q and S highlight correct position of tendon condensations for medial rectus muscle. Blue arrowhead in R marks a diffuse Scx-GFP+ pattern at the position of the mispatterned medial rectus muscle. (Q’-S’) Single Z-section of the segmented volume. White arrowheads in Q’,S’ show tendon tips and white arrows correct Scx-GFP+ connective tissue pattern along with muscle (Q’) or prospective muscle masses (S’). Blue arrowhead in R’ marks a Scx-GFP+ condensation at the muscle tip. Asterisks mark position of the optic nerve. (n = 3 per condition). a, anterior; ATRA, all-trans retinoic acid; cs, corneal stroma; ctrl, control; d, dorsal; eom, extraocular muscles; l, lateral; le, lens; LysoR, LysoTracker red; m, medial; nlg, nasolacrimal gland; p, posterior; pom, periocular mesenchyme; pom-r, periocular mesenchyme ring; v, ventral; WMIF, whole-mount immunofluorescence.