Non-thalamic origin of zebrafish sensory relay nucleus: convergent evolution of visual pathways in amniotes and teleosts

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homologous relationship is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a visual relay nucleus in zebrafish (a teleost fish). Similarly to the tectofugal visual thalamic nuclei in amniotes, the lateral part of the preglomerular complex (PG) in teleosts receives tectal information and projects to the pallium. However, our cell lineage study reveals that the majority of PG cells are derived from the midbrain, not from the forebrain. We also demonstrate that the PG projection neurons develop gradually until juvenile stage, unlike the thalamic projection neurons. Our data suggest that teleost PG is not homologous to the amniote thalamus and that thalamocortical-like projections can evolve from a non-forebrain cell population. Thus, sensory pathways in vertebrate brains exhibit a surprising degree of variation.


Validation of the tectofugal visual pathway in zebrafish
In order to verify whether the GFP+ projections correspond to the ascending visual pathway in zebrafish, we performed tract tracing studies using DiI, biocytin, or biotinylated dextran amine (BDA).
It has been known that retinal projections terminate in the upper layers of TeO in a wide range of species of ray-finned fish (von Bartheld and Meyer, 1987), and it is also the case in zebrafish (Supplementary file 4). In order to confirm whether the zebrafish PG receives visual inputs from TeO, we injected DiI in the PG of the Tg(279A-GFP) line, targeting the lateral subdivision (PGl) with a guide of GFP ( Figure 4A, asterisk). After 2-3 weeks of incubation, we observed retrogradely labeled cell bodies in a deep layer of TeO ( Figure 4B). The cell extends its dendrites up to the retinorecipient upper layer of TeO (the stratum fibrosum et griseum superficiale; SFGS), and this morphology is identical to the neurons receiving the retinal input in carp and goldfish (Yamamoto and Ito, 2008). BDA injection into TeO (Figure 4C,asterisk) labeled axon terminals in the PGl ( Figure 4D, arrowheads), confirming that PGl receives tectal inputs. Thus, we conclude that the PGl conveys visual information from TeO to Dl, and that the GFP+ projection from PGl to Dl in Tg(279A-GFP) recapitulates this visual pathway.

Mesencephalic progenitors give rise to the GFP+ PGl cells
Based on a cell lineage method using tamoxifen inducible Cre-lox recombination, we recently revealed that some brain structures that have been considered to be of prosencephalic origin (forebrain) are actually of mesencephalic origin (midbrain) (Bloch et al., 2019). PG was one of them ( Figure 5, Supplementary file 5), thus we further investigated the development of PG cells, including those in PGl.
We used a double transgenic line generated by crossing Tg(her5:ERT2CreERT2) and , in which mCherry is expressed in the progenies of her5-expressing cells by a tamoxifen induction. The transcription factor her5 is exclusively expressed in the midbrain-hindbrain boundary (MHB) at 24 hours post-fertilization (hpf) ( Figure 5A), and in ventricular cell clusters in the midbrain at juvenile stages (Galant et al., 2016;Bloch et al., 2019).
By following the mCherry-positive (mCherry+) cells at different developmental stages after induction at 24 hpf, we observe that PG is constituted as the most rostro- To further confirm whether the GFP+ and mCherry+ signals co-localize, we generated a quadruple transgenic line Tg(her5:ERT2CreERT2; (Supplementary file 7A), and performed tamoxifen induction at 24 hpf. We have verified that the quadruple transgenic line is identical to the double transgenic lines in terms of expressions of GFP and mCherry, and that their brain development is unaltered. Observing the PGl in the adult stage (3 mpf), we found that there are PGl cells co-expressing both GFP and mCherry ( Figure 4D-G, arrowheads). This suggests that at least some of the GFP+ pallial projection neurons originate from the MHB.
Since the GFP+ projection neurons become observable after 4 wpf (Figure 3), we hypothesized that they may be generated also during the juvenile stages. Thus, we decided to perform the tamoxifen induction at different developmental stages. However, the fertility of the her5 quadruple transgenic line (number of eggs and survival rate of young larva) were relatively low. For this reason, we used an additional transgenic line for the induction at later developmental stages.
As an alternative to the Tg(her5:ERT2CreERT2), we used Tg (Dr830:ERT2CreERT2) (Heuzé, 2017) (Supplementary file 7B). The enhancer sequence "830" (human enhancer is named "Hs830", and zebrafish enhancer is "Dr830") is a highly conserved regulatory sequence in mouse and in zebrafish, which acts as a putative enhancer of the transcription factor Meis2 (meis2a in zebrafish) selectively in the tectum (Heuzé, 2017). In this line, the expression territory of Cre is larger than Tg(her5:ERT2CreERT2) at 24 hpf, but the expression is limited to the tectal area after 30-48 hpf (Figure 7 and Supplementary file 8). We first generated the double transgenic line Tg(Dr830:ERT2CreERT2;, then crossed with Tg(279A-GFP), in order to generate a quadruple transgenic fish Tg(Dr830:ERT2CreERT2; (Supplementary file 7B).
We performed tamoxifen induction at different developmental stages from 24 hpf up to 8 wpf (Supplementary file 9), and examined the adult brains to verify whether GFP+ pallial projection neurons co-express mCherry. We found GFP/mCherry coexpressing cells consistently in all the induction time-points until 6 wpf ( Figure 8). The abundance of mCherry+ cells became less and less along development. At 6 wpf, there were very few mCherry+ cells in PGl, and we found only one cell co-expressing GFP and mCherry among all the specimens examined ( Figure 8G). We didn't observe any co-expression in the case of induction at 8 wpf.
Thus, our results suggest that GFP+ PGl cells are gradually added throughout the larval/juvenile stages around up to 6 wpf. Considering the small number of GFP+ cells in PGl and the short-term tamoxifen induction time, it would be reasonable to conclude that majority of GFP+ pallial projection neurons are progenies of cells derived from the tectal region.

Ontogeny of the zebrafish PG
By using tamoxifen inducible Cre-lox system in zebrafish, we labeled by mCherry the cells that were located in the midbrain region during development (between 24 hpf and 6 wpf). Abundant mCherry+ cells in the adult PG suggests that majority of PG cells derive from the mesencephalic region. PG cells were consistently labeled with mCherry following the treatments at all the developmental stages examined. This suggests that PG progressively grows by addition of cells migrating from the mesencephalon.
It is difficult to prove whether all the PG cells originate from the mesencephalon, due to the technical limitation of the tamoxifen induction. Long term tamoxifen treatment leads a high mortality rate of the fish during the experiment (Bloch et al., 2019;Yu et al., 2019). Moreover, the Cre-lox system would not allow 100% induction rate (Hayashi and McMahon, 2002). Thus, mCherry labeling of each experiment represents only a small fraction of the cells originating from the mesencephalic region.
Thanks to the Tg(279A-GFP), we could label visual pallial projection neurons in PG. There are only about 200 GFP+ neurons in the adult PG. In the quadruple transgenic lines expressing both GFP and mCherry, we observed consistently at least one or two co-expressing cells whenever the tamoxifen induction was performed before 6 wpf. Considering the short duration of each tamoxifen treatment, it may be reasonable to assume that most of the GFP+ cells originate from the midbrain. Thus, we conclude that the PGl projection neurons to the pallium are mainly composed of cells migrating from the mesencephalon, by gradual accumulation from embryonic to juvenile stages (until around 6 wpf). The absence of mCherry/GFP colocalization at 8 wpf suggests that the PGl reaches maturation around this stage, as indicated by the abundance of the projections to the Dl ( Figure 3C,D).
At early embryonic stages in both quadruple transgenic lines, the Cre expression appears to extend from dorsal to ventral MHB, therefore it is hard to conclude whether the PG cells derive from the alar or basal portion. A recent publication using shh-GFP transgenic line suggests that the adult PG contains shh-expressing cells (GFP+ in the transgenic line), indicating that some PG cells may be of basal origin (Wullimann and Umeasalugo, 2020). However, the expression of Cre at later stages is limited to the alar portion of the mesencephalon (notably tectal area). Thus, PG cells are migrating from the alar mesencephalic region at least at juvenile stages. This is consistent with previous studies claiming the PG is comprised of Pax6-expressing migrating cells (Wullimann and Rink, 2001;Ishikawa et al., 2007): both thalamic and tectal regions are Pax6+ domains. The data of the previous studies were interpreted assuming that the PG cells should come from the forebrain, leading the conclusion that the Pax6+ cells should be thalamic or prethalamic. Our data show that the PG cells are mesencephalic, thus they are probably derived from Pax6+ tectal cells. GFP+ fiber labeling in Dl appears to be prominent only at around 8 wpf ( Figure   3C). Unlike the thalamocortical projections in mammals that are already abundant at late embryonic stages, the visual ascending projections to the pallium in zebrafish are not mature until late juvenile stages. Yet zebrafish larvae can coordinate body orientation against the current, capture food, or escape from predators using relatively simple tectal circuitry (retina  TeO  motor outputs) (Del Bene et al., 2010;Grama and Engert, 2012;Barker and Baier, 2013). Such visuo-motor processing at the level of tectum (without reaching the forebrain) may be comparable to the circuitry involved in saccade in mammals . It is possible that larval and early juvenile zebrafish behaviors are largely dependent on this tectal circuitry, and the visual system involving telencephalic circuitry becomes more important at later stages. Schneider (1969) has proposed the presence of "two visual systems" terminating in the mammalian cortex (Schneider, 1969). One is called the "thalamofugal" or "geniculate" pathway, in which retinal inputs reach the striate visual cortex (V1) via a thalamic nucleus (lateral geniculate nucleus in the case of mammals). The other is called the "tectofugal" or "extra-geniculate" pathway, in which retinal inputs reach the extrastriate visual cortex via two structures, the tectum (superior colliculus in mammals) and another thalamic nucleus (pulvinar in primates and lateral posterior nucleus in other mammals like rodents). Since then, most studies in non-mammals have been interpreted based on this notion of "two visual systems" (Hall and Ebner, 1970;Karten and Hodos, 1970;Riss and Jakway, 1970;Ingle, 1973;Karten et al., 1973;Hagio et al., 2018).

Evolution of ascending visual pathways
However, researchers have never reached a consensus on the evolutionary history of the tectofugal pathways. The debates have often been related to unsolved homology of the target pallial areas. The main issue is which part of the pallium would correspond to the mammalian neocortex in non-mammals such as birds (Karten and Shimizu, 1989;Bruce and Neary, 1995;Striedter, 1997;Puelles et al., 2000;Butler et al., 2011;Dugas-Ford et al., 2012) and teleosts (Braford, 1995;Wullimann and Mueller, 2004;Northcutt, 2006;Yamamoto et al., 2007;Mueller et al., 2011).
Our current study does not solve the entire evolutionary scenario in vertebrates, but at least reveals that the tectofugal pathways in amniotes and in teleosts are not homologous. In tetrapods, the neurons giving rise to the pallial projections are in the thalamus, even if the pallial projection targets are different. In contrast, our data clearly indicate that majority (if not all) of the PG neurons are of mesencephalic origin. Thus, in terms of regional homology, the teleost PG is not homologous to the amniote thalamus that is of forebrain origin. There exists a possibility that some PG cells come from a forebrain territory, as suggested by previous studies (Wullimann and Rink, 2001and Rink, , 2002Ishikawa et al., 2007). Nonetheless, in addition to the developmental criteria, the cladistics analysis also reveals the lack of evolutionary continuity of the thalamo-pallial projections.
Substantial tectofugal visual pathways to the pallium are observed only in amniotes and teleosts, and not in the intermediate taxa ( Figure 9). In amphibians, the major sensory relay nucleus is the dorsal thalamus, but unlike amniotes, there are very few projections to the pallium, and the majority terminates in the subpallium (ventral telencephalon) (Kicliter, 1979;Neary and Northcutt, 1983;Wilczynski and Northcutt, 1983;Butler, 1994a). Similarly, basal groups of the actinopterygians also have a poorly developed pallial connectivity, and afferent projections from the thalamic region seem to terminate in the ventral portion of the telencephalon (Albert et al., 1999;Yamamoto et al., 1999;Holmes and Northcutt, 2003). Furthermore, in Polypterus, visual projection to the pallium is mediated via the nucleus medianus of the posterior tuberculum (MTP; Figure 9), which is considered to be uniquely derived in this group and not homologous to any known pathways in tetrapods (Northcutt et al., 2004;Northcutt, 2009). Indeed, there are more than two ascending pathways, with a variable abundance of each pathway depending on species (Riss and Jakway, 1970;Graybiel, 1972;Benevento and Standage, 1983;Gamlin and Cohen, 1986;Albert et al., 1999;Wild and Gaede, 2016;Heap et al., 2017).
In combination, these observations strongly suggest that the teleost and amniote pathways are not homologous to one another, but have evolved independently.

Thalamocortical-like functions by midbrain neurons in teleosts?
The amniote thalamo-pallial projection (thalamocortical projection in mammals) is a intra-forebrain projection, from the dorsal diencephalon to the dorsal telencephalon.
Due to the enlargement of the forebrain in mammals especially in humans, the forebrain evolution has drawn much attention for the study of sensory processing and cognitive functions.
In contrast, our data demonstrate that the PG-pallial projection in teleosts is a midbrain-forebrain projection. This shows that a non-forebrain cell population can play an equivalent role to the thalamocortical projection neurons (conveying sensory information to the pallium). We here focused on the tectofugal visual pathway because of the availability of a zebrafish transgenic line, but PG also receives inputs of other sensory modalities (auditory, lateral line, gustatory etc.) (Striedter, 1991(Striedter, , 1992Yoshimoto et al., 1998;Yamamoto andIto, 2005, 2008;Northcutt, 2006;Ito and Yamamoto, 2009). We have previously shown that the external part of the IL, another teleost-specific multi-sensory integration center (Rink and Wullimann, 1998;Ahrens and Wullimann, 2002), is also derived from the mesencephalon (Bloch et al., 2019).
More studies are needed to determine the functions of PG and IL, but these data indicate that the teleost lineage have taken an evolutionary path different from amniotes, recruiting more mesencephalic structures for sensory processing.
In addition to the variation in neuronal connectivity that we reveal here, we have already demonstrated unexpected diversity of dopamine systems, despite their involvement in similar physiological/behavioral properties across vertebrate groups Yamamoto et al., 2015.
Recently, Striedter and Northcutt (2020) have also pointed out a number of examples of convergent evolution across vertebrate taxa (Striedter and Northcutt, 2020), in agreement with our hypothesis. Taking all these data into consideration, vertebrate brains have tremendously diversified across taxa during evolution, and many similarities may be due to convergent evolution than previously thought. This zebrafish line was maintained either by incross or by crossing with AB.

Fish maintenance and staging
Zebrafish used for the biocytin tract tracing were maintained at Nagoya University (Japan) in aquaria at 22-26ºC. For the rest of the experiments, zebrafish were raised in the animal facility in Neuro-PSI (Gif-sur-Yvette, France). Embryos/larvae up to 5 days post-fertilization (dpf) were maintained and staged as described (Kimmel et al., 1995). After larval stages, zebrafish were raised in a fish facility (maintained at 26-28°C). Zebrafish at 3 mpf or older is considered as adult. In all experiments performed in this study, we randomly used both male and female.
The experimental protocols and care of laboratory animals were conducted in compliance with the official regulatory standards and approval of the French Government (reference document n°APAFIS#1286-2015062616102603 v5), the official Japanese regulations for research on animal, and the regulations on Animal Experiments in Nagoya University.

DiI tract-tracing
To examine brain connectivity in the adult zebrafish, we placed crystals of DiI (1, The brains were dissected out and a small crystal was inserted in the brain using a glass pipette. The crystal was left in the brain for dye diffusion from 10 days to 2 weeks at 37°C in PBS, or PBS containing 0.05% sodium azide to avoid fungal contamination. The brains were embedded in 3% agarose, and sectioned at 80µm (in frontal and sagittal) using a vibratome (Leica VT 1000 S).

Biocytin and BDA tract-tracing
Biocytin (Sigma-Aldrich, B4261) was injected into adult zebrafish brains (n = 40), both in vivo and in vitro. For some in vivo injection into TeO, BDA (molecular weight 3000; Thermo Fisher Scientific-Molecular Probes, D7135) was also used (n=3), because labeled terminals in the PGl were clearer than that with biocytin. Postoperative fish were maintained in aquaria for 1-5 hours. After the survival period, the fish were deeply anesthetized with MS222 (over 200 mg/L) and perfused through the heart with 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were removed from the skull and post-fixed in fresh solution of the same fixative at 4ºC for 1 to 2 days.
We also injected biocytin into the TeO, PGl, and Dl in vitro because it was difficult to maintain postoperative fish in aquaria for hours following injections in vivo. A detailed in vitro tract-tracing method has been reported previously (Yamamoto and Ito, 2008).
Fish were deeply anesthetized with MS222 (over 200 mg/L). We quickly dissected the brain from the skull and then injected crystals of biocytin into TeO, PGl, and Dl with a minute insect pin. The brain was kept in a container filled with 50 mL normal artificial cerebrospinal fluid solution for marine teleosts (126 mM NaCl, 4.0 mM KCl, 1.0 mM MgSO4, 1.7 mM CaCl2 mM, 26 mM NaHCO3, 1.0 mM NaH2PO4, and 10 mM glucose; ( Tsutsui et al., 2001)) at room temperature. The solution was aerated and changed every 30 minutes. After 3-4.5 hours, we fixed the brain by immersion in 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M PB for 1-3 days at 4ºC.

Tissue processing following the biocytin and BDA injection
The fixed brains were cryo-protected by immersion in 0.1 M PB containing 20% sucrose at 4ºC overnight. Cryo-protected brains were embedded in 5% agarose (type IX, ultra-low gelling temperature; Sigma-Aldrich, A2576) containing 20% sucrose and

Tamoxifen treatment
Tamoxifen treatments were performed in quadruple transgenic fish (see above) as described previously (Galant et al., 2016;Bloch et al., 2019). 4-Hydroxytamoxifen (Sigma-Aldrich, T176) was dissolved in ethanol at a concentration of 10 mg/ml and stored at -20°C until use. The working solution was freshly prepared before the treatment, then further diluted with embryo medium (for 24 hpf, 30 hpf, and 7 dpf) or fish water (for 2-6 wpf). The animals were incubated in the tamoxifen working solution at 28°C in the dark.
Embryos at 24 hpf and 30 hpf were dechorionated with Pronase (1 mg/ml; Sigma-Aldrich, P5147) prior to the tamoxifen treatment. Embryos were placed into the six-well culture plate (Thermo Fisher Scientific) and were incubated with embryo medium containing tamoxifen. 24 hpf embryos were treated with 10 µg/ml tamoxifen for 6 hours, and 30 hpf embryos were treated with 5 µg/ml tamoxifen for 24 hours. After the incubation, the fish were washed 3 times with embryo medium, then put back to the incubator. 7 dpf larvae were treated in a large petri dish (around 100 ml embryo medium) with 5 µg/ml tamoxifen on 2 consecutive days, with an incubation time of 4 hours each.
For juveniles (2-6 wpf), fish were placed in a beaker (100-200 ml fish water depending on the number of fish) with an air pump, and incubated with 2 µg/ml tamoxifen on 4 consecutive days. The incubation time per day was 2-4 hours, and the treatment was interrupted whenever the fish looked sick. At the end of each incubation, the fish were gently washed 3 times with fish water, placed back to a clean fish tank and fed.
The tamoxifen treatments were performed at least twice per each developmental stage (see Supplementary file 9), and each treatment contained at least 10 individuals.
The tamoxifen-induced mCherry expression was systematically observed at 3 mpf.
The fish were sacrificed and double-immunofluorescence anti-GFP and anti-dsRed were performed (see below).

Tissue preparations for immunofluorescence or in situ hybridization (ISH)
Zebrafish embryos up to 48 hpf were fixed in ice-cold 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in 0.01 M PBS containing 0.1% Tween 20 (PBST) overnight at 4°C. Zebrafish older than 5 dpf were deeply anesthetized using 0.2% tricaine methanesulfonate (MS222; Sigma-Aldrich) diluted in fish water. The fish were fixed in 4% PFA in PBST overnight at 4°C, then brains were dissected out.
Samples used for ISH were dehydrated in ethanol gradient series, and kept at −20°C in methanol at least for a couple of days. They were rehydrated prior to ISH.
For immunolabeling, samples were conserved in a stocking solution containing 0.5% PFA and 0.025% sodium azide. Adult brains were sectioned in a frontal plane (80 µm) with a vibratome.
For a whole-brain imaging for zebrafish younger than 2 wpf (14 dpf), a simplified clearing protocol was applied as previously described (

Image acquisition
A Leica TCS SP8 laser scanning confocal microscope was used to image adult sections with a 25x or 40x water immersion objective. For clarified brains, the same microscope was used with a Leica HC Fluotar L 25x/1.00 IMM motCorr objective. For all these acquisitions, fluorescence signal was detected through photomultipliers (PMTs) after sequential laser excitation of fluorophores at 405, 488, 552 nm. Steps along the Z-axis were set at 1 µm. Epifluorescence images were acquired using a Multizoom AZ100 (Nikon).
Bright-field images were acquired with upright microscopes, either BX43 or BX60 (Olympus). Acquired images were adjusted for brightness and contrast using ImageJ/FIJI software (Schindelin et al., 2012).

Quantification of mCherry positive cells in PG
The mCherry-positive cells in the adult PG were counted from confocal images using the ImageJ cell counter plugin. We used stacks of 5 μm from frontal sections containing the medio-posterior levels of PG. The total number of cells was determined with DAPI nuclear labeling, and proportion of mCherry positive cells was calculated. The cell count was performed in the brains induced at 4 different time points: 24 hpf, 48 hpf, 2 wpf, and 3 wpf, and the average from two specimens was presented as data for each time point.

3D image reconstruction of young zebrafish brains
In order to visualize the global distribution of mCherry+ cells in the brains of larval/juvenile (5, 7, 14 dpf) zebrafish, 3D reconstruction of confocal images was performed as described in Bloch et al. (2019). A whole brain imaged in confocal microscopy was reconstructed in 3D, using Imaris 8.0.1 software (Oxford Instruments) using the "3D view" visualization tool on a Dell T3610 workstation.

Selective visualization of PGl fiber projections in Tg(279A-GFP)
The signal of the Tg(279A-GFP) was selectively visualized by manual segmentation of the fiber projections in Amira (Thermo Fisher Scientific, FEI). For the classification of the staining of the specimen, we broke down the staining pattern into four categories: background, specimen background, specimen signal, and specimen auxiliary signal that contains a widely distributed population of cells outside the scope of this study.
The process of manual segmentation is an iterative succession of initial freehand