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FIG. 1. Trigeminal neurites’ outgrowth in Xenopus embryos. The mandibular trigeminal nerve innervates a subset of cells located in
the posterior end of the cement gland. (A) Lateral view of a stage 28 tailbudembryo stained with the 3A10 monoclonal antibody. (B)
Lateral view of the head showing neurons stained by the 3A10 monoclonal antibody in tailbudembryo. The mandibular trigeminal
ganglion is indicated by an arrow. (C) Whole-mount in situ hybridization of tailbudembryo using an antisense XA-1 probe to highlight
the target of the mandibular branch of the trigeminal neurites, the cement gland (arrow). The anterior (a) and posterior (p) of the cement
gland are indicated. (D) Double whole-mount immunochemistry with the 3A10 antibody (brown staining) and whole-mount in situ
hybridization with antisense XA-1 probe (purple blue staining). The growth cones contact the posterior end of the cement gland. (E–H)
Developmental profile of cement gland innervation by trigeminal neurites. (E) Stage 24 embryo. (F) Stage 25 embryo. (G) Stage 26 early
tailbudembryo. (H) Stage 28 embryo. In all panels, anterior is to right and dorsal is at top. In D, the scale bar represents 100 mm and in
E–H, 50 um.
FIG. 2. Deletion of the cement gland impairs the formation of the trigeminal nerve arborization formation. Embryos were double stained
as whole-mounts with the 3A10 antibody (brown staining) and with the antisense XA-1 probe (purple blue staining). (A) Lateral view of
the cement gland of an unperturbed embryo control at tailbud stage. (B) Trigeminal neurites grow toward the eye of the tadpole. (C)
Trigeminal neurites grow toward the heart. (D) Trigeminal neurites grow toward the initial location of the target and stop without forming
arborization. All embryos are albinos except for (C). In this embryo the eye is visible. In all panels, dorsal is at top and anterior is to right.
FIG. 3. Rotation of the cement gland alters trigeminal nerve behavior. Rotation experiments demonstrate that the posterior cells of the
cement gland control the behavior of the mandibular trigeminal neurites. (A) Lateral view of a stage 28 tailbud control embryo stained
with both the 3A10 antibody (brown staining) and the antisense XA-1 probe (purple blue staining). (B) Stage 28 embryo in which the
cement gland was rotated by 1807 and reimplanted at stage 19. The neurites innervate the anterior end of the target. (C) Control embryo
in which the cement gland was removed and reimplanted without rotation at stage 19. Innervation occurs at the original posterior end.
In all panels anterior is to right and dorsal is at top.
FIG. 4. Ectopically implanted cement gland competes with the endogenous target. (A–C) and (E, F) are two embryos in which an ectopic
lineage-traced cement gland was implanted on one side of the embryos in the path of the mandibular branch of the trigeminal nerve and
stained with 3A10. (A–C) Lateral view of the head. (E, F) Ventral view of the head. (A) Control side without implant showing the normal
connection of the nerve to its target. (B) Experimental side where the implantation of an ectopic cement gland was performed. (C) The
same view under UV illumination showing the implant revealed by the lineage tracer. Trigeminal neurites innervate a subset of cells in
the ectopic cement gland and do not contact the endogenous cement gland of the host embryo. (D) Ventral view of the head of an
unmanipulated control embryo. (E) Ventral view of the head of an embryo in which an ectopic cement gland is implanted on the path of
the trigeminal nerve (right side). (F) The same view under UV illumination showing the implant revealed by the lineage tracer. The ectopic
connection does not influence the behavior of the nerve on the unmanipulated side. The arrow points to the natural target of the host
embryo.
FIG. 5. Schematic of the animal cap substitution assay. This depicts the strategy used for the assay. In vitro transcribed RNAs are
injected in the animal pole of both blastomeres of 2-cell stage embryos. Animal caps of blastula stage embryos (stage 8) are cut and
cultured in 0.51 MMR until sibling embryos reached neurula stage 18. Animal caps are then bisected and substituted for the cement
gland in stage 18 embryos. These experimental embryos are fixed at tailbud stage 28; 3A10 staining and XA-1 in situ hybridization are
then performed.
FIG. 6. Follistatin-expressing caps mimic the endogenous cement gland in controlling the mandibular trigeminal nerve behavior. Expression
of the activin antagonist follistatin in animal cap induces cues modulating mandibular trigeminal nerve behavior. (A) Lateral view
of tailbud stage 28 embryo stained with the 3A10 antibody, in which the cement gland was substituted by a control animal cap (arrow).
The trigeminal neurites do not recognize this tissue as a target. (B) Embryo in which the cement gland is substituted with an animal cap
expressing 2 ng total b-galactosidase RNA. Staining for b-galactosidase activity is shown in blue. Injection of this control RNA does not
change the behavior of the neurites in the substitution assay. (C, D) The same as (B) with an animal cap expressing 2 ng total follistatin
RNA. In these embryos, trigeminal neurites innervate with 50% frequency either the posterior (C) or the anterior (D) part of the implanted
animal cap. (E) Double staining for 3A10 antigen and the cement gland marker XA-1. The animal explants expressing follistatin induce
the cement gland marker XA-1 (blue staining). In all panels, dorsal is at top and anterior to right.
FIG. 7. Quantification of neurite behavior. (A) Trigeminal neurite
behavior in embryos lacking their target. Percentage of neurites
(whole nerve) with no arborization and extending toward the heart
or the eye or stopping at the original site of the cement gland are
shown after a complete deletion (deletion, n  39) or after partial
posterior deletion (deletion P, n  18) of the cement gland. (B)
Consequence of rotation performed at different embryonic stages
on the behavior of the mandibular trigeminal neurites. Arborization of trigeminal neurites in the posterior (p) and the anterior (a)
sides of the cement gland are indicated. (n) Embryos with no terminal arborization. (control) Embryos from which the cement gland is
removed at stage 19 and reimplanted in its initial A–P orientation.
Numbers of embryos are indicated at the top of the histogram. (C)
Follistatin induces ectodermal explants to express cues controlling
trigeminal neurite behavior. b-Galactosidase RNA is used as con-
trol for RNA injection. Percentage of neurites without arborization
extending toward the heart (heart), the eye (eye), or the animal
cap (cap no arborization) are shown. The percentage of neurites
forming a terminal arborization in the animal cap is shown (cap arborization). Numbers of embryos are indicated to the left of the
legend.
