Jacox L , Sindelka R , Chen J , Rothman A , Dickinson A , Sive H .

1R01 DE021109-01 NIDCR NIH HHS, F30 DE022989 NIDCR NIH HHS, F30DE022989 NIDCR NIH HHS, R01 DE021109 NIDCR NIH HHS, T32 GM007287 NIGMS NIH HHS

	Graphical Abstract
	Figure 1. Mammalian Kinin-Kallikrein Pathway and Putative Pathway Genes Are Expressed in the Developing Face (A) Adult mammalian Kinin-Kallikrein pathway (Kakoki and Smithies 2009). (B–G′) In situ hybridization for kng (B, B′, E, and E′), cpn (C, C′, F, and F′), and nNOS RNA (D, D′, G, and G′) (RNA is purple). Cement gland marker (xcg) is red. Arrow: presumptive mouth. cg, cement gland. (B–G) frontal views; (B′–G′) sagittal sections. Scale bars, 200 μm.
	Figure 2. kng, cpn, and nNOS Are Required for Mouth Opening and Face Formation (A–D′) kng, cpn, and nNOS loss of function (LOF) using antisense morpholinos. Embryos assayed at stage 40, in four independent experiments. Arrow: mouth region. Bracket: unopened mouth. cg, cement gland. Scale bar in (A–D), 2,000 μm. Scale bar in (A′–D′), 200 μm. (A and A′) Control morphants (100% normal, n = 97). (B–D′) kng, cpn, and nNOS morphants (kng [B′] 0% normal, n = 102; cpn [C′] 2% normal, n = 105; nNOS [D′] 0% normal, n = 129). (E–H′′′) Kinin-Kallikrein pathway morphants at stage 22 express presumptive mouth markers, frzb1 and xanf1. Scale bars, 200 μm. (I–P and I′–L′) Histology of kng, cpn, and nNOS LOF. Coronal sections (I–L, control morphant 100% normal, n = 5; each Kinin-Kallikrein morphant, 0% normal, n = 9) assayed at stage 26 in two independent experiments with β-catenin immunolabeling. Parasagittal sections with anterior to the left (I′–L′, control morpholino 100% normal, n = 5; each Kinin-Kallikrein pathway morpholino, 0% normal, n = 12). (M–P) Parasagittal sections with laminin immunolabeling (M–P) assayed at stage 26 in two independent experiments (control morphant 100% normal, n = 10; each Kinin-Kallikrein morphant 8% normal, n = 12). β-catenin: green; laminin: green; nuclear propidium iodide: red. Bracket: presumptive mouth region. cg, cement gland. Scale bar, 170 μm. (Q–X′) kng, cpn, and nNOS morphants showed reduced expression of neural crest markers sox10 and sox9. (Q–T′) sox10 in situ hybridization at stage 22. (U–X′) sox9 in situ hybridization at stage 26. Bracket: cranial NC-free midline region. Arrow: normal extent of first arch cranial NC. Scale bars in (Q)–(X), 200 μm; scale bars in (Q′)–(T′), 800 μm; scale bars in (U′)–(X′), 400 μm. (Y–d) kng morphants showed reduced expression of slug at stage 22, whereas cpn and nNOS morphants and kng morphants coinjected with kng mRNA showed control slug levels. Arrow: specified neural crest. Dorsal view. Scale bars, 800 μm. (e–m) Cell proliferation and death in cranial NC cells. (e–g) sox10 in situ hybridization at stage 22 in axial section. Control and cpn morpholino plus cpn mRNA embryos showed normal expression, whereas cpn morphants had reduced expression. Scale bar, 200 μm. (h) Schematic demonstrating axial section. (i–k) Ph3 staining of axial sections show increased positive cells in control (i) relative to cpn morphants (j). Embryos injected with cpn morpholino plus cpn mRNA (k) had more Ph3-positive cells than cpn morphants. Scale bar, 170 μm. (l and m) Quantification of Ph3 and TUNEL staining, with SD included. p value: one-way ANOVA with multiple comparisons.
	Figure 3. Bradykinin-like Peptides Prevent cpn and kng Loss-of-Function Phenotypes (A) Amino acid sequence alignment of region around Bdk-l peptide. Gray highlights: Bdk-l peptide sequence; red: conserved amino acids; black arrows: Kallikrein and Cpn cleavage sites. Bdk-l (9AA) and Des-Arg Bdk-l peptides (8AA) used. (B) Experimental design. (C–H) Abnormal mouth phenotype after kng LOF prevented by 9AA and 8AA peptides, whereas in cpn morphants was prevented only by the 8AA peptide. (C) kng morphants implanted with 9AA scrambled (9AAscr) peptide bead (28% normal, n = 60). Embryos scored as abnormal if mouth failed to open, was tiny or asymmetric, nostrils failed to form, pigment was absent, or face was abnormally narrow. (D and E) kng morphant implanted with 9AA (D, 60% normal, n = 105) or 8AA bead (E, 57% normal, n = 75). (F) cpn morphants implanted with 9AAscr bead (mouth: 43% normal, n = 67; face: 27% normal, n = 67). (G and H) cpn morphants implanted with 9AA bead (mouth: 41% normal, n = 54; face: 44% normal, n = 54) or 8AA bead (mouth: 65% normal, n = 79; face: 51% normal, n = 79). Scale bar, 200 μm. (I) Graph depicting percent of morphants implanted with beads, displaying normal mouth and face formation. p values: one-tailed Fisher’s exact test. (J–L) Expression of neural crest marker sox9 in kng morphants implanted with 9AA bead. Arrow: normal extent of first arch cranial NC. (J) Wild-type expression of sox9 (100% normal, n = 13). kng morphant with a 9AAscr bead (K, 8% normal, n = 12) and 9AA bead (L, 39% normal, n = 13). Scale bar, 200 μm.
	Figure 4. kng, cpn, and nNOS Loss-of-Function Phenotypes Are Prevented by the NO Donor, SNAP, and Kinin-Kallikrein Morphants Show Reduced Nitric Oxide Production that Is Increased by xBdk (A–D′) Facial morphology of kng, cpn, and nNOS loss of function (A–D) and with SNAP (A′–D′). Embryos assayed at stage 40 in three independent experiments and scored as abnormal if mouth failed to open, was tiny or asymmetric, nostrils failed to form, pigment was absent, or face was abnormally narrow. Arrow: mouth region. Bracket: unopened mouth. cg, cement gland. (A) Control MO injected (98% normal, n = 427) (B–D) kng, cpn, or nNOS MO injected. (A′) SNAP plus control MO. (B′–D′) kng, cpn, or nNOS MO plus SNAP coinjection (kng [B′] 85% normal, n = 105; cpn [C′] 86% normal, n = 98; nNOS [D′] 90% normal, n = 87). Scale bars, 100 μm. (E–L′) Histology of kng, cpn, and nNOS LOF embryos after SNAP treatment. Parasagittal sections with anterior to left assayed at stage 26 with β-catenin (E–H′) and laminin immunolabeling (I–L′). β-catenin: green. Laminin: green, with nuclear propidium iodide: red. cg, cement gland. (E–H′) β-catenin in control embryos (E and E′), LOF embryos (F–H), and LOF embryos coinjected with SNAP (F′–H′) (kng [F′] 100% normal, n = 5; cpn [G′] 100% normal, n = 5; nNOS [H′] 100% normal, n = 5). (I–L′) Laminin staining in control embryos (I and I′), LOF embryos (J–L), and LOF embryos coinjected with SNAP (J′–L′) (kng [J′] 75% normal, n = 4; cpn [K′] 80% normal, n = 5; nNOS [L′] 100% normal, n = 4). Scale bars, 170 μm. (M–P′) Expression of sox9 RNA (in situ hybridization) after SNAP injection into kng (N, N′), cpn (O, O′), and nNOS (P, P′) LOF embryos. Lateral view. Scale bar, 100 μm. (Q–R′) NOS inhibitor TRIM prevents mouth formation and reduces sox9 expression. (Q and Q′) Wild-type embryos (100% normal, n = 6). (R and R′) TRIM-treated embryos (17% normal, n = 6). (Q and R) Frontal view at stage 40. (Q′ and R′) Lateral view of sox9 in situ hybridization at stage 26. Scale bars in (Q) and (R), 100 μm; scale bars in (Q′) and (R′), 400 μm. (S–T′) Extirpated heads show open mouth and normal pigmentation at swimming tadpole (stage 41). (S and S′) Control heads (96% normal, n = 27). (T and T′) Isolated heads (92% normal, n = 26). (S and T) frontal view. (S′ and T′) side view. Scale bar, 100 μm. (U–X) Fluorescence after incubation with NO sensor DAF-FM in control embryos (U), kng (V), cpn (W), and nNOS (X) LOF embryos. cg, cement gland. Sagittal view. Scale bar, 170 μm. (Y–c) Control morphant with no bead (Y). kng morphant with no bead (Z), with 9AA xBdk scrambled bead (a) or 9AA xBdk bead (b). Images collected with same exposure, gain, and fluorescent illumination. kng morphants implanted with 9AA xBdk bead displayed 50% of control florescence compared with 23% of control fluorescence in morphants treated with 9AAscr xBdk peptide. Frontal view. Scale bar, 100 μm. (c) Graph showing morphant fluorescence as percentage of control fluorescence; cpn morphants: 49%, kng morphants: 24%, and nNOS morphants: 64%. Each dot represents average of three biological replicates from independent experiments. p values: one-tailed t test.
	Figure 5. Local cpn Expression Is Required for Mouth Opening Local requirement of kng, cpn, and nNOS expression tested with EAD transplants. (A) Experimental design: donor morphant tissue was transplanted to uninjected sibling recipients. (B–E′) EAD transplant outcome from control, cpn, kng, or nNOS morphant donor tissue (control [B] 100% normal, n = 11; cpn [C] 28% normal, n = 14; kng [D] 83% normal, n = 24; nNOS [E] 61% normal mouth phenotype, 72% normal facial phenotype, n = 18). (B′–E′) Overlay of (B)–(E) with GFP fluorescence indicating donor tissue. Dots surround open mouths. Bracket: unopened mouth. Frontal view. Scale bar, 100 μm. (F) Quantification of structure depending on morphant background of facial tissue. (G–H′) sox9 expression in cpn morphant donor tissue transplants, compared with control morphant transplants. sox9 in situ hybridization in control morphant transplants (G, G′, 70% with normal expression, n = 10) and cpn morphant transplants (H and H′, 36% with normal expression, n = 11). Two representative embryos shown. Scale bar, 100 μm. (I and J) (I) Summary of urea assay for analysis of Cpn activity. (J) Chart summarizing level of urea derived from free Arg in cpn morphants or morphants coinjected with cpn RNA, as percent of urea derived from free Arg in control morphants. Urea levels in control morphants and wild-type embryos were equivalent. Each dot represents an independent experiment. p value: one-tailed t test.
	Figure 6. Global and local cpn Expression Is Required for Cranial Neural Crest Migration Global requirement for cpn expression tested with cranial NC transplants. Embryos scored as normal if three or four distinct branchial arches formed and migrated normally. (A) Experimental design: donor wild-type cranial NC transplanted into cpn morphant sibling recipients. (B–C′) (B and C) Cranial NC transplant outcomes in control and cpn morphant recipients with GFP fluorescence overlay, indicating location of donor transplant at stage 28 (control [B] 69% normal, n = 36; cpn [C] 27% normal, n = 29). (B′ and C′) GFP fluorescence of cranial NC in control and cpn morphant recipient. Numbers indicate branchial arches. Side view. cg, cement gland. Scale bar, 200 μm. (D) Experimental design: donor cpn morphant EAD transplanted into control morphant sibling recipients with fluorescent cranial NC. (E–H′′) (E, F, G, and H) Bright-field view of control and cpn morphant transplants at stages 28 and 40. (E′, F′, G′, and H′) Cranial NC in control and cpn morphant EAD recipients at stages 28 and 40 with GFP fluorescence overlay, indicating location of cranial NC and mCherry fluorescence overlay, indicating location of EAD transplant. (control stage 28 [E′] 85% normal, n = 41; cpn stage 28 [F′] 57% normal, n = 42; control stage 40 [G′] 63% normal, n = 38; cpn stage 40 [H′] 17% normal, n = 35). (E′′, F′′, G′′, and H′′) GFP fluorescence of cranial NC in control and cpn morphant EAD recipients at stage 28 and 40. Arrow: Open mouth. Bracket: unopened mouth. Frontal view. cg, cement gland. Scale bar, 100 μm. (I–J′) (I and J) Cranial NC outcome in control and cpn morphant EAD recipients with GFP fluorescence overlay, indicating location of cranial NC at stage 28. (I′ and J′) GFP fluorescence of cranial NC in control and cpn morphant EAD recipients. Numbers indicate branchial arches. Side view. cg, cement gland. Scale bar, 200 μm. (K–L′) Cartilage in control morphant EAD recipients (K, K′ 78% normal, n = 14) and cpn morphant EAD recipients (L, L′ 6% normal, n = 16). (K and L) Ventral view. (K′ and L′) Dorsal view. M, Meckel’s cartilage. C, ceratohyal cartilage. Scale bar, 100 μm.
	Figure 7. Function of kng in Craniofacial Development Is Conserved in Zebrafish (A and A′) Camera lucida of facial cartilages. E, ethmoid plate; C, ceratohyal cartilage; M, Meckel’s cartilage. (B–E′) kng loss of function using splice morpholinos and rescue with zebrafish (zf) kng mRNA. Embryonic cartilage scored at 5 dpf after Alcian blue staining in three independent experiments. Scale bar, 250 μm. (B and B′) Control morphants coinjected with mRNA were normal (88% normal, n = 50). (C, C′, E, and E′) kng morphants and kng morphants coinjected with 200 ng Xenopus kng mRNA showed abnormal facial cartilage. Meckel’s cartilage was truncated, boxy, and pointed at an abnormal angle. The ceratohyal cartilage was positioned at an abnormal angle, perpendicular to the midline. (kng [C and C′] 3% normal, n = 61; kng mo plus frog mRNA [E and E′] 0% normal, n = 65). (D and D′) kng morphants coinjected with 200 ng zebrafish (zf) mRNA showed partial rescue. Embryos scored as partially rescued if Meckel’s cartilage was longer, more rounded, and pointed dorsally and if ceratohyal cartilage pointed more anteriorly, compared to kng morphants (54% partial rescue, n = 89). (F) Quantification of phenotypes. p values: one-tailed Fisher’s exact test. N, normal or partially rescued phenotype. A, abnormal phenotype. (G–I) Ventral views of mApple-injected embryos at 48 hpf. White arrow: open mouth. White bracket: closed mouth. Scale bar, 100 μm. (G) Control morphants (100% normal, n = 5). (H) kng splice morphants failed to form open mouths (0% normal, n = 6). (I) kng splice morphants coinjected with 200 ng zf mRNA had open mouths (67% normal, n = 6). (J–Q′) Confocal images of Sox10::GFP zebrafish coninjected with 75 pg mApple and 4 ng control morpholino (100% normal, n = 5) or 4 ng kng splice morpholino (0% normal, n = 5). Paired images of the same embryo show GFP signal alone and GFP with mApple. Numbers indicate pharyngeal arches (PA). Bracket: uncondensed/disorganized cartilage. Lateral view. M, Meckel’s cartilage. Scale bar, 100 μm. (J–K′) At 36 hpf, NC has migrated into the face of both morphant and control embryos to form first and second PA. (L–M′) At 48 hpf, the first PA has begun to extend under eye to form the lower jaw in both morphant and control embryos. (N and N′) At 60 hpf, first PA has condensed into Meckel’s cartilage in control embryos. (O and O′) At 60 hpf, first PA remains disorganized in morphants and does not condense. (P and P′) At 72 hpf, Meckel’s cartilage is prominent in control embryos. (Q and Q′) At 72 hpf, cartilage of the lower jaw remains disorganized and uncondensed in morphants.
	Figure S1. Temporal expression profiles, expression in the presumptive mouth and homology of protein sequences, related to Figure 1. (A) Three tissues were dissected from stage 26 heads: cg - cement gland, mouth, and brain - neural and expression of kng, cpn and nNOS were determined by microarrays - m (Dickinson and Sive, 2009) and qPCR - q. kng, cpn, and nNOS displayed 4-fold enrichment, 5-fold enrichment, and 4-fold enrichment, respectively, in the presumptive mouth region relative to surrounding tissue. (B-D) Percentage values correspond to sequence identity of protein sequences among human, mouse, zebrafish, Xenopus laevis and Xenopus tropicalis. Protein sequences were obtained from NCBI Gene. Homologs were aligned using several different algorithms (t-coffee, mafft, probcons, and muscle), with the final alignment determined by consensus as implemented by t-coffee. Phylogeny was estimated with a maximum likelihood method (proml), using default parameters, in the phylip package. (E-G) 5 embryos per biological replicate (error bars: 3 biological replicates) were collected for developmental stages from oocyte (stage 1) to swimming tadpole (stage 42). Total RNA was extracted and expression profiles of kng, cpn and nNOS were measured by qPCR. All genes showed low maternal level of expression and rapid increase of expression after mid-blastula and between stages 20 and 26.
	Figure S2. Morpholino specificity and efficiency, related to Figure 2. (A-D, B’-D’, B’’) The specificity of the morphant phenotypes was confirmed by rescue with 800 pg of kng mRNA (B’), 800 pg of nNOS mRNA (D’), and 1ng of cpn mRNA (C’), which does not hybridize to the start site cpn morpholino. (B’’) kng LOF phenotype was also overcome with human KNG mRNA. Embryos were co-injected using a mixture of morpholino and mRNA at the one cell stage. The craniofacial phenotype of kng, cpn and nNOS LOF was completely rescued by coinjection with mRNAs, i.e. kng (B’) 70% normal, n= 162; cpn (C’) 74% normal, n=112; nNOS (D’) 44% normal, n=94 and human kng (B’’) 32% normal, n=40. Scale bar: 200 m. Phenotypes of kng (B), cpn (C), and nNOS (D) loss of function using antisense morpholinos are described in detail in Fig. 2. When injected into control embryos alone, the kng, cpn, and nNOS RNA did not alter mouth morphology, despite the Kinin-Kallikrein GOF (data not shown). (E-F) Using qPCR, levels of kng and nNOS mRNA in LOF embryos were determined to be 6-10% and 25-34% of control levels, respectively. Two independent experiments are shown. Three independent groups of embryos (n=5 for each group) were analyzed by qPCR (biological replicates).
	Figure S3. Phenotypic comparison at early stages and TUNEL, PH3 staining, related to Figure 2. (A-L) Frontal views of control and LOF embryos at earlier stages 22, 26 and 30, complement to Fig.2. Scale bar: 200 m. (A’ - L’) Lateral views of control and LOF embryos at earlier stages 22, 26 and 30. Scale bar: 500 m. Whole body phenotypes vary from morphant to morphant. kng morphants displayed a normal head size (B, F, J). Pockets of edema frequently formed in the presumptive heart region starting at stage 26. cpn morphants displayed a larger head relative to the posterior, most obvious at stage 22 (C, G, K). These morphants showed a tendency to disintegrate at high MO levels after stage 30 (hatching), suggesting abnormal epithelial integrity. nNOS morphants showed a diminished overall head size and expanded posterior (D, H, L). All morphants had reduced pigment and diminished movement at low MO levels, and no ability to move at high levels. Cell death was increased in LOF embryos. (M-P) Histological coronal sections showing TUNEL labeling in the presumptive mouth (brackets) at stage 26. Scale bar: 100m. (Q-T) Histological coronal sections with PH3 labeling in the presumptive mouth (brackets) at stage 26. Scale bar: 100m. (U) Graph showing TUNEL data. kng (average n=13.3 positive cells), cpn (average n=23.0 positive cells), and nNOS (average n=17.0 positive cells) morphants have an increase in cell death, respectively, compared with control morphants (average n = 1 positive cell), n = 8 embryos counted per condition, p values < 0.05. (V) Graph showing PH3 positive cells (green) compared to negative cells (red). kng (5% of total cells), cpn (16% of total cells), and nNOS (8% of total cells) morphants have a similar level of cell proliferation, respectively, compared with control morphants (10% of total cells), n = 8 embryos per condition, p values > 0.2.
	Figure S4. Laterally located Bradykinin-like peptides promote cranial NC migration but do not restore mouth opening in kininogen (kng) morphants, related to Figure 3. (A) Schematic. (B,B’) kng morphant with no beads (35% with right side expression, n=17). (C,C’) kng morphant plus implanted beads (74% with right side expression, n=19). kng morphants had 9AA peptide implanted on the right side of the head, and 9AAscr peptide implanted on the left side of the head. Bracket: normal location of branchial arch 2. Scale bar: 120 m. (D) kng morphant with 9AAscr peptide (28% with a normal mouth, n=14). (E) kng morphant with 9AA peptide (21% with a normal mouth, n=14). (F) kng morphant with 8AA peptide (28% with a normal mouth, n=14). Bracket: unopened mouth. Scale bar: 100 m.
	Figure S5. NO donor (SNAP) prevented cpn, kng and nNOS LOF phenotypes when injected late, and SNAP rescue is a specific effect, related to Figure 4. (A-D) Embryos were injected with morpholino at the one cell stage. NO donor (SNAP) was injected into the presumptive mouth region at stage 20. Phenotypes of kng (B), cpn (C), and nNOS (D) loss of function using antisense morpholinos are described in detail in Fig. 2. (B’-D’) Embryos injected with NO donor late, at stage 20. The craniofacial phenotype of kng, cpn and nNOS LOF was prevented by injection of NO donor into the EAD. (kng (B’) 54% normal, n= 48; cpn (C’) 79% normal, n=34; nNOS (D’) 53% normal, n=15) Scale bar: 100 m. (E-J) NO specificity for the Kinin-Kallikrein pathway was tested by co-injecting SNAP and morpholino at the one cell stage. Embryos were scored at stage 26. (E) Control morphants. (F-G) The phenotype of cpn LOF was prevented by injection of NO donor SNAP with 82% normal embryos. (H-I) No rescue was obtained when SNAP was injected with par1 morpholino with 0% normal embryos. Par1 morpholino sequence and phenotype were published by Ossipova et al. 2005. (J) Quantification of results.
	Figure S6. kininogen (kng) is expressed throughout zebrafish craniofacial development and loss of function results in craniofacial cartilage abnormalities, related to Figure 7. (A-C, A’-C’) kng loss of function using splice site and start site morpholinos. Embryonic cartilage was observed at 5dpf using alcian blue staining in three independent experiments. E, Ethmoid plate. C, Ceratohyal cartilage. M, Meckel’s cartilage. Scale bar: 250m. (A,A’) Control morpholino injected embryos appeared normal (95% normal, n=43). (B-B’, C-C’) Splice and start morphants showed abnormal facial cartilage. Meckel’s cartilage is truncated and lies at an abnormal angle. The ceratohyal cartilage points at an abnormal angle, perpendicular to the midline. (kng splice morpholino (B,B’) 7% normal, n=95; kng start morpholino (C,C’) 6% normal, n=47). (D) Quantification of morphant phenotypes. P-values: one-tailed Fisher Exact test. (E) Non-quantitative RT-PCR. kng expression is present at bud stage and extends until 72hpf. Expression spans mouth opening at 48hpf and formation of facial cartilage at 72hpf. (F) Non-quantitative RT-PCR. RNA was extracted at 24hpf. Controls yield normal length kng transcripts, while splice morphants yield fewer normal length transcripts and a truncated transcript. Arrow: truncated transcript band.

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