PCNA and Proliferation Patterns
(A) PCNA pattern as scored during the screen. In a dorsal view at 40 hpf, PCNA staining is observed in the medial and posterior part of the tectum, and in the cerebellum, the neural crest (arrowhead), and the pectoral fin (arrow).
(B) In a sideview (42 hpf), a ring of positive cells is visible around the lens.
(C) In a whole-mount BrdU labeling from day 3.5 to 4.5, similar regions are labeled indicating that PCNA RNA expression prefigures where BrdU will be incorporated.
(D and E) Sibling and hu418B mutants, respectively, showing increased PCNA labeling in the CMZ, but most prominently in the tectum.

Phenotypes of uki^hu418B Mutant Embryos
(A and B) Lateral view of a wild-type (wt) and uki^hu418B mutant.
(B) Showing an increased volume of the head. The size of the pupil is reduced in the uki^hu18B mutant without affecting the size of the eye.
(C–E) The size of the pupil is reduced in the uki^hu18B mutant compared to wild-type, without affecting the size of the eye. Measurements revealed that the length and width of the pupil is significantly reduced in the uki^418B mutant (n = 5, *p < 0,001).
(F–H) Pectoral fins showing the increased size of an uki^418B mutant in which the dorsal/ventral (D/V) size of the pectoral fin is increased by 50%, (n = 3, *0.01 < p < 0.02).
(I) The anterior/posterior size is not significantly affected, but the fin area has increased by 65%; (n = 3, p < 0.001).
(J and K) uki^hu418B mutants lack the dorsolateral septum in the ear (arrowhead). Scale bar is 100 µm.

Patterning of the Branchial Arches in a 5-Mo-Old dre Mutant
(A and B) The strict organization of the brachial arch into primary (p) and secondary (s) lamellae in a wild-type situation (100× magnification). Higher magnification shows stacks of single chondrocytes in the primary lamellae.
(C and D) Sectioning of a dre mutant shows disturbed patterning, resulting in the absence of secondary lamellae and the presence of foci of chondrocyte-like cells in the primary lamellae (arrowsHE, hematoxylin and eosin stain; wt, wild-type.
(E and F) Alcian Blue staining reveals the presence of differentiated chondrocytes in the wild-type (wt) primary lamellae, but not in the dre mutant, indicating that the differentiation of these chondrocytes is affected (200×).
(G and H) Branchial arches of uki and lep mutants appear to be wild-type (wt).

MO Injection Experiments against Su(fu), Hip, and Ptc2
(A) Dorsal view of the eye showing the lens in the eye chamber.
(B–D) Dorsal view of embryos injected with the indicated MOs, resulting in a phenocopy of dre, uki, and lep mutants.
(E) A wild-type ear showing the presence of the dorsolateral septum (arrow), which is not present after injections with the indicated MOs (F–H, arrow).
(I) Injections with control MOs against the initiation codon of Su(fu) results in chevron-shaped somites with an angle of 97°.
(J) Injection of MOs against Su(fu) results in a more obtuse angle of the somite (126°).

Premature Stop Codons Were Identified in the uki and lep Mutant in Hip and Ptc2, Respectively
(A) Schematic representation of the genomic organization of the Hip gene. All three alleles of the uki mutation contain premature stop codons positioned in exon 5 and exon 7.
(B) Representation of the protein structure of Ptc2 shows that the identified nonsense mutation is positioned after the sixth transmembrane domain, probably resulting in a malfunctioning protein.
(C) ISH experiments show that Ptc1 expression is increased in uki and lep (arrow), confirming the aberrant activation of the Hh signaling pathway.

Phenotypic Analysis of dre/uki/lep Triple Mutants
(A) Wild-type (wt) embryo at 96 hpf.
(B–E) The indicated double and triple mutants do not show severe enhancement of the phenotype.
(F–I) dre/lep double mutants have an ear phenotype comparable with a single mutant. In the uki/lep and dre/uki/lep triple mutants, the epithelial projections (arrows) fail to grow out to fuse in the middle of the ear to form the ear lumen.

Expression Pattern of Hip in Wild-Type Embryos
(A and B) At 24 hpf, Hip is expressed in the brain and in two lines of adaxial cells in the developing tail.
(C and D) At 42 hpf, Hip expression is reduced in the somites. The mid-hindbrain boundary, some branchial arches, and the pectoral fins are Hip positive. Hip expression is also detected in the tectum (arrowhead).

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