Adrenergic activation modulates the signal from the Reissner fiber to cerebrospinal fluid-contacting neurons during development

The cerebrospinal fluid (CSF) contains an extracellular thread that is conserved in vertebrates and referred to as the Reissner fiber. Genetic ablation in the zebrafish revealed that the Reissner fiber controls body axis morphogenesis in the embryo. Yet, the signaling cascade originating from this fiber to ensure body axis straightening is not fully understood. Here, we explore the functional link between the Reissner fiber and undifferentiated spinal neurons contacting the CSF (CSF-cNs). First, we show that the Reissner fiber is required for the expression of urp2, a neuropeptide expressed in CSF-cNs. Using in vivo calcium imaging, we show that the Reissner fiber is also required for embryonic calcium transients in these spinal neurons. Finally, we study how local adrenergic activation directly in the CSF can substitute for the Reissner fiber-signaling pathway to CSF-cNs and rescue body axis morphogenesis. Our results suggest that the Reissner fiber acts on CSF-cNs to establish body axis morphogensis by controlling the availability of a chemical signal in the CSF.


Introduction 27
One of the major questions in the study of multicellular organism development is to 28 understand how precise morphogenesis is ensured during embryonic and postembryonic 29 stages while the animal grows into an adult. In particular, this process requires a coordination 30 between cell specification signals and the control of the tissue shape (Chan et al., 2017). It 31 has recently emerged that the cerebrospinal fluid (CSF) contains many signals important for 32 cell differentiation, and is an important route for the control of morphogenesis (Fame and 33 Lehtinen, 2020). The CSF is a complex liquid filling the central nervous system cavities 34 containing a set of diffusible signaling cues guiding neurogenesis (Lehtinen et al., 2011) and 35 brain morphology in a tissue autonomous manner (Kaiser et al., 2019;Langford et al., 2020). 36 In numerous vertebrate species, the CSF circulation is in part generated by the coordinated 37 In this study, we aimed to better understand the signal linking the Reissner fiber to 77 body axis morphogenesis. To investigate whether the Reissner fiber is required for a signal 78 towards the immature CSF-cNs, we sought to decipher the nature of this signal and how it 79 affects at long distance body axis straightening. We show that the Reissner fiber is required 80 for a signal controlling urp2 but not urp1 expression. Using in vivo calcium imaging, we report 81 that the Reissner fiber is also required for calcium signaling in urp2-expressing ventral CSF-82 cNs, confirming the existence of a crosstalk between the Reissner fiber and undifferentiated 83 CSF-cNs at the embryonic stage. Using the pkd2l1 mutant, we show that the loss of calcium 84 signaling in ventral CSF-cNs does not affect urp2 expression nor body axis curvature. 85 Finally, we show that local injections of monoamines in the brain ventricles of scospondin 86 mutants restore the Reissner fiber-dependent signal together with body axis straightening. 87 Our work demonstrates that the Reissner fiber-dependent signal to ventral CSF-cNs 88 contributes to body axis straightening and is modulated by adrenergic ligands.

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See also   The gene urp2 encodes for a secreted neuropeptide belonging to the Urotensin-II-134 related-peptide family (Tostivint et al., 2014). Together with urp1, these transcripts have 135 recently been identified as both strongly downregulated in curled-down mutants (Lu et al., 136 2020;Zhang et al., 2018). To ascertain our RNAseq results and confirm the difference we 137 observed with previous results, we carried out qRT-PCR analysis of urp1 and urp2 138 expression levels in the hypomorphic scospondin icm15 allele. Interestingly, we observed that 139 urp1 expression level is not significantly decreased in scospondin homozygous mutants 140 compared to their control siblings, neither at 30 hpf nor at 48 hpf ( Figure 1B). Consistently 141 with transcriptomic results, urp2 expression level shows a strong decrease at 48 hpf in 142 mutant embryos compared to their control siblings (3.6 ± 0.2 fold decrease; mean ±SEM; 143 Figure 1C). This is also true at the onset of the curled-down phenotype (30 hpf: 4.2 ± 0.5 fold 144 decrease; mean ±SEM; Figure 1C) indicating that urp2 gene expression level is affected 145 when embryos start to develop an abnormal morphogenesis of the posterior axis. Taken 146 together, these data show that in zebrafish embryo the presence of the Reissner fiber in the 147 CSF is required for the normal expression level of urp2, but not urp1. 148

The Reissner fiber is required for calcium signaling in urp2 expressing CSF-cNs 149
The expression of Urotensin-II-related peptides is restricted to the ventral population 150

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As previously described, we observed that ventral CSF-cNs exhibit spontaneous 186 calcium transients in wild type embryos (scospondin +/+ , Figure 2B   Early calcium transients in CSF-cNs require the Pkd2l1 channel in vivo (Sternberg et 199 al., 2018). The reduction in calcium activity when the Reissner fiber is lacking could therefore 200 be due to a defect in the Pkd2l1 channel localization or opening probability. In toto 201 immunohistochemistry for Pkd2l1 protein showed that this channel is enriched at the 202 differentiating apical extension of CSF-cNs in curled-down scospondin icm15/icm15 mutants as 203 well as in control embryos ( Figure 2D). In order to assess Pkd2l1 channel properties, we 204

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Thanks to the viability of pkd2l1 icm02/icm02 zygotic mutants, we generated genetically-244 related clutches that were either fully wild type or fully maternal & zygotic (MZ) homozygous 245 mutant ( Figure 3A). We confirmed that pkd2l1 icm02/icm02 MZ mutant embryos did not display 246 any defect in body axis morphogenesis and were morphologically undistinguishable from wild 247 type embryos ( Figure 3B). Using immunohistochemistry, we observed that pkd2l1 icm02/icm02 248 mutants form a normal Reissner fiber in the central canal of the spinal cord at 30 hpf ( Figure  249 3C). Next, using qRT-PCR, we tested whether urp1 and urp2 gene expression levels were 250 diminished by the absence of calcium transients in CSF-cNs ( Figure 3D). As the mutation in 251 the icm02 allele generates a premature stop codon in the pkd2l1 gene (Böhm et al., 2016), 252 one can predict that the pkd2l1 mRNA would be degraded by nonsense-mediated decay. 253 Indeed, mutant clutches displayed a 3.4 folds decrease in pkd2l1 transcripts level compared 254 to wild type counterparts ( Figure 3D). Interestingly, we observed that urp2 gene expression 255 was not decreased in pkd2l1 icm02/icm02 embryos compared to wild types, as well as urp1 256 expression that also showed no decrease (if anything, it showed a non-significant increase). 257 Altogether, these data show that the loss Pkd2l1-driven calcium transients in CSF-cNs does 258

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We first analyzed the effect of 2.5 hours bath applications of epinephrine and 317 norepinephrine on control embryos at 30 hpf ( Figure 4A). Epinephrine and norepinephrine 318 have a moderate impact on the shape of the head-to-tail axis of initially straight embryos, 319 which display a slight curled-up phenotype after exposure to monoamines ( Figure 4B). To 320 estimate the straightness of the posterior axis, we quantified the angle formed between the 321 ear, the caudal limit of the yolk extension and the tip of the tail. This angle is distributed 322 around 190.6° in control embryos exposed to a vehicle solution (median value; Figure 4C). 323 Comparatively, control embryos exposed to monoamines exhibit a distribution of ear-to-tail 324 angles shifted towards slightly higher values (median values: 220.4° and 213.6° for 325 epinephrine and norepinephrine, respectively; Figure 4C). Next, we analyzed the effect of 326 epinephrine and norepinephrine on scospondin icm15/icm15 embryos. While mutant animals 327 exposed to a vehicle solution display a typical curled-down body axis, scospondin icm15/icm15 328 mutants treated with monoamines exhibit a reduction in the downward curvature of the 329 posterior axis ( Figure 4B). Quantifications of ear-to-tail angles in scospondin icm15/icm15 mutants 330 show a large increase of the median angle value after exposure to monoamines, compared 331 to embryos treated with a vehicle solution (106.5° for vehicle, 167.1° for epinephrine and 332 158.5° for norepinephrine; median values; Figure 4C). Altogether, these data show that 333 epinephrine and norepinephrine can partially restore the posterior axis geometry of 334 scospondin icm15/icm15 mutants, suggesting that monoamines can rescue the Reissner fiber-335 dependent signal required for a straight embryonic body axis. 336 Next, we analyzed the effect of epinephrine and norepinephrine on urp1 and urp2 337 gene expression using qRT-PCR. In control siblings, urp1 expression remains comparable 338 after vehicle, epinephrine or norepinephrine exposure ( Figure 4D). Consistently with our 339 previous results, urp1 expression is not significantly modified in curled-down 340 scospondin icm15/icm15 embryos compared to their control siblings receiving the same treatment 341 (1.01 +/-0.17 fold change; mean ± SEM; Figure 4D). However, we observed a slight 342 increase of urp1 expression in curled-down mutant embryos treated with epinephrine and 343 norepinephrine compared to vehicle treatment (1.58 ± 0.43 and 1.28 ± 0.23 fold changes for 344 epinephrine and norepinephrine respectively; mean ± SEM). As expected, urp2 expression 345 level is significantly decreased in curled-down scospondin icm15/icm15 mutants compared to 346 straight siblings (0.22 ± 0.06 fold change; mean ± SEM; Figure 4E). Similarly to what we 347 observed for urp1, epinephrine and norepinephrine treatments do not change urp2 348 expression in control siblings, but significantly increase it in curled-down homozygous mutant 349 embryos (7.42 ± 4,23 and 4.67 ± 1.15 fold changes for epinephrine and norepinephrine 350 respectively; mean ±SEM; Figure 4E).     Figure 5F). Interestingly, we observed that norepinephrine positive 421 signals follow similar patterns of distribution than densely packed material labelled by the 422 Tg(scospondin-GFP) transgene ( Figure 5F). This observation suggests that norepinephrine 423 is a ligand endogenously present in the embryonic CSF that is associated with the Reissner-424 positive material in the CSF within the central canal. To address the question of the receptor 425 associated to this signal, we performed immunostainings against the adrenergic receptor 426 Adrb2 that is described to be transiently expressed in the zebrafish nervous system at early 427 stages of embryonic morphogenesis (Wang et al., 2009). We observed a signal that was 428 localized ventrally in the neural tube in a pattern that suggests a membrane location in both 429 control siblings and scospondin icm15/icm15 mutants ( Figure 5G). Thus, Adrb2 localization is 430 suitable for binding endogenous ligands in the CSF. Altogether, these results indicate that 431 endogenous adrenergic signals could modulate the Reissner fiber-dependent signaling 432 pathway that instructs body axis straightening during embryonic development. therefore not be detected using our transcriptomic strategy may be involved. 463 The discovery of a new hypomorphic allele for scospondin recently revealed an 464 inflammatory signature induced by the loss of the Reissner fiber at the embryonic stage 465 (Rose et al., 2020). In our work, we did not detect such a signature in our transcriptomic 466 analysis (see Table Supplement 1). We can speculate that these differences are due to 467 difference in the fish genetic background or husbandry conditions. Our work therefore begs 468 for further studies in order to decipher the molecular pathways downstream and/or in parallel 469 to Urp2 that regulate the morphogenesis of the embryonic axis. cord. This is reinforced by our results showing the localization of the Adrb2 receptor at the 504 interface with CSF in the ventral part of the neural tube, ideally located to bind these ligands 505 that we found closely distributed to Reissner-positive material. Interestingly Adrb2 belongs to 506 the G protein coupled receptors family and was identified to trigger cytoplasmic calcium raise 507 in vitro (Galaz-Montoya et al., 2017). In zebrafish, the adrenergic system plays important 508 roles in the control of wakefulness (Singh et al., 2015) and of cardiac contractions (Steele et 509 al., 2011). However, no morphological defects have been reported in animal missing the 510 rate-limiting enzyme for the synthesis of epinephrine and norepinephrine (Singh et al., 2015). 511 This might reflect the masking of the phenotype due to transcriptional adaptation to the 512 genetic mutation (Rossi et al., 2015). Alternatively, redundant signaling pathways might mask 513 the role of monoamines in axis curvature control. Nonetheless, our results support the idea 514 that the cross-talk between the Reissner fiber and undifferentiated CSF-cNs is likely to be of 515 chemical nature, possibly through monoamines themselves. Future investigations will allow 516 to fully delineate the contribution of endogenous monoamines on ventral CSF-cNs signaling 517 and body axis curvature, and whether they act through a direct or indirect mechanism. 518 Altogether, our study unravels a signal from the Reissner fiber to the developing CSF-519 contacting neurons. We also show that adrenergic activation can modulate this signal during 520

RNA sequencing analysis 551
The SmartSeq v4 (Clontech/Takara) was used to generate double-stranded cDNA from each 552 replicate; these pools were fragmented and tagged for sequencing using the Nextera DNA 553 Library Preparation Kit (Illumina). Finally, the prepared library was sequenced on an Illumina 554 NextSeq 500. Quality of raw data was evaluated with FastQC (Andrews et al., 2010). Poor 555 quality sequences was trimmed or removed using Trimmomatic software (Bolger et al., 2014) 556 to retain only good quality paired reads. Star v2.5.3a (Dobin et al., 2013) was used to align 557 reads on GRCz11 reference genome using standard options. Quantification of gene and 558 isoform abundances was achived using with Rsem 1.2.28, prior to normalization with the 559 edgeR bioconductor package (Robinson et al., 2009). Finally, differential analysis was 560 conducted with the GLM framework likelihood ratio test from edgeR. 561 The quality control and PCA analysis (not shown) indicated that one sample was not 562 reaching the same reproducibility as the two others, even after trimming and normalization. 563 We therefore used the average value of the two most reliable replicates to calculate the 564 average expression level (count per million, cpm) of all the 28 214 genes either in straight 565 controls or in curled-down embryos (average of the expression in the two alleles icm13 and 566 icm15). We then filtered out low-expression genes that had an expression level below 1 cpm 567 in the average of the straight control replicates. We then kept genes that had an average fold 568 change between curled embryos and controls of 0.75 for down-regulated genes (120 genes), 569 and 1.45 for up-regulated genes (94 genes). We turned back to the original raw cpm values 570 of the three replicates in the two alleles for these two short-lists. We tested the consistency of 571 the fold change across replicates and across alleles with a general linear model (GLM) 572 (Nelder et al., 1972). The design matrix included 2 regressors of interest (encoding whether 573 the sample is a straight control or curled embryos) and 2 confounding variables (the 574 unwanted variability that might be associated with the two different genetic environments of 575 the two families of fish carrying the icm13 and the icm15 allele). Statistical significance of the 576 effect of interest (above and beyond confounding factors) was tested using a t-test. Because 577 of the potential false discovery associated with multiple testing, we then used the  Hochberg procedure (Benjamini, et al., 1995). 579 Potential genes of interest were sorted according to their increasing p-values. To shorten the 580 list of potential genes of interest, we determined at which rank i the p-value became higher 581 than the Benjamini-Hochberg criterion using (i/total nb)* 0.2. This method allows for up to 582 20% of false-positives, but avoids the rejection of true-positives that was manifest with a 583 more stringent correction as Boniferri. For example, the scospondin mRNA level in the icm13 584 allele is highly decreased, presumably through non-sense mediated decay: the Boniferri 585 correction would have rejected this result as not significant, whereas the Benjamini-586 Hochberg procedure keeps it in the list of potentially interesting genes. However, this 587 procedure requires a post-hoc validation with an independent technique for each potential 588 gene of interest, as we did by qRT-PCR (see below). 589 Quantitative RT-PCR 590 1.2 µg of each RNA sample was retro-transcribed using the Verso cDNA Synthesis Kit 591 (ThermoFischer Scientific, AB1453B) following the provider's instructions. A 1:3 ratio of 592 random hexamer:polydT primers was used to favor mRNA amplification without a strong bias 593 for the 3' end of messengers. qPCR experiments were performed using the LightCycler® 480 594 SYBR Green I Master kit (Roche, 04707516001) on a LightCycler® 96 machine (Roche). 595 Each pair of primers (see Key Resource Table) was tested beforehand on a given cDNA 596 stock that was diluted in a series of 4 points. Linearity of CT variation with the cDNA dilution 597 as well as single peaks in the melting curves corresponding to single amplicons were 598 assessed. For each new RNA extraction, we tested that no amplification was detectable 599 when the PCR was performed directly on the RNA stock (-RT). We used the housekeeping 600 gene lsm12b as an internal reference in each experiment. qPCR were repeated two to three 601 times for each cDNA stock and the average CT was used for further calculations. The 602 relative abundance of the gene of interest was evaluated using the CT comparison formula: 603 2e -ΔCT . All results were obtained on at least three biological replicates (except for pkd2l1 604 mutant extracts), each originating from a single mating. 605

Pharmacology and quantification of body axis curvature 606
30 hpf embryos from scospondin icm15/+ incrosses were treated during 2.5 hours with E3 607 medium alone (vehicle), epinephrine hydrochloride (Sigma, E4642) or norepinephrine 608 hydrochloride (Sigma, A7256) both diluted to 3 mM in E3 medium. To ensure a proper 609 quantification of ear-to-tail angles, embryos were then fixed over-night in 4% PFA 6-well 610 plates, rinsed in 1X PBS, mounted laterally in 1.5% low-melting point agarose and imaged 611 using a AZ100M macroscope (Nikon). For each embryo, the angle between the ear, the 612 caudal limit of the yolk extension and the tip of the tail was quantified using Fiji (Schindelin et

RNA microinjections 662
To produce mRNA, urp2 CDS was amplified from cDNA by PCR and cloned (BamHI -XbaI) 663 into pCS2+. Messenger RNA were produced with the mMESSAGE mMACHINE™ kit 664 (Ambion™). 1 nL RNA-containing solution was injected into 1-to 2-cell stage embryos 665 obtained from scospondin icm15/+ incrosses. Each clutch was separated into three groups: 666 uninjected and injected either with a control mRNA (100 ng/µL, ras-eGFP encoding for a 667 membrane tagged GFP, (Ségalen et al., 2010) or with a mix containing control mRNA and 668 urp2 mRNA (100 ng/µL total, 1:1 ratio). To assess for injection quality, GFP-positive embryos 669 were first sorted out at 1 dpf and then scored at 48 hpf for body axis curvature defects. 670

Statistics 671
All values are represented as boxplots (median ± interquartile range) or mean ± SEM (stated 672 for each in the figure legend). All statistics were performed using MATLAB and Excel. In the 673 figure panels, asterisks denote the statistical significance calculated using the appropriate 674 test (stated for each test in the legends): *, p<0.05; **, p<0.01; ***, p<0.001; ns, p>0.05. 675 After filtering for low expression, we kept a list of genes that were differentially expressed in 873 null scospondin icm13/icm13 and hypomorphic scospondin icm15/icm15 mutant embryos at 48 hpf. The 874 table shows the average fold changes in the two alleles and their mean. Genes are ranked 875

Supplementary Figure 870
based on their p-value that compare the reproducibility of the change inside and between the 876 two alleles using a GLM framework. We adjusted the false discovery rate due to multiple 877 comparisons using the Benjamini-Hochberg procedure (see Material and Methods for 878 details).