Sequence of steps involved in periodic formation of protoneuromasts.

A. Interactions between Wnt and FGF signaling determine initiation of a center-biased domain of Fgf activity (blue) in the trailing part of the primordium in the context of a gradient of Wnt activity (yellow). B. Center-biased Fgf activity initiates center-biased atoh1a expression, which becomes restricted by Notch mediated lateral inhibition to a central cell, which becomes specified as the sensory hair cell progenitor. C. The central atoh1a-expressing cell becomes a source of Fgf10 and DeltaD. They activate Fgf and Notch signaling, respectively in neighboring cells to promote reorganization of neighboring cells to form epithelial rosettes. D. Maturation of the protoneuromasts is associated with establishment of atoh1b expression and maintenance of atoh1a through positive autoregulation. It is also associated with suppression of Wnt signaling in the trailing zone such that the initially broad domain of Wnt activity shrinks, creating conditions for periodic formation of new protoneuromasts in its wake. Sequential formation and maturation of protoneuromasts is associated with changes in the pattern of Fgf signaling; from center-biased in a newly formed protoneuromast, to broader as the central cell becomes a source of Fgf, to finally, donut shaped in the most mature protoneuromast, as Fgf signaling is lost in the central atoh1a-expressing cell.

Regulation of soxB1 family members by Wnt and Fgf activity in the pLL primordium A-C.

Expression of soxB1 family members in the pLL primordium of claudinB-GFP transgenic embryos. sox1a in the leading domain (bracket)(A), sox2 and sox3 in the trailing domain (B, C). Arrows in B indicate center-biased (white), broad (blue) and donut-shaped (black) sox2 expression domains. D-M. Effects of Heat shock induced ΔN-TCF. Expression of Wnt-responsive lef1 and Fgf-responsive pea3 following heat shock in wt siblings (D, F) and hs: ΔN-TCF (E, G) embryos. Expression of sox1a, sox2 and sox3 following heat shock in wt siblings (H, J, L) and hs: ΔN-TCF (I, K, M) embryos. N-R. Effects of heat shock induced dnFGFR expression. Expression of lef1 and pea3 following heat shock in hs: dn-FGFR embryos (N,O). Expression of sox1a, sox2 and sox3 following heat shock in hs: dn-FGFR embryos (P-R). S-U. Expression of lef1, pea3 and sox2 in following heat shock in hs: ΔN-TCF/ hs: dn-FGFR double transgenic embryos. V, W. sox2 expression in control and lef1 morphant claudinB-GFP embryos. Bracket indicates leading domain. X, Y. lef1 expression in control and sox2/3 morphant claudinB-GFP embryos. Bracket indicates trailing domain. Z. Regulation of soxB1 family member expression. All embryos were fixed at 32hpf. Heat shock performed at 26-28 hpf after pLLP had clearly separated from the otic vesicle. Dotted lines demarcate outer edge of pLLP as determined by immunofluorescence(anti-GFP) or DAPI staining.

Deposition pattern of neuromasts in soxB1 family morphants and mutants in claudinB-GFP embryos A-D.

Deposition pattern of neuromasts (arrows): in a control morpholino (ctrl MO) and 2ng sox2 MO1 morpholino injected embryo (A,B), and in wild type sibling (wt) and sox2-/- mutant (C,D). E-H. Distances of L1-L6 neuromasts from the otic vesicle in embryos represented in A-D, respectively. I, J, K. Neuromast deposition pattern in a sox2-/-, sox1a-/-, and sox1a-/-/sox2-/- double mutant embryo. L, M, N Neuromast deposition pattern in a sox2-/-, sox3a-/-, and sox2-/-/sox3-/- double mutant. Arrows in I-N indicate position of 1st neuromast (L1). All imaged at 52 hpf. O, P. Live imaging of early deposition events in control and sox2MO1/sox3 MO injected embryos. Numbered Arrowheads indicate position of deposited neuromast or forming protoneuromasts. Dotted oval outlines disassembled deposited neuromast 375 minutes after initiation of image acquisition at 25hpf. Q-T. Expression of atoh1a in control and sox2MO1/ sox3 MO co-injected embryos at 26hpf.

soxB1 family members inhibit Wnt activity in the pLL primordium A, B.

Expression of lef1 in control and sox2 MO1 morpholino injected embryos. C-H. Expression of lef1 in soxB1 family single and double mutants. I-N. Expression of pea3 in soxB1 family single and double mutants. O, P. Expression of sox1a in wt and sox2 mutant embryos. Dotted lines demarcate outer edge of pLLP. Q. Time-lapse images of Wnt activity in control and sox2/sox3 morphants in Tg[tcf/lef1-miniP:d2GFP] / CldnB:Lyn-mScarlet transgenic embryos. GFP reporting Wnt activity visualized with Fire LUT (Yellow to white-high, purple -low) and cell membranes with Lyn-mScarlet (inverted grey scale). Arrowhead indicates depositing neuromast. Dotted oval outlines disassembled deposited neuromast 420 minutes after initiation of image acquisition at 25hpf.

Inhibition of Wnt activity in sox2/sox3 morphants restores timely L1 deposition A-D.

Expression of lef1 in control and sox2 MO1-injected embryos treated with DMSO (A, B) or Wnt signaling antagonist IWR-1(C, D). E-H. Deposition pattern of neuromasts for same treatment groups. Arrow indicates position of 1st deposited neuromast L1. I. Quantification of distance of L1 from otic vesicle for treatment groups. Asterisks indicate significance by Mann-Whitney test (**** p< 0.0001, ** p= 0.0079).

Inhibition of Wnt activity in sox2/3 morphants restores timely neuromast maturation A-D.

Expression of dkk1b and atoh1b, which, respectively, mark nascent and mature protoneuromast, in control and sox2 MO1morphant embryos, in the absence or the presence of the Wnt signaling antagonist IWR-1. E. Schematized phenotypes with corresponding examples of patterns of dkk1b (large red circle) and atoh1b (small green dot) expression. Each pattern is coded by a color. F. Frequency distribution of phenotypic categories in treatment groups using the color code. Asterisks indicate significance by Fisher’s exact test (**** p< 0.0001)

Summary of regulatory interactions and Steps to self-organization of neuromasts in the Posterior Lateral Line primordium

A. Regulatory interactions between SoxB1 family members and the Wnt and FGF signaling in the primordium B. Steps to self-organization of neuromasts in the Posterior Lateral Line primordium. See text for details.

The relationship between sox1a and sox2 expression and Wnt activity, as indicated by lef1

A, B, D. sox1a expression (cyan) typically overlaps with lef1 expression (yellow). A, C, E. sox2 expression (magenta) is complementary to lef1 expression (yellow). F. sox1a (cyan) and sox2 (magenta) expressions are complementary to each other. In C, white arrow shows center-biased expression, blue arrow -broad expression, and black arrow shows donut shaped expression.

Cross-reactivity of sox2 MO1 with sox3 and enhanced phenotype compared to sox2-specific MO

A. sox2 and sox3 antibodies labeling control, sox2 MO1 and sox3 morphant pLLP. B. L1 deposition in sox2 MO1 and sox2-specific morphants. Asterisks indicate significance by Mann-Whitney test (**** p< 0.0001, * p< 0.05).

L1 deposition in sox1a/sox2 and sox2/sox3 heterozygous mutant incross

A, B. Deposition distance of L1 neuromast in 52hpf double mutant embryos. sox2+/- and sox1a+/- or sox3+/- were crossed to generate heterozygous progeny for imaging followed by genotyping. Asterisks indicate significance by Mann-Whitney test (*** p< 0.001, ** p< 0.01, * p< 0.05).

CRISPR mutagenesis of sox2y589 and sox1ay590

A. sox2 insertional mutant with site of frame shift and predicted translation. B. sox1a deletional mutant with site of frame shift and predicted translation.