a cacnb1 ts25/ts25 Supplemental Figure 1

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1 ccn1 ts/ts α -ungrotoxin prlyzed 0.6 ΔF/F ΔF/F 2 s stimulus α -ungrotoxin ccn1 ts/ts Supplementl Figure 1 CSF-cNs recorded from lrv prlyzed with α-ungrotoxin nd ccn1 mutnt lrv show no difference in ctivity., Clcium trnsients recorded in CSF-cNs from lrv prlyzed with α- ungrotoxin (red) nd in CSF-cNs from ccn1 ts/ts mutnt lrv (ornge) during fictive escpes; stimulus indictes when the wter jet ws triggered, verge response in colored lines, the α-ungrotoxin dtset is the sme s shown in Figure 2., Quntifiction of clcium trnsient ΔF/F mplitude (ech dt point represents one recording from one cell; plots use medin s the mesure of centrl tendency). Responses show no significnt difference etween oth popultions (p = 0.8, 19 CSF-cNs from 12 ccn1 ts/ts mutnt lrve: men ΔF/F = 0.029; 192 CSF-cNs from 7 α-ungrotoxin prlyzed lrve: men ΔF/F = 0.02). 1

2 ΔF/F tgrfp ΔF/F GCMP ΔR/R t 1 t 2 t 3 ΔF/F * 2 s t 1 t 2 t 3 GCMP tgrfp with ROI trtcking tgrfp * 2

3 Supplementl Figure S2 Motion rtifct removl for imging clcium trnsients from CSF-cNs ssocited with ctive nd pssive til ends., Smple trces of responsive dorsl ipsilterl CSF-cNs from the externl pressure dtset. Shown re the ΔF/F trces of the red chnnel (tgrfp) nd the green chnnel (GCMP) s well s the ΔR/R of the comintion of oth chnnels to ccounting for the motion rtifct. The gp represents the time points immeditely fter the onset of stimultion where lrge movements preclude imging of the cell. The ΔF/F is used only for illustrtion purposes nd the ΔR/R is clculted from the rw, ckground corrected signl., Smple imges of the three time points indicted in. At t 1, GCMP fluorescence is very low while incresed fluorescence cn e seen t t 3 for the cell ody tht returns to the focl plne. The chnge in fluorescence due to shifts in focl plne is estimted from the tgrfp signl. The tgrfp signl is lso used to trck the position of the cell ody in order to move the corresponding region of interest (ROI). During the stimultion (t 2 ) cells move out the focl plne nd trcking fils. These dt points re omitted in the nlysis. The sterisk mrks the ROI, which corresponds to the smple trce in. The smple dt shown is from pssive externl pressure experiments ut the principle of nlysis is the sme for the til free experiments. Scle r: µm. ltency 00 durtion 3 distnce (mm) 0.16 speed (mm/ms) 13 oscilltions (n) c-end (degrees) c tril tril tril tril tril tril Supplementl Figure 3 Additionl ehviorl prmeters re not ffected in pkd2l1 icm02/icm02 mutnt lrve., Ltency, durtion, distnce, speed, numer of oscilltions nd C end mplitude were not ffected in the 3

4 mutnt., Two minute inter tril intervls led to n increse in ltency (p =.1x -3 ) nd reduction of distnce (p =.1x -3 ), speed (p = 0.0), nd numer of oscilltions (p = 8.1x - ). Escpe durtion nd C end mplitude were the only prmeters not ffected y this pprent hitution in our ssy. c, Sme s in with wildtypes (20 escpes from 73 lrve) in red nd pkd2l1 mutnts (217 escpes from 66 lrve) in lue. ltency durtion distnce (mm) speed (mm/ms) oscilltions (n) TBF* (Hz) c-end* (degrees) c siling siling siling siling tril tril tril tril tril tril tril 2 siling siling 60 siling Supplementl Figure Silencing of vesiculr relese in CSF-cNs y Botulinum toxin () cuses ehviorl deficits reminiscent of pkd2l1 icm02/icm02 lrve, in prticulr, reduction of TBF., TBF is significntly reduced in LC-GFP + lrve (p = 0.002), s is C end mplitude (siling men: ± 1.17º, LC-GFP + men: 6.13 ± 1.9º, p = )., Two minute inter tril intervls led to significnt decrese in speed (p = 0.01) nd TBF (p = 0.01). Other prmeters exhiit trends suggesting hitution, ut none re sttisticlly significnt. c, Sme s in, silings represented in lck (368 escpes from 128 lrve) nd LC-GFP + lrve in green (177 escpes from 7 lrve). Accounting for the hitution effects oserved in, significnce increses for the TBF effect (p = 0.00).

5 wt pkd2l1 sensory Pkd2l1 I-In E-In C-In ccsf-cn icsf-cn MN CC motor Supplementl Figure Cererospinl fluid-contcting neurons (CSF-cNs) re locted on either side of the centrl cnl in the spinl cord. These neurons re recruited during innte locomotion when the spinl cord is ending, specificlly on the compressed side. During movement, CSF-cNs provide GABAergic mechnosensory feedck input to the locomotor centrl pttern genertors tht modultes locomotor frequency.

6 Supplementl Tle 1 Trnsgenic lines nd mutnts generted for nd used in this study. Trnsgenic/mutnt Affected gene Description Originl reference Tg(pkd2l1:GCMPG)icm07 A promoter frgment of the pkd2l1 gene drives expression of the clcium indictor GCMPG in CSF-cNs. Generted in this study Tg(UAS:GCMP6f;cry:mCherry)icm06 The expression of the clcium indictor GCMP6f is dependent of the ctivtion of upstrem ctivtor sequence (UAS). A crystlline promoter frgment (cry) induces expression of mcherry in the lens. Generted in this study Tg(pkd2l1:Gl)icm A promoter frgment of the pkd2l1 gene drives expression of GFF (optimized Gl) trnsctivtor in CSF-cNs 1 pkd2l1 icm02 pkd2l1 The mutnt pkd2l1 icm02 ws generted with TALE nucleses engineered ginst the second exon of pkd2l1. +1 frmeshift. Generted in this study Tg(mnx1:Gl)icm23 The Tg(mnx1:gl) line driving selective expression in spinl motor neurons sed on the injection of the mnx1 construct 2. Generted in this study Tg(UAS:tgRFP-cx;cmcl2:eGFP)icm22 The expression of tgrfp loclized to the memrne with CAAX motif is dependent of the ctivtion of upstrem ctivtor sequence (UAS). A crystlline promoter frgment (cry) induces expression of egfp in the lens. Generted in this study Tg(pkd2l1:tgRFP)icm17 A promoter frgment of the pkd2l1 gene drives expression of tgrfp in CSF-cNs. Generted in this study Tg(βct:Arl13-GFP) A promoter frgment of the βct gene drives expression of the mouse Arl13 open reding frme fused to GFP. 3 relxed ts ccn1 A point mutnt of ccn1 generted y ENU mutgenesis cusing premture stop codon in exon 13. Tg(UAS:BLC-GFP)icm21 Botulinum toxin light chin serotype B codon-optimized for zerfish with C-terminl GFP fusion. Generted in this study 6

7 Supplementl References 1. Fidelin, K. et l. Stte-Dependent Modultion of Locomotion y GABAergic Spinl Sensory Neurons. Curr Biol, (). 2. Zelenchuk, T. A. & Brusés, J. L. In Vivo leling of zerfish motor neurons using n mnx1 enhncer nd Gl/UAS. genesis 9, 6 (11). 3. Borovin, A., Superin, S., Vosks, D. & Cirun, B. Vngl2 directs the posterior tilting nd symmetric locliztion of motile primry cili. Nt Cell Biol 12, ().. Grnto, M. et l. Genes controlling nd mediting locomotion ehvior of the zerfish emryo nd lrv. Development 123, (1996). 7

Supplementary Figure 1

Supplementary Figure 1 Supplementry Figure (nesthetized) (wke) Normlized mplitude.5 Pek width (ms).6.4.2 4 2 2 x 3 Wveform slope Normlized mplitude.5 Pek width (ms).6.4.2 x 3 3 2 Wveform slope c (nesthetized) d (wke) Normlized