# An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain

Alessandro Alunni, Constance Pierre, Jorge Torres-Paz, Natacha Clairet, Auriane Langlumé, Marie Pavie, Thomas Escoffier-Pirouelle, Michael Leblanc, Maryline Blin, Sylvie Rétaux

<figure><img src="image_1.png" /></figure>

► To cite this version:

Alessandro Alunni, Constance Pierre, Jorge Torres-Paz, Natacha Clairet, Auriane Langlumé, et al.. An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain. Development, Growth and Differentiation, 2023, 65 (9), pp.517-533. 10.1111/dgd.12896. hal-04265637

HAL Id: hal-04265637

https://hal.science/hal-04265637v1

Submitted on 20 Nov 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
1

Submission for Development Growth and Differentiation

2

Special issue

3

4

5

6

7

An *Astyanax mexicanus mao* knockout line uncovers the
developmental roles of monoamine homeostasis in fish brain

8

9

Alessandro Alunni*, Constance Pierre, Jorge Torres-Paz, Natacha Clairet, Auriane Langlumé, Marie Pavie, Thomas Escoffier-Pirouelle, Michael Leblanc, Maryline Blin, Sylvie Rétaux*

10

11

12

13

14

Paris-Saclay Institute of Neuroscience, CNRS, Université Paris-Saclay, 91400 Saclay,
France

15

16

*

17

Corresponding authors: alessandro.alunni@cnrs.fr and sylvie.retaux@cnrs.fr

18

19

Short title: MAO and fish brain development

20

21

Key words: monoamine oxidase, serotonin, dopamine, locomotion, proliferation, cell cycle

22

23
24

**Abstract**

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, *MAO-A* and *MAO-B*, fish possess one single *mao* gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a *mao* knockout line in *Astyanax mexicanus* (surface fish), by CRISPR/Cas9 technology. Homozygote *mao* knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of *mao* function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by *mao* loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of *mao* knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on *Astyanax mexicanus* brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth.

44

45
46

**Introduction**

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

The characiform fish, *Astyanax mexicanus*, exists under two very different forms (Mitchell et al., 1977; Elliott, 2018): a surface morphotype ("Surface Fish", SF), which lives in Central American rivers, and a cave blind and depigmented morphotype ("Cavefish"), which has colonized northeastern Mexican caves about 20,000 years ago (Fumey et al., 2018; Policarpo et al., 2020). During their adaptation to life in darkness, cavefish have evolved both regressive and constructive features. The comparison between the two forms of the species allows unravelling changes in genetic networks underlying adaptive modifications that occurred within a relatively short period of evolution (Keene et al., 2016; Gross et al., 2023; Retaux et al., 2023).

One of the known genetic differences between the two morphotypes is the existence of a partial loss-of-function mutation in the coding sequence of the Monoamine oxidase (MAO P106L), which reduces its enzymatic activity by 50%, and that is shared by several cavefish populations (Elipot et al., 2014; Pierre et al., 2020). Consequently, as MAO is the enzyme responsible for the degradation of monoamines, cavefish carrying the mutation in the homozygous state have high brain serotonin, dopamine and noradrenaline levels (Pierre et al., 2023).

MAO is a mitochondrial enzyme that catalyzes the oxidative deamination of biogenic amines (monoamine neurotransmitters and dietary amines). Mammals possess two MAO isoenzymes, *MAO-A* and *MAO-B*, in their genomes. In humans, only two families with *MAO-A* mutations are known, and they show severe behavioral and cognitive deficits with borderline
mental retardation and maladaptive regulation of impulsive aggression (Brunner et al., 1993; Piton et al., 2013). In mice also, MAO-A/B knockouts display developmental alterations with weight and size at birth significantly lower than wildtype littermates. On the behavioral side, MAO knockout mice show aberrant emotional response to novelty, high levels of anxiety-like behaviors in select tasks and low latency to attack in the resident-intruder paradigm (Chen et al., 2004). These findings underscore the importance of MAO function for developmental, neurophysiological and behavioral control.

Interestingly, only one *mao* gene is present in the genomes of other eukaryotes, from fungi (Schilling and Lerch, 1995) to invertebrates (Boutet et al., 2004) and teleost fish (Chen et al., 1994; Setini et al., 2005; Anichtchik et al., 2006; Elipot et al., 2014). This is despite the teleost-specific genome duplication that usually confers two gene paralogs in this vertebrate group (Jaillon et al. 2004). Whereas mammalian MAO-A and MAO-B have preferences for serotonin (5-HT) and noradrenaline (NA) versus dopamine (DA) substrates, respectively, the single fish MAO displays a strong affinity for 5-HT but a modest one for DA (Anichtnik 2016; Pierre 2023). In larval zebrafish, the inhibition of MAO increases 5-HT levels, which causes decreased locomotion, increased heart rate and altered vertical positioning (Sallinen et al., 2009).

A zebrafish knockout line for *mao* was recently generated. The *mao* knockout larvae are hypoactive at 6 days post-fertilization (dpf) and they have stronger serotonin immunoreactivity in their brains at 10 dpf (Baronio et al., 2021). In *A. mexicanus* on the other hand, the *mao P106L* mutation, which affects brain monoamines levels (Pierre et al., 2023), has a tendency to decrease basal anxiety in a familiar environment, but strongly increases the
amplitude of stress response after environmental change. This is associated to changes in locomotion and food intake in juveniles and young adults (Pierre et al., 2020).

To explore further the role of MAO enzyme on brain and behavioral homeostasis in *A. mexicanus*, here we have generated a surface fish line knockout for *mao*, by CRISPR/Cas9 technology. This line will be useful to characterize the drastic effects of complete loss of MAO function, to compare with the partial loss-of-function *mao P106L* mutation, and to observe the consequences on fish development and physiology. Here, we adopted an integrated approach to analyze the effects of *mao* knockout in *A. mexicanus* throughout the available developmental time (*mao* knockout fry die before reaching 2 weeks). First, we explored the development of monoaminergic neurons and their neurotransmitter contents through immuno-labelling. Next, we analyzed the locomotor activity of fish over the same period. Finally, we quantified proliferation of neural progenitors and cell cycle kinetics in the fry telencephalon and hypothalamus. We found that *mao* knockout has profound effects on brain developmental homeostasis.

107

108
109

110

# Materials and Methods

111

112

## Fish husbandry

113

114

115

116

117

118

119

120

121

122

123

124

125

126

## Fish lines

127

128

129

Laboratory stocks of *Astyanax mexicanus* (De Filippi 1853) surface fish (origin: Texas) were obtained in 2004–2006 from the Jeffery Laboratory at the University of Maryland, College Park, MD, USA and were then bred in our local facility (Gif-sur-Yvette and Saclay, France). Fish were maintained at 23–26°C on a 12 h:12 h light:dark cycle and they were fed twice a day with dry food. The fry were raised in Petri dishes and fed with an unlimited quantity of micro-worms (*Panagrellus* sp., obtained from the local amateur aquarist network), after opening of the mouth. Animals were treated according to the French and European regulations for handling of animals in research. SR's authorization for use of *A. mexicanus* in research is 91-116 and the Paris Centre-Sud Ethic Committee protocol authorization number related to this work is 2017-05#8604. The animal facility of the Institute received authorization 91272105 (for Gif-sur-Yvette) in 2015 and authorization B91272108 (for Saclay) in 2021 from the Veterinary Services of Essonne, France.

The knockout (KO) lines for *mao* were obtained using CRISPR-Cas9 technology. RNA sgRNAs were designed to target the *mao* genomic sequence containing the polymorphic SF/CF position (Fig. 1A), using the CRISPOR-TEFOR website (http://crispor.tefor.net/). Two sgRNAs
130 were tested, and both generated indels at the targeted sequence as follows: Sg1 5'-
131 AGGGGCCTTTCCCACCGATGTGG-3'; Sg3 5'-CCCACCGATGTGGAACCCTCTAG-3'.

132 A mix of Cas9 mRNA (200ng/µL), sgRNA1 or sgRNA3 (500 ng/µL) and phenol red (0.05%) was
133 injected into 1-cell stage A. mexicanus surface embryos. Injected embryos (G0; founders) were
134 reared to adulthood and their progeny (G1) screened and genotyped for indel mutations (Fig.
135 1B). Genotyping for screening of these progenies was performed on Labchip GX touch (Perkin
136 Elmer). The mao KO line analyzed herein had an 11pb deletion in exon 4.

137 The only rigorous and definitive way to control off-target is genome sequencing, which we
138 didn't perform. We generated two different *mao* KO lines by CRISPR. The first one that we
139 used in the manuscript induced a deletion of 11 nucleotides. The second one induced one
140 nucleotide deletion and frame shift. Our preliminary results with the second line confirm the
141 results obtained with the first one, suggesting that the phenotype we observed are specific
142 to *mao* KO.

143 We used the second and third generation for all the experiments.

144 To establish the mao KO line we incrossed the heterozygotes *mao* KO.

146 Immunohistochemistry, immunofluorescence and imaging

147 These experiments were performed on embryos (36 hpf, 48 hpf; hpf: hours post-fertilization)
148 and fry (4 to 12 days post-fertilization (dpf)) wild-type (WT), knockout (KO) and heterozygote
149 (het) surface fish. All samples were fixed in 4% paraformaldehyde in phosphate-buffered
150 saline (PBS), gradually dehydrated in methanol and stored at -20°C. For the
151 immunohistochemistry, the fry were permeabilized for 30 min in 1:1 ethanol: xylene at room
temperature (RT), then 10 min in 1:2 ethanol: acetone at -20°C. The samples were rehydrated
in PBS and 1% Tween (PBST). Next, brains were dissected out and incubated in PBST with 6%
$H_2O_2$ for 30 min at RT. The samples were incubated in blocking buffer [10%sheep serum, 1%
Triton, 1% Tween 20, 1% DMSO, 1% bovine serum albumin (BSA) in PBST] for 2 hours, then
incubated with the primary antibody (rat anti-serotonin, MAB352, 5/1000, Chemicon; rabbit
anti-tyrosine hydroxylase, SAB2701683, 2/1000, Sigma Aldrich) in buffer (1% sheep serum, 1%
Triton, 1% DMSO, 1% BSA in PBST) for 3 days, followed by the secondary antibodies
(biotinylated goat anti-rat antibody, 112-066-072, 1/500, Jackson ImmunoResearch). The
revelation was performed using an avidin/biotin complex coupled with peroxidase (ABC kit,
Universal Vectastain kit PK6200) and diaminobenzidine (Sigma Aldrich). Brains were mounted
in glycerol. Images were taken on a Nikon Eclipse 800. Images were corrected for brightness
and contrast in ImageJ or Photoshop, but no other corrections were made.

For the immunofluorescence experiments, the samples were rehydrated in PBS and 1% Tween
(PBST). Embryos were processed *in toto*. For the fry samples, we dissected the brains. The
samples were incubated in blocking buffer [10%sheep serum, 1% Triton, 1% Tween 20, 1%
DMSO, 1% bovine serum albumin (BSA) in PBST] for 2 hours, then incubated with the primary
antibody (rabbit anti-PH3, Thermofisher 1/500; rabbit anti-caspase activated, BD pharmigen
1/500; mouse anti-PCNA, Santa Cruz 1/300) for 3 days, followed by secondary antibodies (goat
anti-rabbit Alexa Fluor 486, 1/1000, goat anti-rabbit Alexa Fluor 546, 1/1000) and DAPI
(4/1000). The samples were mounted in Vectashield (Vector Laboratories). Images were taken
on a Leica confocal SP8 microscope. The images were processed using Fiji software. All labelled
cells were counted over the entire thickness of the tissue structure (telencephalon and
hypothalamus) at 10 µm intervals, i.e. the average thickness of the cells.
175 All the experiments, including the cell counts, were carried out blindly, without knowledge of the larvae' genotype. At the end of all the experiments, samples were retrieved from mounted slides and genotyping was carried out.

178

179 **EdU pulse-chase**

180 A pulse of Edu 10 mg/mL in EM (with 0.01%DMSO) was carried out by bath during 1 hour. The larvae were washed in EM and fixed in 4%PFA after a chase of 6 hours. Edu-positive cells were detected with the Click-iT™ EdU Alexa Fluor™ 647 (Thermo Fisher Scientific, C10340). Samples were whole-mounted in Vectashield medium (Vector, H1000) and imaged with a confocal microscope (Leica TCS SP8). EdU revelation was performed first, followed by the immunofluorescence labelling procedure.

186

187 **In situ hybridization**

188 To clone *A. mexicanus tph1* and *tph2*, total RNA from 48 hpf embryos was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad) and amplified using the following primers: *tph1a* forward, 5'-CATCGGCCTCACTCTGGAAG-3'; *tph1a* reverse, 5'-GCCCTTCGACCCCAAAGTTA-3';
189
190
191 *tph2* forward: 5'-TCCGGGAGCTGACTAAACTGTR-3'; *tph2* reverse : 5'-
192
193
194
195
196
197 GTGATGAGGCACTCCTGGTAG-3'. PCR products were cloned in pGEM-T vector and sequences.
Digoxigenin- riboprobes were prepared using PCR products as templates. ISH was carried out as previously described (Pottin et al., 2011). These experiments were performed on embryos (36 hpf) from WT, KO and het surface fish. All samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), gradually dehydrated in methanol. For probe penetration, embryos were treated with proteinase K (10 µg/ml) for 20 minutes at 37°C. Embryos were
198 incubated at 65°C for 18 h in 2 ng/µl DIG-labelled mRNA probes *tph1* and *tph2*, then with POD-
199 conjugated anti-DIG (sheep, Roche, 1:500). All *in situ* hybridization experiments used
200 NBT/BCIP (Sigma).

201

202 Image acquisition and cell counting

203 For immunohistochemistry and *in situ* hybridization, all images were taken on Nikon Eclipse
204 E800 microscope using 20X and 40X air objective. For immunofluorescence, all images were
205 taken on a Leica SP8 confocal microscope using, 20X air or 40X water objectives, then the
206 images were processed using Fiji software. Sections are presented as single confocal planes
207 except for Fig. 7, which corresponds to maximum intensity projections of two adjacent
208 confocal planes. All labelled cells were counted throughout the thickness of the structure by
209 counting all the cells at 10 µm intervals.

210

211 Aggressiveness assay

212 Behavioral tests were performed on 3 months post-fertilization (mpf) wild-type SF and
213 heterozygotes. Fish were used in a resident-intruder test, as in *Elipot et al.* (2013). Fish were
214 placed individually in tanks (12 cm×9 cm) with 200 ml of water for the night. In the morning,
215 intruders were transferred into tanks of residents, and a video of 1 h was recorded. Attacks
216 between the two fish were counted manually using ODREC software (Observational Data
217 Recorder; https://sourceforge.net/projects/odrec/).

218

219 Locomotion
The measures of locomotion on larvae between 6 and 12 days old were performed in a Zebrabox (Viewpoint), a soundproof box isolated from light in which fish were placed individually in multiwell plates (24 wells up to 8 dpf and 12 wells between 9 and 13 dpf). The plates were then placed on an infrared floor, under an infrared-sensitive camera. This system allows following the movement of the fish thanks to their pigmentation during 30 min every day, during their activity phase. Locomotor activity was recorded using ZebraLab software (Videotrack; ViewPoint Life Sciences). The recording session was preceded by a 2-hour habituation phase in the room. After recording, the fish were transferred to another well plate where the water was changed every day, and they were fed micro-worms. The plates were kept in an incubator at 24°C between recording sessions.

For 2-month-old fish, the experiments were performed on wild-type SF and heterozygote siblings only. Fish were placed individually in 12×9 cm tanks (200 ml). Tanks were placed on an infrared-emitting floor. Videos were recorded with an infrared detecting camera placed above the tanks, after 1 h of habituation. Videos were recorded and locomotion was measured with ViewPoint software. All these experiments was performed on the PSI-CO platform of our Institute (UMR 9197 CNRS NEUROPSI 91400 Saclay, France).

## Gut motility assay

The experiments were performed on 8dpf WT and KO. Larvae were anesthetized in a 140 ppm tricaine solution. After 5 minutes of habituation, we recorded movies with 1 image every second for 15 minutes under a dissection microscope (Olympus SZX 16), as in Riddle et al., 2018.

## Cardiac rhythm
The experiments were performed on 8 dpf WT, het and KO. Larvae were anesthetized in a 140 ppm tricaine solution, and the cardiac beats were counted manually under a binocular microscope. We cannot rule out that tricaine had a bradycardic effect. However, if any, the effect was the same on all fish lines. Note that the concentration of tricaine used here was low and this method was preferred to low temperature for example, which had a clear and immediate effect on cardiac frequency.

## 249 Genotyping

After carrying out the various experiments in blind, the genotype of the samples was determined. The slides were opened and the whole embryos or the dissected brains placed in 25µL of lysis buffer (10 mM Tris-HCl pH8; 50 mM KCl; 0.3% Tween20; 0.3% NP40; 4 mM EDTA). The tubes were heated at 98°C for 10 min before adding 1.25 µL of proteinase K. The tubes were heated at 55°C for 18h, then 98°C for 10 min to denature the proteinase K. 3 µL of this solution was used as template DNA to amplify the *mao* sequence by PCR by using sense (5'-GGGAAATCATATCCATTCAAGGGG-3') and antisense (5'-CTCCATGCCCCATCTTGTCCATAG-3') primers. The PCR products were then purified (NucleoSpin Gel and PCR Clean-up, Mini kit for gel extraction and PCR clean up, REF 740609.50, Macherey-Nagel) and sent to Eurofins Genomics for sequencing.

## 261 MAO enzymatic activity assay

For enzymatic activities: MAO (EC.1.4.3.4.) enzymatic activities were determined on brain homogenates as described (Elipot et al., 2014; Pierre et al., 2023) by radioenzymology using [¹⁴C]-5-hydroxytryptamine creatinine sulphate (serotonin, 5-HT, 1.96 GBq mmol⁻¹, Amersham
GE Healthcare, Saclay, France) as substrate. In brief, 20-µl aliquots of the homogenates were pre-incubated (possibly with an inhibitor) in a total volume of 220 µl at 37 °C for 30 min. At the end of the pre-incubation period, the enzyme reaction was started by addition of 80 µl of MAO substrate and the incubation continued for 15 min at 37 °C. Then, the reaction was stopped by addition of 200 µl of HCl 2N, and the deaminated metabolites were extracted by vigorous shaking for 10 min with 5 ml of diethylether. After centrifugation (1,000 g; 30 s) the water phase was frozen in dry ice and the organic layer poured into plastic vials containing 5 ml of scintillation cocktail. Finally, the radioactivity was determined in a scintillation spectrometer. Reaction mixtures as described above, but lacking the brain homogenate, served as blanks.

275

276 **Statistics**

Data shown are representative of a minimum of two independent biological replicates. Statistical analyses were performed with R studio. All data were analyzed in appropriate pairwise comparisons using non-parametric Mann-Whitney tests. For Fig. 3, Mann-Whitney pairwise comparisons were completed by a Kruskal-Wallis analysis, accounting for all comparisons of independent means across morph and genotypes (p< 0.0001).

282

283

284
285

# Results

286

287

## *A mao knockout (KO) Astyanax mexicanus* surface fish line

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

We used the CRISPR/Cas9 technology to generate a *mao* KO *Astyanax mexicanus* line. The line studied below had an 11bp deletion, creating a shift of the reading frame from the 106th amino acid of the protein. This gave rise to a STOP codon and generated a truncated protein of 127 amino acids (out of 522 amino acids total; **Fig. 1 A and B, CC'**). Accordingly, MAO enzymatic activity in 6dpf embryos *mao*⁻/⁻ KO heads was reduced down to almost zero as compared to WT (**Fig. 1D**). The morphological appearance of the KO individuals was similar to WT and het, and they could not be recognized by any simple phenotypic trait (even though they may be slightly smaller) (**Fig. 1 E**). The *mao*⁻/⁻ KO fry died abruptly at 13 days post-fecundation. Thus, all analyses below were performed between 1 and 12 dpf.

Serotonin is implicated in intestinal function, particularly in the motility of the gut (Bülbring and Crema, 1958; Gershon, 2013; Mawe and Hoffman, 2013). To check whether a deficit in intestinal function could cause the defect of KO fish and their death, we performed gut motility measurements in 8dpf embryos (i.e., 2 days after mouth opening and the beginning of independent feeding in *Astyanax*). The intestinal motility of KO fry was qualitatively indistinguishable from that of WT (**Suppl Fig. 1 A-D**).

Similarly, serotonin affects cardiac rhythm, development and function in mice (Abzalov et al., 2015; Mialet-Perez et al., 2018; Nebigil and Maroteaux, 2001; Nebigil et al., 2000; Stoyek et al., 2017). Yet, the KO fry had the same cardiac rhythm at 8dpf as the WT or heterozygotes
306 (Suppl Fig.1 E). These data suggest that *mao* KO fry die for reasons that are not related to
307 intestinal motility or cardiac beating defects.

308

309 Monoaminergic systems in *mao*⁻/⁻ KO embryos and larvae

310 As the MAO enzyme catalyzes the oxidative deamination of monoamine neurotransmitters,
311 we first assessed the developmental anatomy of dopamine, noradrenalin and serotonin
312 neurons in *mao* KO brains.

313 We first analyzed the effect of the loss of MAO function on the DA and NA system at 6 dpf by
314 immunofluorescence staining against tyrosine hydroxylase (TH), their biosynthesis rate-
315 limiting enzyme. The organization and the number of DA neurons (corresponding to all
316 neuronal clusters in Figure 2 except LC) and NA neurons (LC, locus coeruleus) did not differ in
317 KO as compared to WT and het larvae (Fig. 2A-C). The staining intensity for TH in neurons was
318 not different either in brains from the three genotypes.

319 We next analyzed the serotonergic system by immunostaining against 5-HT. Different spatio-
320 temporal effects of MAO loss of function were observed, in the hypothalamus and in the raphe
321 5-HT neuronal clusters, between 36 hpf and 12 dpf.

322 At all stages, WT and heterozygotes fish showed similar 5-HT labeling and were together
323 assimilated as controls. Early in development at 36 hpf, 5-HT labeling was stronger in KO than
324 in controls, both in the hypothalamus (note that only the anterior paraventricular nucleus, or
325 PVa, is observed at this stage) and in the raphe nucleus (Fig. 3 A, B). Then, labeling intensity
326 progressively decreased in the hypothalamic region in KO individuals, reaching its lowest level
327 at 6 dpf (Fig. 3 F, H, J). This progressive decrease in labeling involved the anterior regions of
328 the hypothalamus, the anterior paraventricular nucleus (PVa) and the intermediate nucleus (PVi) between 4 and 6 dpf, and also affected the posterior nucleus (PVp) at 6 dpf (Fig. 3 F, H, J).

331 At 8 dpf, in *mao* KO brains there was a progressive reappearance of 5-HT immunolabeling in 332 PVp and in PVi (Fig. 3 L), and labeling subsequently returned to normal in the 3 hypothalamic 333 5-HT clusters at 10 and 12 dpf as compared to controls (Fig. 3N, P). Thus, the pattern of 5-HT 334 immunoreactivity in the hypothalamic region seems highly dynamic in *mao* KO, with spatial 335 and temporal variations during development. In the raphe however, at all stages studied, 336 5-HT neurons remained much more strongly labelled in KO than in control fry (Fig. 3A-P). Overall, 337 these data show that serotonergic neurons in the hypothalamus show finely tuned and 338 dynamic regulation of serotonin levels across developmental stages upon loss of MAO 339 function, in contrast to the raphe nucleus where serotonin levels are constantly heightened 340 (Fig. 3 Q).

341 Finally, we have previously found that upon partial loss of MAO function due to the *mao* P106L 342 mutation, the increase in brain 5-HT levels caused by lack of degradation through 343 malfunctioning MAO is partially compensated for by a decrease in the activity of TPH 344 (tryptophane hydroxylase), the 5-HT biosynthetic enzyme (Pierre et al., 2023). Moreover, in 345 surface fish this compensation is observed in the hypothalamus but not in the raphe (Pierre et 346 al., 2023). Thus, we sought to test the possibility of a differential regulation of TPH in the 347 hypothalamus and in the raphe in the context of *mao* KO. In fish, there are two different *tph* 348 genes, *tph1a* and *tph2*, specifically expressed in the hypothalamus and raphe, respectively. 349 We performed *in situ* hybridizations for *tph1a* and *tph2* at 36 hpf, with the aim of determining 350 whether there are variations in *tph* expression related to the *mao* KO genotype (Suppl Fig. 2
G, H). *Astyanax tph1a* was expressed in two discrete bilaterally symmetrical areas in the ventral forebrain, corresponding to the developing hypothalamus, in the position of the PVa nucleus. The localization, the intensity and the number of labeled cells did not vary according to the different *mao* genotypes at 36 hpf (Suppl Fig. 2 G). *Tph2* was expressed more posteriorly than *tph1a*, in two discrete areas in the hindbrain at the level of the raphe nucleus. Again, no difference was observed between *mao* genotypes at 36 hpf (Suppl Fig. 2 H).

357

## 358 Locomotion in *mao*⁻/⁻ KO embryos and larvae

Serotonin and MAO have demonstrated roles in the control of locomotor activity in 6 dpf zebrafish larvae (Sallinen et al., 2009; Baronio et al., 2022). As 5-HT content in neurons followed important variations as a function of age in *Astyanax mao* KO fry (Fig. 3Q), we recorded the locomotion 30 min per day, at the time of their activity phase, throughout the available developmental time between 6 and 12 dpf, to gain insight into the evolution of locomotion as a function of development. Of note, we initially wished to carry out this experiment on the whole duration of the developmental period studied above, however before 6 days *Astyanax* larvae were not sufficiently large and pigmented for proper tracking in the Zebrabox system. Moreover, before this stage the larvae had a rather limited locomotion, independently of their genotype.

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

The distances traveled by WT and het fry were identical at all developmental stages recorded. Between 7 and 10 dpf, the KO fish swam significantly less than WT and het, covering about 100 cm during the 30 min of recording, compared to about 250 cm for the controls over the same period (Fig. 4 A, B, C). The comparison of trajectories illustrates well the differences in locomotion between the different genotypes (Fig.4 B, C). At 11 and 12 dpf however, the
locomotion of *mao* KO was not different from controls anymore (**Fig. 4 A**). We also analyzed locomotion in juveniles and compared the available het and WT fish. At 2mpf, the het covered a longer distance in 10 minutes than the WT (**Fig. 4 D**). In sum, MAO inactivation affected locomotion in a stage-dependent manner in *Astyanax*.

Finally, as the double MAO-A/MAO-B KO mice are very aggressive (Chen et al., 2004), we analyzed the aggressiveness in 3 month old fish, using a resident-intruder assay. There was no difference between WT and het in the number of attacks performed in one hour between two fish of the same genotype (**Fig. 4 E**).

382

### 383 Proliferation of brain progenitors

The distribution of serotonergic neuronal nuclei and their fibers overlap with highly proliferative areas of fish brain (Grandel et al., 2006). Moreover, studies on zebrafish have shown that the inhibition of serotonin synthesis modifies the proliferation pattern in the periventricular hypothalamic regions (Perez et al., 2013). Thus, we hypothesized that *mao* KO and associated neuronal 5-HT level variations may influence the proliferation or, more generally, the biology of brain progenitors.

Fluorescent immunostaining against Phospho-histone H3 (PH3), which labels all cells in M phase of the cell cycle, was performed to measure the amount of proliferation in the brain. In 36 hpf and 48 hpf embryos, the number of proliferating cells did not differ between the three genotypes (WT, het, KO), whether the analysis was performed globally in the brain or focused on specific regions (telencephalon and hypothalamus) (Suppl Fig. 2 A-C).
At later larval stages (6dpf, 8 dpf and 10 dpf), we next monitored the number of PH3-positive cells in the telencephalon, a richly proliferative region receiving neuronal projections from the raphe, and in the hypothalamus (which contains 3 groups of serotoninergic neurons). The proliferative PH3-positive cells were present in the ventricular zone of the telencephalon (Fig. 5A-C) and in a large and continuous proliferation zone lining the periventricular hypothalamus (Fig. 6A-C). At 6dpf, the number of PH3 cells was identical in KO and WT larvae, both in the telencephalon (Fig.5A-C) and in the hypothalamus (Fig. 6A-C). At 8dpf, in KO we observed a reduction of the number of proliferative cells in the telencephalon (Fig.5A-C) and in the hypothalamus (Fig. 6A-C) as compared to the WT. At 10 dpf, KO fry showed a dramatic reduction of proliferative cells in both the telencephalon (Fig. 5 A – C) and hypothalamus (Fig. 6 A-C). In fact, at this stage the number of proliferative cells in both regions was almost zero.
These results suggested a potential link between MAO and brain proliferation in *Astyanax* fry.

Next, we reasoned that the reduction of the number of cells in M phase (PH3-positive) in the 8-10 dpf KO brains might be due either to a longer cell cycle length, or to fewer cells entering the cell cycle, or both. To discriminate between these possibilities, we performed an experiment in which 8 dpf larvae were incubated for 1 hour with EdU (which incorporates in cells during the S phase of the cell cycle), followed by a chase of 6 hours. We estimated that within 6 hours the cells in S phase should have progressed in the cell cycle to reach the M phase. In addition to the EdU marker, we used a general proliferation marker (PCNA) to label all the cells in proliferative state as well as a specific marker to label the cells in M phase (PH3).

In *mao* KO, we observed a drastic reduction of the number of cells labelled for all three markers of proliferation: EdU (S phase), PH3 (M Phase) and PCNA (entire cell cycle), both in the telencephalon (Fig.7 A-B) and in the hypothalamus (Fig.7 D-E). These results suggest a
418

strong overall decrease in brain proliferation in *mao* KO larvae, including very few cell entering the cell cycle.

419

420

Then, to assess a potential difference in cell cycle length, we calculated how many cells that have incorporated the EdU 6 hours before fixation of the larvae, have reached the M phase.

421

422

In the telencephalon, there was a strong tendency, albeit non-significant, towards a reduction of this number in the KO fry as compared to WT and het (Fig. 7 C). In the hypothalamus, the percentage of cells in M phase (PH3+) 6 hours after they were in S phase (EdU+) was significantly decreased in KO compared to WT and het (Fig.7 F). These data suggest that cell cycle length is also affected and is longer in *mao* KO larvae.

423

424

425

426

427

428

Cell death in the brain of *mao*⁻/⁻ embryos

429

Finally, in parallel to an effect of loss of MAO function on proliferation, we tested a potential effect of MAO on apoptosis, which we assessed through activated caspase 3 immunofluorescence. In 36 hpf embryos, we observed no overall difference in cell death between WT, het and KO brains (Suppl Fig. 2 D). The same was true at 48 hpf (Suppl Fig. 2 E).

430

431

432

433

Looking more specifically at the different forebrain regions, the telencephalon and the hypothalamus, there was also no difference between the three genotypes, neither at 36 hpf nor at 48 hpf (Suppl Fig. 2F). These results suggest that the loss of MAO has no effect on cell death in the brain of *A. mexicanus* surface fish embryos at these stages.

434

435

436

437
438

439

Discussion

440

441

Here, we have investigated the developmental outcomes of loss of MAO function in embryos and larvae of the fish *Astyanax mexicanus*. Although the effects on larval locomotion could have been anticipated (see Baronio et al., 2021), those we discovered on the fine regulation of serotonin content in different brain neuronal clusters on one hand, and on the control of brain progenitor proliferation on the other hand, were unexpected and open novel research opportunities.

447

448

*mao* knockout is lethal in fish

449

450

*Astyanax* individuals that are KO for *mao* die abruptly at larval stage, between 13 dpf and 15 dpf. This observation is similar to what was reported for zebrafish *mao* KO, i.e., death within 20 days post-fertilization (Baronio et al., 2021). MAO KO mice (*MAO-A*, *MAO-B*, or double *MAO-A/B* knockouts) do not die but display several abnormalities. MAO-A KO mice show elevated levels of 5-HT and hyperactivation of 5-HT1B receptor which induce neurodevelopmental alterations (Cases et al., 1995; Vitalis et al., 1998; Salichon et al., 2001). Further, MAO-A KO mice display elevated aggressiveness (Cases et al., 1995; Scott et al., 2008) and reduction in exploratory activity (Godar et al., 2011). MAO-B KO mice show increased novelty-seeking and reduced anxiety (Bortolato et al., 2009). Double MAO-A/B knockouts exhibit aberrant emotional response to novelty, high levels of anxiety-like behaviors and low latency to attack in the resident-intruder paradigm (Chen et al., 2004). All these adult phenotypes cannot be assessed in *mao* KO fish which die prematurely.

451

452

453

454

455

456

457

458

459

460

461
Although serotonin is implicated in the control of intestinal function and cardiac rhythm, we did not find any evidence for an involvement of these processes in the lethality of the loss of MAO function in our model. Death might be due to multiple dysfunctions, some unknown and some related to the major brain homeostasis defects that we have uncovered and that are discussed below.

467

468

mao knockout affects differentially the fish serotonergic and dopaminergic systems

469

In zebrafish, pharmacological experiments had suggested that 5-HT is the preferred substrate for MAO, and that DA and NA would be poor substrates for the enzyme (Sallinen 2009; Anichtnikov 2006). In *Astyanax*, direct measurements of the affinity of MAO for 5-HT and DA further demonstrated a ~30-fold difference, the Km for 5-HT being in the order of 12µM and the Km for DA being ~370 µM (Pierre et al., 2023). Thus, the affinity of the single fish MAO enzyme for its preferred substrate 5-HT is similar to the affinities of MAO-A for 5-HT and NA, and of MAO-B for PEA and DA, respectively, in mammals (Bortolato et al., 2011; Peters et al., 2011; Darryle et al., 1981). The lower affinity of the fish MAO for DA and NA poses the unsolved question of the catabolic pathway that is used in fish to degrade these catecholamines. Furthermore, based on these features, one can anticipate or predict that the effects of MAO perturbation in fish should be stronger on the serotonergic than on the dopaminergic and noradrenergic systems.

482

Accordingly, a complete loss of MAO enzyme activity after *mao* knockout in fish is expected to give rise to a dramatic increase in the amount of serotonin levels in the brain. This is what we consistently observed in the raphe nucleus throughout the developmental period where
we had access to homozygote knockout specimens, and using the intensity of 5-HT immunoreactivity as a proxy of neurotransmitter content. However and surprisingly, in contrast to those in the raphe, the serotonergic neurons of the hypothalamus showed very fine and dynamic serotonin level regulation upon loss of MAO function. While at 36hpf hypothalamic 5-HT neurons contained more serotonin in KO than in WT (consistently with expectations), at 48hpf the difference disappeared. Further, between 4dpf and 8dpf a drastic and unexpected reduction of 5-HT levels occurred in the three serotonergic nuclei composing the PVO in KO fish. In addition and surprisingly, starting at 8dpf, the serotonin signal re-increased to reach control levels at 12dpf. Of note, Baronio et al. (2021) described a decrease in the number of hypothalamic serotonergic neurons in 10dpf zebrafish in *mao* knockouts. Our detailed time-course analysis rather suggests that 5-HT neurons are transiently not detectable anymore because their neurotransmitter content is extremely low, but that they are still present, as demonstrated by their "re-appearance" a few hours later. Our results further suggest that loss of MAO function does not affect the development and the number of 5-HT neurons in the hypothalamus, but that it strongly affects the serotonergic content and signaling pathway. The highlighting, thanks to the *mao* KO line, of such fine regulation strongly suggests that the homeostasis of 5-HT signaling in the hypothalamus is crucial for proper brain and behavior development. The mechanisms underlying the rapid fluctuation in 5-HT content in the hypothalamus (but not the raphe) are unknown and can only be discussed.

The hypothesis that 5-HT is able to regulate its own biosynthetic pathway by negative feedback on the expression or the activity of the TPH enzyme would explain the observed wave profile of hypothalamic 5-HT immunoreactivity. As soon as serotonin reaches higher levels than the "control" level (at 36 hpf), a negative feedback loop would start functioning
and the 5-HT content would rapidly return to "normal" (here at 48 hpf). The inertia of this regulation could lead to a continuation of the decrease in 5-HT level until 6 dpf, the stage at which 5-HT labelling is the lowest in the three hypothalamic nuclei. Finally, from 8 days onwards, the very low level of 5-HT in neurons would stop the negative feedback, allowing the production of serotonin again and explaining the progressive re-increase in 5-HT staining observed between 8 and 12 dpf. This hypothesis can be implemented at two main levels: either at the level of regulation of the transcription of the enzyme, or at the level of its activity.

Regarding transcriptional control, here we have some preliminary evidence that *tph* expression is unchanged in *mao* KO, at least at 36hpf, a stage when the expression of *tph1a* would be expected to decrease in response to elevated levels of 5-HT. Later stages should be examined as well. Regarding enzymatic activity control, we have not attempted yet to assay TPH activity due to technical limitations on the amount of tissue and samples needed to measure enzymatic activity on tiny parts of embryonic brains. However, we favor this hypothesis of a feedback loop controlling MAO enzymatic activity as a function of 5-HT neuronal levels, because we have recently demonstrated the existence of this mechanism at work in adult *Astyanax* brains carrying the partial loss-of-function *mao* P106L mutation (Pierre *et al.*, 2023).

Finally, the striking difference in serotonergic regulation in the hypothalamus and in the raphe nucleus after *mao* knockout adds to the many differences already described between 5-HT neurons in these two regions of the fish brain – at the difference of mammalian brain where 5-HT neurons are exclusively located in the raphe. At all levels - specification, metabolism, regulation - raphe and hypothalamic 5-HT neurons appear distinct (Lillesaar, 2011). Their development requires the function of different ETS-domain transcription factors: *pet1* in the
hindbrain, etv5b and fezf2 in the forebrain (Bosco et al., 2013; Rink and Guo, 2004; Berberoglu et al., 2009). They express different tph enzymes and serotonin transporter (SERT): tph2 and SERTA in the raphe, tph1a and SERTB in the hypothalamic clusters (Herculano and Maximino, 2014; this paper; Norton et al., 2008; Wang et al., 2006; Wen et al., 2008; Yamamoto et al., 2011). Finally, they respond differently to metabolic or genetic perturbations (Pierre et al., 2023; this paper). In sum, 5-HT neurons of the hypothalamus and the raphe, although they all use serotonin as a neurotransmitter, are distinct in identity (and probably in function). They highlight the idea of convergent mechanisms leading to monoaminergic specification and differentiation in the fish brain.

The dopaminergic system on the other hand was unchanged between the *mao* KO and WT larvae at 6dpf, neither in the organization of neuronal clusters nor in the intensity of TH immunostaining. The same holds true for noradrenergic neurons. This is in line with the idea discussed above of DA and NA being poor substrates for fish MAO. In zebrafish too, a reduction in the number of Th1-immunoreactive cells in only one DA cluster was reported in *mao* knockouts, without overall difference in *th1* transcript levels in whole larvae at 10 dpf (Baronio et al., 2021). Although the assessment of 5-HT itself (i.e., the neurotransmitter) to study serotonin is not equivalent to the assessment of TH (i.e., the biosynthetic enzyme) immunoreactivity or expression to study DA and NA, the TH "tool" was used to investigate catecholamines in both species, zebrafish (Baronio et al., 2021) and Astyanax (this paper) because immunohistochemistry against DA is technically challenging. Nevertheless, the definitive proof for an absence (or not) of major change in DA or NA signaling after *mao* knockout in fish will need further investigation, using HPLC for example.
558 *mao* knockout and locomotion in larval fish

559

560 In zebrafish, modulation of the serotonergic system (pharmacological treatment with
561 serotonin, serotonin agonists, serotonin reuptake or synthesis inhibitors) from the fourth day
562 of development onwards induces changes in locomotion (Airhart et al., 2007; Airhart et al.,
563 2012; Brustein and Drapeau, 2005; Brustein et al., 2003a; Brustein et al., 2003b; Drapeau et
564 al., 2002; Sallinen et al., 2009). Moreover, zebrafish *mao* knockouts are hypoactive at 6dpf
565 (Baronio et al., 2021). In *Astyanax* too, we found that *mao* KO individuals swim shorter
566 distances than controls between 7 and 10 dpf. However, our time-course analysis revealed
567 that at 11 and 12 dpf locomotor activity was back to values similar to controls. Strikingly, this
568 variation is parallel to the changes we observed for serotonin levels in the hypothalamus, with
569 a slight delay (Fig. 8). Indeed, the stages at which serotonin levels are lowest in PVO are
570 between 5 and 8 dpf, and then they increase again at 10 and 12 dpf. It is therefore tempting
571 to speculate that the two phenomena are related. Furthermore, if the causal relationship
572 between hypothalamic 5-HT levels and locomotor activity were true, it would mean that the
573 locomotion in fish larvae is controlled by the serotonergic neurons in the hypothalamus, not
574 those of the raphe.

575

576 We also performed locomotion assays in 2 months old juveniles, and observed hyperactivity
577 of the heterozygotes compared to the WT. These results are different from those obtained in
578 adult zebrafish *mao* knockouts, where no difference was found (Baronio et al., 2021). In adult
579 fish, treatment with a serotonin reuptake inhibitor (citalopram, fluoxetine) or directly with
580 serotonin shows an inhibitory effect of serotonin on locomotion (Fingerman, 1976; Gabriel et
581 al., 2009; Perreault et al., 2003; Winberg et al., 1993). Other studies, however, show no effect
582 of serotonin on locomotion (Winberg and Thörnqvist, 2016), or even an activating effect
583 (Clements et al., 2003). In mammals as well, the effects of 5-HT signaling on locomotion are
584 variable among studies. For example, treatment with a serotonin reuptake inhibitor
585 (citalopram) reduces locomotion in neonatal mice (Dunbar et al., 2010). On the contrary, the
586 double MAO-A/B KO mice are hyperactive (Chen et al., 2004). Thus, the contribution of
587 serotonin in the control of locomotion appears complex, and probably depends on both the
588 developmental stage (adult, juvenile, larva) and the model studied. The effect of serotonin in
589 controlling locomotion is probably also modulated by other factors.

590

591 *mao knockout affects neural progenitor proliferation and cell cycle kinetics*

592

593 Three pieces of evidence prompted us to investigate a potential influence of MAO function on
594 embryonic and larval brain proliferation: (1) the anatomical correlation between the 5-HT
595 clusters and their projections with highly proliferative areas of fish brain (Grandel et al., 2006);
596 (2) a pioneer study suggesting a functional link between 5-HT signaling and proliferation in the
597 hypothalamic ventricular region (Perez et al., 2013); (3) the recent report that in the
598 embryonic ferret and fetal human neocortex, 5-HT stimulates the proliferation of basal
599 progenitors, and notably of radial glial cells, through HTR2A receptor signaling (Xing et al.,
600 2020). Thus, in the *Astyanax mao* KO model, we hypothesized that MAO loss-of-function and
601 associated neuronal 5-HT level variations may influence the proliferation or, more generally,
602 the biology of brain progenitors.

603

604 In line with our hypothesis, we discovered a dramatic loss in the number of proliferating cells
605 in both the hypothalamus and the telencephalon, associated to an increase in cell cycle length
606 (hence a slowdown of the cell cycle) in *mao* knockout larvae, from 8dpf onwards. We do not  
607 know whether these two effects are linked or independent. The *mao* KO larvae have fewer  
608 EdU-positive cells that reach the M phase, which suggests that cells remain longer in G2 phase  
609 and therefore enter mitosis less quickly. We can only speculate that slowing down of the cell  
610 cycle progression may in part contribute to a decrease in the re-entry of progenitors into the  
611 cell cycle.

612
613 Importantly, our results demonstrate that MAO function on monoamine homeostasis controls 614 larval fish brain proliferation. This process is probably mediated by 5-HT signaling (see above). 615
616 As for locomotion, the deregulation of serotonin levels and proliferation dynamics are slightly 617 shifted in time but parallel (Fig. 8). Thus, there would be a delay between 5-HT signaling and 618 its influence on proliferative cells. The proliferative phenotype observed from 10 dpf in mao 619 KO larvae would therefore be the result of the decrease in hypothalamic serotonin level 620 observed at 6 and 8 dpf. This result is consistent with the idea that 5-HT signaling stimulates 621 progenitor proliferation in the hypothalamus, as proposed in zebrafish, in which acute 622 pharmacological inhibition of serotonin production induces a decrease in proliferation in the 623 adult PVO (Pérez et al., 2013).

624
625 The results we observed in the telencephalon are more surprising. Indeed, our experiments  
626 show a reduction in the number of proliferating cells in the telencephalon that is comparable  
627 to that observed in the hypothalamus. Yet, 5-HT signaling in the telencephalon mostly  
628 originates from the raphe (Lillesaar, 2011), a cluster that shows very high 5-HT  
629 immunoreactivity in the *mao* KO throughout development. Could drastic 5-HT deregulation,  
630 whether positive or negative, induce the same type of phenotype? These seemingly
630 inconsistent results could be more easily explained by the specific expression of distinct
631 serotonin receptors in the telencephalon and hypothalamus, which would have an opposite
632 effect on proliferation.

633
634 The serotonin receptors HTR2A that stimulates the proliferation of radial glia in the ferret and
635 human embryonic cortex is not expressed in the lissencephalic mouse embryonic neocortex;
636 consequently, 5-HT does not modulate mouse basal progenitor proliferation (Xing et al, 2020).

637 This study illustrates an evolutionarily relevant function of 5-HT in the control of brain
638 proliferation. This interspecies difference seems to be related to a different mode of
639 progenitor division. In mice, basal progenitors undergo consumptive division to give rise to
640 two neurons. In humans, basal radial glia are more abundant and capable of giving rise to
641 more basal progenitors. In the zebrafish telencephalon, the mode of division of radial glia
642 progenitors is more similar to that of humans than to that of mice (Than-Trong et al, 2018).

643 Thus, from an evolutionary point of view, it will be very interesting to explore the effect of
644 MAO and serotonin on progenitor and stem cell biology in fish. There are 23 serotonin
645 receptors in the zebrafish genome, and their specific spatiotemporal expression patterns
646 description is fragmentary (Zfin database). It will be important to determine exactly where
647 these receptors are expressed and how they are involved in the control of proliferation in fish
648 brains.

649
650 **Conclusion**
651

651
Altogether, our study shows that the loss of MAO function strongly affects the homeostasis of serotonergic neurons, the locomotor activity and the proliferation of brain progenitors in *Astyanax* surface fish larvae (Fig. 8). The lethality of *mao* knockout at larval stages in the two fish species where it has been achieved so far (zebrafish and *Astyanax*) renders impossible the study of older animals and the further deciphering of MAO functions in mature brains.

Interestingly, some populations of cavefish morphs of the *Astyanax* species carry a natural, partial loss-of-function mutation in *mao* that should allow investigating, probably with more subtle and physiologically-relevant effects than the complete gene inactivation, the outcomes of compromised MAO function on neural progenitors homeostasis as well as their consequences on behavior.

662

## 663 Authors' contributions

Conceptualization: AA, SR; Methodology: AA, CP, JT-P, NC, AL, MP, TE-P, ML, MB.

Writing - original draft: AA, SR; Funding acquisition: SR.

666

## 667 Acknowledgements

We thank Frederic Sohm (UMS Amagen) for help in CRISPR-Cas9 design and founder screening and genotyping. Our *Astyanax* colony has benefited from the zootechnical services of the TEFOR unit.

Work supported by Equipe FRM (Fondation pour la Recherche Médicale, grants DEQ20150331745 and EQU202003010144) and IDEEV (Institut Diversité Evolution Ecologie du Vivant).

672
673 **Competing interests**

674 The authors declare that they have no competing interests.
675 References :

676 Abzalov, R. A., Valeev, A. M., Abzalov, N. I. and Gulyakov, A. A. (2015). Role of 5-HT2B serotonin
677 receptor agonist in the regulation of pumping function of the heart. Bull. Exp. Biol. Med. 159, 302–304.

678 Airhart, M. J., Lee, D. H., Wilson, T. D., Miller, B. E., Miller, M. N. and Skalko, R. G. (2007). Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC).
679 Neurotoxicol. Teratol. 29, 652–664.
680

681 Airhart, M. J., Lee, D. H., Wilson, T. D., Miller, B. E., Miller, M. N., Skalko, R. G. and Monaco, P. J. (2012).
682 Adverse effects of serotonin depletion in developing zebrafish. Neurotoxicol. Teratol. 34, 152–160.

683 Anichtchik, O., Sallinen, V., Peitsaro, N., Panula, P. (2006). Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio). J Comp Neurol. 498(5):593-610.

685 Baronio, D, Chen, Y.-C., Panula, P., (2022). Abnormal brain development of monoamine oxidase mutant poisson zèbre and impaired social interaction of heterozygous fish. Dis. Model. Mech. dmm.049133. https://doi.org/10.1242/dmm.049133

688 Berberoglu, M.A., Dong, Z., Mueller, T., Guo, S. (2009). fezf2 expression delineates cells with proliferative potential and expressing markers of neural stem cells in the adult zebrafish brain. Gene Expr Patterns 9(6):411-22.

691 Bortolato, M., Godar, S.C., Davarian, S., Chen, K., and Shih, J.C. (2009). Behavioral disinhibition and reduced anxiety-like behaviors in monoamine oxidase B-deficient mice. Neuropsychopharmacology 34(13), 2746-2757.

694 Bortolato, M., Shi, JC. (2011). Behavioral outcomes of monoamine oxidase deficiency: preclinical and clinical evidence. International review of neurobiology 100, 13-42.

696 Bosco, A., Bureau, C., Affaticati, P., Gaspar, P., Bally-Cuif, L., Lillesaar, C. (2013). Development of hypothalamic serotoninergic neurons requires Fgf signalling via the ETS-domain transcription factor Etv5b. Development 140(2):372-84.

699 Boutet, I., Tanguy, A., and Moraga, D. (2004). Molecular identification and expression of two nonP450 enzymes, monoamine oxidase A and flavin-containing monooxygenase 2, involved in phase I of xenobiotic biotransformation in the Pacific oyster, Crassostrea gigas. Biochim. Biophys. Acta 1679, 29-36.

703 Brunner, H.G., Nelen, M., Breakefield, X.O., Ropers, H.H., van Oost, B.A. (1993). Abnormal behavior associated with a point mutation in the structural gene for MAOA. Science 262(5133):578-80.

705 Brustein, E. and Drapeau, P. (2005). Serotoninergic modulation of chloride homeostasis during maturation of the locomotor network in Zebrafish. J. Neurosci. 25, 10607-10616.

707 Brustein, E., Saint-Amant, L., Buss, R. R., Chong, M., McDearmid, J. R. and Drapeau, P. (2003a). Steps during the development of the zebrafish locomotor network. J. Physiol. Paris 97, 77-86.

709 Brustein, E., Chong, M., Holmqvist, B. and Drapeau, P. (2003b). Serotonin Patterns Locomotor Network Activity in the Developing Zebrafish by Modulating Quiescent Periods. J. Neurobiol. 57, 303-322.

711 Bülbring, E. and Crema, A. (1958). Observations concerning the action of 5-Hydroxytryptamine on the peristaltic reflex. Br. J. Pharmacol. Chemother. 13, 444-457.

713 Cases, O., Seif, I., Grimsby, J., Gaspar, P., Chen, K., Pournin, S., Muller, U., Aguet, M., Babinet, C., and Shih, J.C. (1995). Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA. Science 268, 1763-1766.
716 **Chen, K.**, Wu, H.F., Grimsby, J., and Shih, J.C. (1994). Cloning of a novel monoamine oxidase cDNA from trout liver. Mol. Pharmacol. 46, 1226-1233.

718 **Chen, K.**, Holschneider, D.P., Wu, W., Rebrin, I., and Shih, J.C. (2004). A spontaneous point mutation produces monoamine oxidase A/B knock-out mice with greatly elevated monoamines and anxiety-like behavior. J. Biol. Chem. 279, 39645-39652.

721 **Clements, S.**, Moore, F. L. and Schreck, C. B. (2003). Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha). Horm. Behav. 43, 214-221.

724 **Darryle, D.**, Schoepp and Albert J. Azzaro (1981). Specificity of Endogenous Substrates for Types A and B Monoamine Oxidase in Rat Striatum. Journal of Neurochemistry 36(6):2025-2031.

726 **Drapeau, P.**, Saint-Amant, L., Buss, R. R., Chong, M., McDearmid, J. R. and Brustein, E. (2002). Development of the locomotor network in zebrafish. Prog. Neurobiol. 68, 85-111.

728 **Dunbar, M. J.**, Tran, M. A. and Whelan, P. J. (2010). Endogenous extracellular serotonin modulates the spinal locomotor network of the neonatal mouse. J. Physiol. 588, 139-156.

730 **Elipot, Y.**, Hinaux, H., Callebert, J., Launay, J.M., Blin, M., Rétaux, S. (2014). A Mutation in the Enzyme Monoamine Oxidase Explains Part of the Astyanax Cavefish Behavioral Syndrome. Nature Communications, 10; 5:3647.

733 **Elliott, W. R.** (2018). The Astyanax caves of Mexico. Cavefishes of Tamaulipas, San Luis Potosi, and Guerrero. Assoc. Mex. cave Stud. Bulletin 2.

735 **Fingerman, S. W.** (1976). Circadian rhythms of brain 5-hydroxytryptamine and swimming activity in the teleost, Fundulus grandis. Comp. Biochem. Physiol. Part C, Comp. 54, 49-53.

737 **Fumey, J.**, Hinaux, H., Noirot, C., Thermes, C., Rétaux, S. and Casane, D. (2018). Evidence for late Pleistocene origin of Astyanax mexicanus cavefish. BMC Evol. Biol. 18, 1-19.

739 **Gabriel, J. P.**, Mahmood, R., Kyriakatos, A., Söll, I., Hauptmann, G., Calabrese, R. L. and El Manira, A. (2009). Serotonergic modulation of locomotion in zebrafish - Endogenous release and synaptic mechanisms. J. Neurosci. 29, 10387-10395.

742 **Gershon, M. D.** (2013). 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Curr. Opin. Endocrinol. Diabetes Obes. 20, 14-21.

744 **Godar, S.C.**, Bortolato, M., Frau, R., Dousti, M., Chen, K., and Shih, J.C. (2011). Maladaptive defensive behaviours in monoamine oxidase A-deficient mice. Int. J. Neuropsychopharmacol. 14(9):1195-207.

746 **Grandel, H.**, Kaslin, J., Ganz, J., Wenzel, I., Brand, M. (2006). Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Biol.295(1):263-77.

748 **Gross, J. B.**, Boggs, T. E., Rétaux, S. and Torres-Paz, J. (2023). Developmental and genetic basis of troglomorphic traits in the teleost fish Astyanax mexicanus. Chapter 11, The Groundwater Ecology and Evolution Book, 2023. Editors: Florian Malard, Christian Griebler, Sylvie Rétaux. Elsevier

751 **Hendricks, T.**, Francis, N., Fyodorov, D. and Deneris, E. S. (1999). The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes. J. Neurosci. 19, 10348-10356.

754 **Herculano, A.M.**, Maximino, C. (2014). Serotonergic modulation of zebrafish behavior: towards a paradox. Prog Neuropsychopharmacol Biol Psychiatry. 55, 50-66.

756 **Jailon, O** et al. (2004). Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 21; 431 (7011): 946-957.
758 **Keene, A. C.**, Yoshizawa, M. and McGaugh, S. E. (2016). Editors. *Biology and evolution of the Mexican cavefish*. Academic Press, San Diego. Elsevier.

760

761 **Lillesaar, C.**, Tannhäuser, B., Stigloher, C., Kremmer, E. and Bally-Cuif, L. (2007). The serotonergic phenotype is acquired by converging genetic mechanisms within the zebrafish central nervous system. *Dev. Dyn.* 236, 1072-1084.

764 **Lillesaar, C.** (2011). The serotonergic system in fish. *J Chem Neuroanat.* 41(4):294-308.

765 **Liu, C.**, Maejima, T., Wyler, S. C., Casadesus, G., Herlitze, S. and Deneris, E. S. (2010). Pet-1 is required across different stages of life to regulate serotonergic function. *Nat. Neurosci.* 13, 1190-1198.

767 **Mawe, G. M.** and Hoffman, J. M. (2013). Serotonin signalling in the gut-functions, dysfunctions and therapeutic targets. *Nat. Rev. Gastroenterol. Hepatol.* 10, 473-486.

769 **McLean, D.L.**, Fetcho, J.R. (2004). Ontogeny and innervation patterns of dopaminergic, noradrenergic, and serotonergic neurons in larval zebrafish. *J Comp Neurol.* 480(1):38-56.

771 **Mialet-Perez, J.**, Santin, Y. and Parini, A. (2018). Monoamine oxidase-A, serotonin and norepinephrine: synergistic players in cardiac physiology and pathology. *J. Neural Transm.* 125, 1627-1634.

773 **Mitchell, R.**, Russell, W. and Elliott, W. (1977). Mexican eyeless characin fishes, genus Astyanax: environment, distribution, and evolution. *Spec. Publ. Mus. Texas Tech. Univ.* 12, 1-89.

775 **Nebigil, C. G.** and Maroteaux, L. (2001). A novel role for serotonin in heart. *Trends Cardiovasc. Med.* 11, 329-335.

777 **Nebigil, C. G.**, Choi, D. S., Dierich, A., Hickel, P., Le Meur, M., Messaddeq, N., Launay, J. M. and Maroteaux, L. (2000). Serotonin 2B receptor is required for heart development. *Proc. Natl. Acad. Sci. U. S. A.* 97, 9508-9513.

780 **Norton, W. H.**, Folchert, A. and Bally-Cuif, L. (2008). Comparative analysis of serotonin receptor (HTR1A/HTR1B families) and transporter (slc6a4a/b) gene expression in the zebrafish brain. *J. Comp. Neurol.* 511, 521-542.

783 **Perez, MR.**, Pellegrini, E., Cano-Nicolau, J., Guegeun, MM., Menouer-LeGuillou, D., Merot, Y., Vaillant, C., Somoza, MG., Kah, O. (2013). Relationships between radial glial progenitors and 5-HT neurons in the paraventricular organ of adult zebrafish- potential effects of serotonin on adult neurogenesis. *Dev. Neuroscience* 38, 3292-3301.

787 **Perreault, H. A. N.**, Semsar, K. and Godwin, J. (2003). Fluoxetine treatment decreases territorial aggression in a coral reef fish. *Physiol. Behav.* 79, 719-724.

789 **Peters, TMA.**, Lammerts van Bauren, I., Geurtz, BPBH., Coene, KLM., de Leeuw, N., Brunner, HG., Jonsson, JJ., Willemse, MAAP., Wevers, RA., Verbeek, MM. (2021). Monoamine oxidase A activity in fibroblasts as a functional confirmation of MAOA variants. *JIMD Reports.* 2021;58:114-121.

792 **Pierre, C.**, Callebert, J., Launay, JM., Retaux, S. (2023). The enzymatic and neurochemical outcomes of a mutation in Mexican cavefish MAO reveal teleost-specific aspects of brain monoamine homeostasis. *J Exp Biol.* Jul 1;226(14):jeb245448. doi: 10.1242/jeb.245448. Epub 2023 Jul 28. PMID: 37309675.

795 **Piton, A.**, Poquet, H., Redin C., Masurel, A., Lauer, J., Muller, J., Thevenon, J., Herenger, Y., Chancenotte, S., Bonnet, M., Pinoit, J.M., Huet, F., Thauvin-Robinet, C., Jaeger, A.S., Le Gras, S., Jost, B., Gérard, B., Peoc'h, K., Launay, J.M., Faivre, L., Mandel, J.L. (2013). 20ans après: a second mutation in MAOA identified by targeted high-throughput sequencing in a family with altered behavior and cognition. *Eur J Hum Genetics* 22(6):776-83.
800
801

**Policarpo, M.**, Fumey, J., Lafargeas, P. and Naquin, D. (2020). Contrasted gene decay in subterranean vertebrates: insights from cavefishes and fossorial mammals. *bioRxiv* 978213.

802

**Popova, N.K.** (2006). From genes to aggressive behavior: the role of serotonergic system.

803

Bioessays. 28(5):495-503. Review.

804
805
806

**Pottin, K.**, Hinaux, H., Rétaux, S. (2011). Restoring eye size in Astyanax mexicanus blind cavefish embryos through modulation of the Shh and Fgf8 forebrain organising centres. *Development*. 138(12):2467-76.

807

**Puglisi-Allegra, S.**, Andolina, D. (2015). Serotonin and stress coping. *Behav Brain Res.* 277:58-67.

808
809
810

**Rétaux, S. and Jeffery, W. R.** (2023). Voices from the underground: Animal models for the study of trait evolution during groundwater colonization and adaptation. Chapter 10, The Groundwater Ecology and Evolution Book, 2023. Editors: Florian Malard, Christian Griebler, Sylvie Rétaux. Elsevier.

811
812

**Riddle, M.R.**, Boesmans, W., Caballero, O., Kazwiny, Y., Tabin, J., C. (2018). Morphogenesis and motility of the Astyanax mexicanus gastrointestinal tract. *Dev Biol.* 441: 285-296.

813
814

**Rink, E.**, Guo, S. (2004). The too few mutant selectively affects subgroups of monoaminergic neurons in the zebrafish forebrain. *Neuroscience* 127(1):147-54.

815
816
817
818

**Salichon, N.**, Gaspar, P., Upton, A.L., Picaud, S., Hanoun, N., Hamon, M., De Maeyer, E., Murphy, D. L., Mossner, R., Lesch, K.P., Hen, R., and Seif, I. (2001). Excessive activation of serotonin (5-HT) 1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-ht transporter knock-out mice. *J. Neurosci.* 21, 884-896.

819
820
821

**Sallinen, V.**, Sundvik, M., Reenilä, I., Peitsaro, N., Khrustalyov, D., Anichtchik, O., Toleikyte, G., Kaslin, J. and Panula, P. (2009). Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish. *J. Neurochem.* 109, 403-415.

822
823

**Schilling, B.**, and Lerch, K. (1995). Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger. *Mol. Gen. Genet.* 247(4), 430-438.

824
825

**Scott, A.L.**, Bortolato, M., Chen, K., and Shih, J.C. (2008). Novel monoamine oxidase A knock out mice with human-like spontaneous mutation. *Neuroreport* 19, 739-743.

826
827

**Setini, A.**, Pierucci, F., Senatori, O., and Nicotra, A. (2005). Molecular characterization of monoamine oxidase in zebrafish (Danio rerio). *Comp. Biochem. Physiol. B Biochem. Mol. Biol.* 140, 153-161.

828
829

**Stoyek, M. R.**, Jonz, M. G., Smith, F. M. and Croll, R. P. (2017). Distribution and chronotropic effects of serotonin in the zebrafish heart. *Auton. Neurosci. Basic Clin.* 206, 43-50.

830
831
832

**Than-Trong, E.**, Ortica-Gatti, S., Mella, S., Nepal, C., Alunni, A. and Bally-Cuif, L. (2018). Neural stem cell quiescence and stemness are molecularly distinct outputs of the Notch3 signalling cascade in the vertebrate adult brain". *Development* 15; 145(10).

833
834
835

**Vitalis, T.**, Cases, O., Callebert, J., Launay, J.M., Price, D.J., Seif, I., and Gaspar, P. (1998). Effects of monoamine oxidase A inhibition on barrel formation in the mouse somatosensory cortex: determination of a sensitive developmental period. *J. Comp. Neurol.* 393, 169-184.

836
837

**Wang, X.**, Lee, J. E. and Dorsky, R. I. (2009). Identification of Wnt-responsive cells in the zebrafish hypothalamus. *Zebrafish* 6, 49-58.

838
839
840

**Wen L**, Wei W, Gu W, Huang P, Ren X, Zhang Z, Zhu Z, Lin S, Zhang B. (2008). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. *Dev Biol.* 314(1):84-92.
841 **Winberg, S.** and Nilsson, G. E. (1993). Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish. *Comp. Biochem. Physiol. Part C Comp.*
842 106, 597-614.

844 **Winberg, S.** and Thörnqvist, P. O. (2016). Role of brain serotonin in modulating fish behavior. *Curr. Zool.* 62, 317-323.

846 **Yamamoto, K.**, Ruuskanen, J. O., Wullimann, M. F. and Vernier, P. (2011). Differential expression of dopaminergic cell markers in the adult zebrafish forebrain. *J. Comp. Neurol.* 519, 576-598.

848 **Xing, L.**, Kalebic, N., Namba, T., Vaid, S., Wimberger, P., Huttner, WB. (2020). Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex. *Neuron.* 108(6):1113-1129.

851
852 **Legends**

853 **Figure 1:** Generation of a *mao* KO *Astyanax mexicanus* surface fish line with CRISPR-Cas9.

854 A, Genotyping by Labchip on F1 fish (progeny of Fo zero injected fish). Red asterisks indicate
855 heterozygote fish with an indel in exon 4, visible by the presence of several bands
856 corresponding to heteroduplexes of PCR fragments. The black arrow indicates the expected
857 band size.

858 B, Chromatograms showing the sequence of *mao* exon 4 in WT, KO and heterozygotes (het).
859 The 11 nucleotides indel is indicated. The shift in the reading frame is visible on the het
860 chromatogram.

861 CC', Protein alignment and functional domain comparison. WT MAO is a 531 aminoacid
862 protein. TheKO form the protein is affected after amino acid 106, which results in the almost
863 complete truncation of the amino-oxidase functional domain.

864 D, MAO enzymatic activity in the heads of 6dpf larvae that are WT (grey) or KO (yellow) for
865 *mao*. Each sample (n=1) was formed with 15 heads. P-value after Mann-Whitney test.

866 E, Morphological aspect of 6dpf larvae from the 3 genotypes, as indicated.

867 **Figure 2:** Anatomy of TH-positive cells in the brain of 6dpf larvae.

868 A-B, Whole brain confocal projections showing a general view of TH-immunopositive cells
869 (cyan) in WT (A) and KO (B) fish. Eyes were removed. Scale bar: 100µm.

870 C, Quantification of the number of TH⁺ cells in the preoptic anterior parvocellular nucleus
871 (Papc), the suprachiasmatic nucleus (Sup), the posterior tuberculum (Pt), the posterior tuberal
872 nucleus (Ptn), the olfactory bulbs and subpallium (OB/Sp), the locus coeruleus (LC) and the
873 medulla oblongata (MO) of WT (grey), het (orange) and KO (yellow) fish. The number of brains
874 analysed is indicated. All pairwise Mann-Whitney comparisons retrieved p-values > 0.05,
875 except one in the Sup.

876

877 **Figure 3:** Anatomy of 5-HT-positive cells in the brain during the development of 5-HT-positive
878 cells in the brain during the development.

879 A-D, ventral view of serotonin immunostaining in WT and KO, 36 and 48 hpf embryos. The
880 serotoninergic neurons are present in the hypothalamus (hyp) and in the raphe (ra). Scale bar:
881 40 µm.

882 E-P, ventral view of serotonin immunostaining in WT and KO dissected brains (4dpf-12dpf).

883 The three regions of the hypothalamus were the serotoninergic neurons are present are
884 indicated: the anterior paraventricular nucleus (pva), the intermediate paraventricular nucleus
885 (pvi) and the posterior paraventricular nucleus (pvp). The serotoninergic neurons are also
886 present in the raphe (ra) indicated on the picture. Scale bar: 40 µm.

887 Q, Schematic representation illustrating variations of serotonin immunostaining during brain
888 development in *mao* KO *Astyanax mexicanus*.

889
890 **Figure 4:** Behavioral experiments.

891 A, quantification of distance covered in 30 minutes at the seven stages studied and for the three genotypes. *N* = 11 (WT) ; *N* = 25 (het) ; *N* = 14 (KO).

892 B-C, tracking the trajectories of fish according to their genotype. Two representative examples are shown for each genotype (7dpf and 10 dpf).

893 D, quantification of distance moved in 10 minutes in 2 months old fish (WT and heterozygotes). *N* is indicated on the graph.

894 E, quantification of attacks in 3 months old fish (WT and heterozygotes). *N* is indicated on the graph.

899

900 **Figure 5:** PH3 immunostaining in the telencephalon.

901 A, ventral view of confocal optical plane (3 µm) of WT telencephalon at 6, 8 and 10 dpf stained for the proliferative marker PH3 (green). DAPI is in blue. Scale bar: 20µm.

902 B, ventral view of confocal optical plane (3 µm) of KO telencephalon at 6, 8 and 10 dpf stained for the proliferative marker PH3 (green). DAPI is in blue.

903 C, quantification of PH3-positive cells in the three genotypes at 6, 8 and 10 dpf. *N* is indicated on the graph. \*Padj<0.05, \*\*Padj<0.005.

907

908 **Figure 6:** PH3 immunostaining in the hypothalamus.

909 A, ventral view of confocal optical plane (3 µm) of WT hypothalamus at 6, 8 and 10 dpf stained for the proliferative marker PH3 (green). DAPI is in blue. Scale bar: 20µm.

910 B, ventral view of confocal stacks (3 µm) of KO hypothalamus at 6, 8 and 10 dpf stained for the proliferative marker PH3 (green). DAPI is in blue.

911 C, quantification of PH3-positive cells in the three genotypes at 6, 8 and 10 dpf. *N* is indicated on the graph. \*Padj<0.05, \*\*Padj<0.005, \*\*\*Padj<0.00005.

915

916 **Figure 7:** Proliferation kinetics.

917 A, ventral view of confocal optical plane (3 µm) of KO telencephalon at 8 dpf stained for the EdU (magenta), phase M marker PH3 (green) and general marker of the cell cycle PCNA (blue). DAPI is in gray. Scale bar: 20µm.

918 B, quantification of EdU-positive cells, PH3 positive cells and PCNA positive cells in the three genotypes at 8 dpf in the telencephalon. *N* is indicated on the graph. \*P<0.05, \*\*P<0.005.

919 C, quantification of the number of PH3-EdU-positive cells on the EdU total number of positive cells in the three genotypes at 8 dpf in the telencephalon. *N* is indicated on the graph. Padj<0.1

920 D, ventral view of confocal optical plane (3 µm) of KO hypothalamus at 8 dpf stained for the EdU (magenta), phase M marker PH3 (green) and general marker of the cell cycle PCNA (blue). DAPI is in gray. Scale bar: 20µm.

921 E, quantification of EdU-positive cells, PH3 positive cells and PCNA positive cells in the three genotypes at 8 dpf in the hypothalamus. *N* is indicated on the graph. \*Padj<0.05, \*\*Padj<0.005.

922 F, quantification of the number of PH3-EdU-positive cells on the EdU total number of positive cells in the three genotypes at 8 dpf in the hypothalamus. *N* is indicated on the graph. \*Padj<0.05.

932
933
934
**Figure 8: Summary**
935
936 Graphical and schematic representations of the variations we have observed on various
937 phenotypes in *mao* KO larvae across developmental stages (indicated in dpf). WT are in grey
938 and KO are in yellow.

939 A, serotonin in the raphe.

940 B, serotonin in the hypothalamus.

941 C, locomotion.

942 D, proliferation.

943
944 **Supplemental Figure 1.** Lack of effect of *mao* KO on gut motility and heart beat rhythm at 8dpf.

945 A-D, Examples of kymographs from WT (A and B) and KO fish (C and D). The ordinate represents
946 time, the abscissa the position along the digestive tract from the stomach to the rectum.

947 Waves of peristaltism are well visible on images A and C. Fish of both genotypes (WT and KO)
948 have phases where peristaltism is intense (A and C), and other phases where it is less intense
949 (B and D).

950 E, Quantification of heart beat in WT, het and KO fish at 8dpf.

951
952
**Supplemental Figure 2:**
953
954

955 A, ventral view of confocal optical plane (3 µm) of WT and KO embryos at 36hpf stained for
956 the proliferative marker PH3 (green). DAPI is in blue. Scale bar: 40 µm.

957 B, ventral view of confocal optical plane (3 µm) of WT and KO embryos at 48hpf stained for the
958 proliferative marker PH3 (green). DAPI is in blue.

959 C, quantification of PH3-positive cells in the three genotypes at 36hpf and 48hpf in the total
960 brain, in the telencephalon and in the hypothalamus.

961 D, ventral view of confocal optical plane (3 µm) of WT and KO embryos at 36hpf stained for
962 the activated caspase (green). DAPI is in blue. Scale bar: 40 µm.

963 E, ventral view of confocal optical plane (3 µm) of WT and KO embryos at 36hpf stained for the
964 activated caspase (green). DAPI is in blue.

965 F, quantification of caspase-positive cells in the three genotypes at 36hpf and 48hpf in the total
966 brain, in the telencephalon and in the hypothalamus.

967 G, *in situ* hybridization for *tph1a* at 36 hpf in WT and KO. *Tph1a* is expressed in the hypothalamus.
968 In high magnification, we can see that it is expressed in a few number of cells. Scale bar: 40 µm.

969 H, *in situ* hybridization for *tph2* at 36 hpf in WT and KO. *Tp2* is expressed in the raphe. In high
970 magnification, we can see that it is expressed in two areas.

971