Morphogenesis is transcriptionally coupled to neurogenesis during olfactory placode development

Morphogenesis of sense organs occurs concomitantly with the differentiation of sensory cells and neurons necessary for their function. While our understanding of the mechanisms controlling morphogenesis and neurogenesis has grown, how they are coordinated remains relatively unknown. The earliest wave of neurogenesis in the zebrafish olfactory placode requires the bHLH proneural transcription factor Neurogenin1 (Neurog1). To address whether Neurog1 couples neurogenesis and morphogenesis in this system, we analyzed the morphogenetic behavior of early olfactory neural progenitors. Our results indicate that the oriented movements of these progenitors are disrupted in neurog1 mutants. Morphogenesis is similarly affected by mutations in the chemokine receptor, cxcr4b, making it a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-boxes cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development. One Sentence Summary Neurog1 controls olfactory placode morphogenesis via cxcr4b

movements shape placodal progenitors and newly born neurons into a rudimentary cup (Whitlock and Westerfield, 2000;Miyasaka et al., 2007;Breau et al., 2017). We hypothesized that in parallel to its role in olfactory neurogenesis Neurog1 is ideally placed to control the cell movements that underlie morphogenesis of the olfactory cup, thus coupling morphogenesis and neurogenesis. To address this possibility, we analyzed morphogenesis of the olfactory cup by time-lapse imaging neurog1 mutant or wild-type embryos carrying a Tg (-8.4neurog1:gfp) transgene (Golling et al., 2002;Blader et al., 2003); this transgenes recapitulates the expression of endogenous neurog1 in the developing placode/cup and can be used as a short-term lineage label for the progenitors of early olfactory neurons, referred to hereafter as EON Breau et al., 2017). As recently described by Breau and colleagues, we found that EON reach their final position in wild-type embryos by converging towards a point close to the center of the future cup (as represented in Figure 1A; (Breau et al., 2017)). Considering overall antero-posterior (AP) length of the placode, this convergence appears to happen quickly between 12 and 18 hours post-fertilization (hpf), after which it slows ( Figure S1A,B). In neurog1 hi1059 mutants, we observed a delay in convergence, which translates into a longer AP length spread of EON than seen in wild-type embryos ( Figure S1B). This delay is overcome, however, with the final cup in neurog1 hi1059 mutant embryos attaining AP length of control embryos around 27 hpf ( Figure S1B).
To assess the morphogenetic phenotype of neurog1 hi1059 mutant embryos at cellular resolution, we injected synthetic mRNAs encoding Histone2B-RFP (H2B-RFP) into Tg (-8.4neurog1:gfp) transgenic embryos, which were again imaged from 12 to 27 hpf. Morphogenetic parameters of individual EON located in the anterior, middle and posterior thirds of the neurog1:GFP+ population were then extracted from datasets generated by manually tracking H2B-RFP positive nuclei. The position of each tracked EON was then plotted according to their medio-lateral (X) and (Y) position. As for the global analysis, the behavior we observe for single EON in control embryos largely recapitulates those already reported ( Figure 1B; (Breau et al., 2017)). Comparing the behavior of EON in neurog1 hi1059 mutants and siblings, we found that whereas EON in the middle and posterior regions of mutant placodes migrate similarly to their wild-type counterparts, the migratory behavior of anterior EON is profoundly affected in neurog1 hi1059 mutant embryos from 12 to 18 hpf ( Figure 1B,C and Figure S2A); morphogenetic movements of individual skin cells showed no obvious differences in control versus neurog1 mutants suggesting that the effect is specific to EON ( Figure   S3). Principal component analysis (PCA) of the morphometric datasets confirmed that the major difference between olfactory placode morphogenesis in control and neurog1 hi1059 mutant embryos (PC1) lies in the migratory behavior of anterior EON along the AP axis; PCA revealed a more subtle difference in migration of the middle EON population along the same axis ( Figure 1D and Figure S2B) and between the posterior EON populations along the medio-lateral axis ( Figure S2B). These migratory defects are not due to a decrease in cell mobility as EON in neurog1 mutants displayed increase displacement over time compare to controls (Figure S1C,D); little or no difference was detected in the displacement of skin cells between control and neurog1 hi1059 mutant embryos ( Figure S1C,E). Taken together, our results indicate that Neurog1 is required for an early phase of morphogenesis of the olfactory placode.
The chemokine receptor Cxcr4b and its ligand Cxcl12a (also known as Sdf1a) have been implicated in olfactory placode morphogenesis in the zebrafish (Miyasaka et al., 2007). To address whether the behavior of EON in neurog1 hi1059 mutants resembles that caused when the activity of this guidance receptor/ligand pair is abrogated, we analyzed the morphogenetic parameters of EON migration in cxcr4b t26035 and cxcl12a t30516 mutants (Knaut et al., 2003;Valentin et al., 2007). As previously reported, olfactory placodes of embryos lacking Cxcr4b or Cxcl12a function display convergence defects, highlighted by an increase in the AP length of the cup relative to controls ( Figure S4A; (Miyasaka et al., 2007)). Analysis of the behavior of individual cells in cxcr4b t26035 and cxcl12a t30516 mutant embryos indicates that defects in EON migration are largely restricted to the anterior cohort of EON (Figure 2A,B and Figure S5A,B); EON show increased displacement over time in both cxcr4b t26035 and cxcl12a t30516 mutants ( Figure S4B,C) and no difference is apparent in the behavior of skin cells in either mutant relative to wild-type siblings ( Figure S4B,D and S6). A combined PCA of morphometric datasets from anterior EON of neurog1 hi1059 , cxcr4b t26035 and cxcl12a t30516 mutants confirms that the major difference in EON behavior lies in their displacement along the AP axis (PC1; Figure 2C). Finally, unbiased clustering of the PCA analysis reveals that there is more resemblance in the behavior of anterior EON between the three mutants than between any single mutant and controls ( Figure 2D).
The similarity between the migration phenotype of EON in neurog1 hi1059 , cxcr4b t26035 and cxcl12a t30516 mutant embryos suggests that the proneural transcription factor and the receptor/ligand couple act in the same pathway. To determine if the expression of either the receptor or its ligand are affected in the absence of Neurog1, we assessed their expression in neurog1 hi1059 mutant embryos. We found that cxcr4b expression is dramatically reduced or absent in EON progenitors at 12 and 15 hpf in this context ( Figure 3A); the expression of cxcr4b recovers in neurog1 hi1059 mutant embryos from 18 hpf, a stage at which we have previously reported that the expression of a second bHLH proneural gene, neurod4, also becomes Neurog1-independent ( Figure   3A; . Contrary to cxcr4b, the expression of cxcl12a is unaffected in neurog1 hi1059 mutant embryos at all stages analyzed ( Figure 3B). Taken together, these results suggest that the EON migration phenotype in neurog1 hi1059 mutant embryos results from the lack of Cxcr4b during the early phase of olfactory cup morphogenesis.
If the absence of early cxcr4b expression in the olfactory placodes of neurog1 hi1059 mutants underlies the morphogenesis defects, we hypothesized that restoring cxcr4b expression should rescue these defects. To test this, we generated a transgenic line where expression of the chemokine receptor is controlled by a -8.4 kb fragment of genomic DNA responsible for neurog1 expression in EON, Tg , and introduced it into the neurog1 hi1059 mutant background (Blader et al., 2003;. Analysis of the migratory behavior of anterior EON in neurog1 hi1059 mutant embryos carrying the transgene indicates that they display oriented posterior migration similar to wild-type siblings carrying the transgene ( Figure 3C,D). The similarity in the behavior of the anterior EON is also evident after PCA analysis ( Figure 3E). Here, neurog1 hi1059 mutant cells carrying the transgene cluster primarily with control cells rather than mutant cells lacking the transgene ( Figure 3E,F). These data lead us to conclude that Cxcr4b is the predominant downstream effector of Neurog1 during the early phase of olfactory cup morphogenesis.
Finally, we asked whether cxcr4b is a direct transcriptional target of Neurog1 by searching for potential Neurog1-dependent cis-regulatory modules (CRM) at the cxcr4b locus. Proneural transcription factors bind CANNTG sequences known as E-boxes, which are often found in clusters (Bertrand et al., 2002). We identified 18 E-boxes clusters in the sequences from -100 to +100 kb of the cxcr4b initiation codon, but only 1 of these clusters contains more than one of the CA A / G ATG E-box sequence preferred by Neurog1 ( Figure 4A and data not shown; ). Coherent with a role for this E-box cluster in the regulation of cxcr4b expression, a transgenic line generated using a 35kb fosmid clone that contains this cluster, TgFOS(cxcr4b:eGFP), shows robust expression of GFP in the olfactory cup ( Figure  suggesting that tagging Neurog1 does not affect its transcriptional activity and that cxcr4b behaves as a Neurog1 target ( Figure 4B). We have previously shown that the deltaA locus contains two proneural regulated CRMs  whereas CRM HI is a Neurog1-dependent CRM, HII underlies regulation of deltaA by members of the Ascl1 family of bHLH proneural factors (Hans and Campos-Ortega, 2002;. We found that ChIP against Neurog1-Ty1 after misexpression effectively discriminates between the Neurog1-regulated HI and Ascl1regulated HII CRM at the deltaA locus, thus providing a control for the specificity of our ChIP experiments ( Figure 4C). Similarly, we were able to ChIP the potential CRM containing the E-box cluster 7 suggesting that this region is also a target for Neurog1 ( Figure 4C).
To confirm the importance of the E-box cluster in the regulation of cxcr4b expression, we employed a Crispr/Cas9 approach to delete this CRM using a pair of sgRNAs flanking the CRM ( Figure S7A). The sgRNA pair efficiently induces deletions in the targeted sequence, as judged by PCR on genomic DNA extracted from injected embryos ( Figure S7B). Injection of the sgRNA pair into TgFOS(cxcr4b:eGFP) transgenic embryos caused mosaic disruption of the eGFP expression pattern ( Figure   4D). Loss of TgFOS(cxcr4b:eGFP) transgene expression is not due to cell death as eGFP-negative cells maintain the expression of the early neuronal marker HuC/D (insert in Figure 4D). Taken together, the results from our ChIP and Cripsr/Cas9 experiments strongly suggest that the CATATG E-box cluster upstream of cxcr4b is regulated directly by Neurog1.
In zebrafish, the proneural transcription factor Neurog1 directly regulates the expression of the neurogenic genes deltaA and deltaD (Hans and Campos-Ortega, 2002;. We have shown that Neurog1 is required for the development of an early wave of neurons in the olfactory placode . The data presented here show that Neurog1 controls an early phase of morphogenesis of the zebrafish peripheral olfactory sensory organ via its target gene cxcr4b. We propose that Neurog1 couples neurogenesis with morphogenesis in this organ via the transcriptional regulation of distinct targets. That members of the Neurog family regulate Delta1 and Cxcr4 expression, as well as development the olfactory epithelium in mouse suggests that this role may be conserved (Beckers et al., 2000;Mattar et al., 2004;Shaker et al., 2012).

Fish Husbandry and lines
Ethics Statement and Embryos: All embryos were handled according to relevant national and international guidelines. French veterinary service and national ethical committee approved the protocols used in this study, with approval ID: A-31-555-01 and APAPHIS #3653-2016011512005922v6.

Establishment of new transgenic lines
The TgFOS(cxcr4b:eGFP) fu10Tg transgenic line was generated using homologous recombination by replacing the second exon of cxcr4b by LynGFP in the Fosmid CH1073-406F3, followed by zebrafish transgenesis (Revenu et al., 2014). The first 5 amino acid encoded by the first exon of cxcr4b are fused to LynGFP, preventing targeting to the membrane. The GFP localizes to the cytoplasm in this transgenic line.
The Tg  transgene was generated by first cloning the coding region of cxcr4b minus its endogenous stop codon in frame upstream of mCherry in pCS2. The resulting cxcr4b-mCherry fusion coding sequence was transferred into the middle entry clone, pME, of the Tol2kit developed in the Chien lab (Kwan et al., 2007). The final transgene vector was generated using LR recombination with a previously described p5'-8.4neurog1 , the pME-cxcr4b-mCherry, and the p3E-polyA and pDestTol2pA/pDestTol2pA2 from the Tol2kit (Kwan et al., 2007). The new line was then generated by co-injecting the transgene with mRNA encoding Tol2 transposase into freshly fertilized zebrafish embryos.

In situ Hybridization, Immunostaining and Microscopy
In situ hybridization was performed as previously described (Oxtoby and Jowett, 1993).
Antisense DIG-labeled probes for cxcr4b and cxcl12a (David et al., 2002) were generated using standard procedures. In situ hybridizations were visualized using BCIP and NBT (Roche) as substrates.
Embryos were immunostained as previously described

Track analysis
Track parameters were extracted from Imaris as Excel files and analyzed using a custom script generated in R (The R Project for Statistical Computing, www.rproject.org). First, tracks were rendered symmetric across the left-right axis for ease of interpretation. Tracks were then color coded according to their genotype and to the phase of migration (early from 12-18 hpf; late from 18-27 hpf) and plotted. Finally, the mean for each set of tracks was generated using the "RowMeans" function and a plot was generated.
Principal component analysis (PCA) and clustering were performed using the built-in R function "prcomp" from the "FactoMineR" package and the "kmeans" function, from the "stats" package, respectively. Finally, the "barplot" function ("graphics" package) was used to represent either the EON cluster composition or the Skin cluster composition.

Chromatin Immunoprecipitation and qPCR
ChIP experiments were performed as previously described using approximately 300 embryos (12 to 15 hpf) per immunoprecipitation (Wardle et al., 2006). Two to four separate ChIP experiments were carried out with corresponding independent batches of either control un-injected embryos or embryos injected with a synthetic mRNA encoding Neurog1-Ty1; ChIP-grade mouse anti-Ty1 (BB2; Sigma-Aldrich, USA) was used.
Primers used for qPCR on ChIPs were: sgRNAs were generated by PCR following previously described protocols (Nakayama et al., 2014). Injection of sgRNAs was performed as described by Burger and colleagues, using a commercially available Cas9 protein (New England Biolabs). The efficiency of creating deletion after co-injection of the sgRNA pair was determined by PCR on genomic DNA extracted from injected embryos using the following primers: While a 500 base-pair PCR fragment is generated from a wild-type locus, an approximately 200 base-pair fragment is amplified if a deletion has been induced.