Doublecortin in the Fish Visual System, a Specific Protein of Maturing Neurons

Simple Summary Doublecortin (DCX) is an essential protein in the development of the central nervous system and in lamination of the mammalian cortex. It is known that the expression of DCX is restricted to newborn neurons. The visual system of teleost fish has been postulated as an ideal model since it continuously grows throughout the animal’s life. Here, we report a comparative expression analysis of DCX between two teleost fish species as well as a bioinformatic analysis with other animal groups. Our results demonstrate that DCX is very useful for identifying new neurons in the visual systems of Astatotilapia burtoni, but is absent in Danio rerio. Abstract Doublecortin (DCX) is a microtubule associated protein, essential for correct central nervous system development and lamination in the mammalian cortex. It has been demonstrated to be expressed in developing—but not in mature—neurons. The teleost visual system is an ideal model to study mechanisms of adult neurogenesis due to its continuous life-long growth. Here, we report immunohistochemical, in silico, and western blot analysis to detect the DCX protein in the visual system of teleost fish. We clearly determined the expression of DCX in newly generated cells in the retina of the cichlid fish Astatotilapia burtoni, but not in the cyprinid fish Danio rerio. Here, we show that DCX is not associated with migrating cells but could be related to axonal growth. This work brings to light the high conservation of DCX sequences between different evolutionary groups, which make it an ideal marker for maturing neurons in various species. The results from different techniques corroborate the absence of DCX expression in zebrafish. In A. burtoni, DCX is very useful for identifying new neurons in the transition zone of the retina. In addition, this marker can be applied to follow axons from maturing neurons through the neural fiber layer, optic nerve head, and optic nerve.


Introduction
Doublecortin (DCX; also known as doublin or lissencephalin-X) is a microtubuleassociated protein which is typically expressed in the early neuronal differentiation stage, both in precursors and immature neurons [1,2]. Due to DCX expression being nearly exclusive to developing neurons, several research groups are using it as a marker for neurogenesis in a wide range of vertebrate species, e.g., mammals [3], lampreys [4], sharks [5], and teleosts [6]. Brain formation depends on microtubules (MTs) and accompanying microtubule associated proteins (MAPs) to regulate specific migration of different neural cell types [7]. Brain development includes nuclear displacement and process formation that require the action of MTs and specific MAPs [8]. Furthermore, MTs are essential in the formation of growth cones [9]. The de-stabilization of MTs leads to the collapse of the migrating cell body and cessation of nuclear translocation [10]. Faulty of DCX expression causes critical brain defects, which implies that other MTs stabilizing proteins cannot compensate for DCX function in the central nervous system (CNS) [7,11]. In newly formed neurons, DCX are involved in the growth of neuronal processes [12,13].
The continuous life-long growth of the visual system of anamniotes (such as teleost fish) has been an intriguing phenomenon [14,15], especially since such extensive growth does not occur in mammals [16,17]. Several studies have used different fish species to understand the mechanism of adult neurogenesis in vertebrates [2,18,19]. The retina has been proposed as an ideal model to study the generation of new neurons in adults due to the presence of well-delimited neurogenic zones [20,21]: the peripheral germinal zone (PGZ), formed by stem cells; the transition zone, occupied by differentiating cells; and the layered retina, harboring completely differentiated cells, except for the generation of new rods which are added to the outer nuclear layer from rod precursors [22]. Among the many markers used to label differentiating neurons, DCX stands out both in mammals and some teleosts such as cichlid fish [6,23]. Given that commercially available anti-DCX-antibodies efficiently label processes from maturing neurons [12], we were interested in testing DCX as a potential marker to study differentiating neurons within the retinal transition zone of the fish retina.
The aim of the present study was to identify newly generated DCX-positive neurons in the adult fish retina and to follow their axonal pathway into the optic nerve. We used two fish, the zebrafish (Danio rerio) and Burton's mouthbrooder fish (Astatotilapia burtoni, formerly known as Haplochromis burtoni). The zebrafish is the main non-mammalian vertebrate animal model used in scientific research including visual studies [24], and A. burtoni is a model organism for behavior showing a substantial growth of retinal tissue throughout its life [15,25].
Here, we report the immunohistological reactivity for DCX in A. burtoni retina, which was conspicuously absent in the zebrafish retina. In addition, we performed in silico and western blotting analyses in order to clearly define the presence of DCX in different models-from flies to mice-to study neurogenesis.

Animals
We used adult specimens of Astatotilapia burtoni (Ostariophysi; Cichlidae) measuring between 2-6 cm standard length and Danio rerio (Acanthopterygii; Cyprinidae) measuring more than 1.5 cm. All animals were bred in our own animal facilities, in the Institute of Clinical Anatomy and Cell Analysis (University of Tübingen, Tübingen, Germany) and in the Institute of Neuroscience of Castilla y León (University of Salamanca, Salamanca, Spain). For protein expression assays, mice tissue was obtained from brains (excluding olfactory bulb and cerebellum) of newborn mice (C57BL/6 strain at p10) kindly provided by Dr. J.R. Alonso's group of University of Salamanca [26].
Both fish species were kept at 28.5 ± 1 • C, with a photoperiod on 12 h light/dark cycle, and without feeding restrictions. All procedures were performed in accordance with the guidelines of the European Union Council Directive (2010/63/EU). Local authorities (Regierungspräsidium Tübingen and the Animal Ethical Committee of The University of Salamanca) approved animal use before experimentation in both institutions.

Tissue Preparation
We used the eyes and optic nerves from at least four animals of each fish species for histological analysis. Adult fish were deeply anaesthetized with tricaine methane For tissue clearing, the entire eyeballs with a short piece of optic nerve were dissected for each species, removing the sclera and outer pigmented tissue. Before tissue clearing, we applied a bleaching protocol to eliminate pigment granules. Samples were incubated in a bleaching solution constituted of 0.5% (v/v) KOH, and 1% (v/v) Na 2 HPO 4 in H 2 O plus 3% H 2 O 2 (Sigma-Aldrich, St. Louis, MO, USA, H1009). Sequential incubations of two hours at room temperature were performed until sample bleaching was achieved, followed by washing in acid acetic for 1 h at room temperature. Then, we applied our adjusted clearing protocol [27]. Briefly, samples were fixed overnight in a hydrogel monomer solution constituted by 10% (v/v) acrylamide, 2.5% (v/v) bis-acrylamide, 4% (wt/v) paraformaldehyde, and 0.25% (wt/v) VA-044 Initiator (FUJIFILM Wako Pure Chemical, Neuss, Germany" 017-19362) in PBS (pH 7.4). The gel was polymerized in a vacuum oven at 40-45 • C for 3 h. Embedded eyes were cleared in an 8% (wt/v) sodium dodecyl sulfate solution (SDS) in PBS at 45-50 • C for 1-2 weeks. Clearing solution was changed every 2 days.
In the clearing samples, after washing SDS solution, we performed immunostains to detect DCX in entire eyes of teleost fish. Pre-incubation was performed overnight by using donkey serum. The following day, samples were incubated in anti-DCX primary antibody for 4 days followed by an overnight incubation with secondary antibody and DRAQ5. Several washing steps were performed to eliminate background fluorescence, followed by an incubation in 80% glycerol for refractive index matching.

Image Acquisition and Analysis
Images were acquired on an LSM Exciter confocal microscope (Zeiss, Jena, Germany) or an LSM510 using laser excitations at 488, 543, and 633 nm in sequential scans with appropriate sets. Images, as well as the three-dimensional reconstructions, were generated with ZEN 2009 software. Once adjusted for contrast and brightness, the image plates were assembled with Photoshop CS6.

Western Blotting
Total proteins were extracted from the brains of wild type adult zebrafish (AB strain), from adult A. burtoni, and from newborn mice (C57BL/6 strain at P10). Tissues were mechanically homogenized with hand-held sterile pestles and insulin syringes and lysed in RIPA buffer (50 mM Tris (VWR International Eurolab S.L, Barcelona, Spain, 0027C481) pH 8; 150 mM NaCl; 1% Igepal; 0.5% sodium deoxycholate; 0.1% SDS) containing a protease inhibitor cocktail (VWR; M222-1ML). Samples were centrifuged (10.000× g for 10 min at 4 • C) to eliminate the debris, and supernatants were transferred to clean Eppendorf tubes. Protein concentration was determined using the Bradford methodology.
Lysates were resuspended in 2× Laemmli buffer (4% SDS; 20% glycerol; 10% βmercaptoethanol; 0.004% bromophenol blue; 0.125M TrisHCl pH 6.8) and boiled for 7 min. Amounts of 40 µg, 60 µg and 80 µg of total proteins were separated using conventional SDS-PAGE in reduced conditions and transferred to polyvinylidene difluoride membrane (PVDF, Amersham). Membranes were blocked with 3% bovine serum albumin ( Protein expression was determined by densitometric analysis using Adobe Photoshop CS6. Total integrated density was obtained for each band (DCX, DCLK, and β-actin) and background integrated density was subtracted to obtain the specific integrated density for each protein.

In Silico Identification and Analysis of DCX in Teleost Fish
Amino acid sequences of DCX and DCLK proteins of teleost fish were obtained by BLASTp analysis using the Homo sapiens DCX (Accession number: O43602) and DCLK (Accession number: AAI52457) protein sequences as queries against the non-redundant protein database of the National Center of Biotechnology Information (NCBI; Available online: www.ncbi.nlm.nih.gov (accessed on 19 April 2021).
In order to set up an accurate protein database, a two-parameter approach was followed: (i) exclusion of all DCX conserved domain-containing protein (DCDC) sequences, and (ii) selection of those proteins containing both the DCX1 (IPR003533; PF03607; cd16109) and DCX2 (IPR003533; PF03607; cd17069) conserved domains identified using InterPro Scan v5.44-79.0 [28] and the NCBI Conserved Domain Database. Finally, a total of 159 proteins were included in the database, containing all the sequences described by Reiner et al. [29] and those obtained here by BLASTp analysis.

DCX Is Present in A. burtoni Retina but Not D. rerio
Antibody stains applied to histological sections enabled the detection of DCX expression in the A. burtoni retina ( Figure 1A,B). In contrast, no specific immunofluorescence was found in the zebrafish retina ( Figure 1C,D). DCX positive cell bodies were located in the transition zone ( Figure 1A,B), next to the PGZ, of the retina of A. burtoni and their axons passing through the nerve fiber layer to the optic nerve head ( Figure 1E). No cell bodies expressing DCX were observed in the differentiated retina and/or in the optic nerve.

DCX Proteins Are Present in All the Genomes Analyzed but Not in Zebrafish
We generated a database containing 159 protein sequences belonging to 14 teleost fish (Triplophysa tibetana, Danio rerio, Betta splendens, Parambassis ranga, Oryzias latipes, Nothobranchius furzeri, Astatotilapia burtoni, Gadus morhua, Anabarilius grahami, Nothobranchius pienaari, Oryzias melastigma, Carassius auratus, Labeo rohita, and Tetraodon nigroviridis), 6 mammals (Mus musculus, Homos sapiens, Pan troglodytes, Canis lupus, Bos taurus, and Rattus norvegicus), one bird (Gallus gallus), and one shark (Callorhinchus milii), as well as D. melanogaster and C. elegans as invertebrate species (Table 1). Alignment of DCX amino acidic sequences showed a high conservation degree within and among analyzed groups (Table 2). Interestingly, shark and human DCX homologues share 96% of similarity, but no DCX coding sequence was found in the genome of zebrafish. Since we could not find DCX in zebrafish, we also considered the DCLK proteins, which have been previously described in zebrafish, and belong to the DCX superfamily [29]. DCLK sequences showed around 50-55% pairwise identity among species (Table 2). No DCLK sequences were found in the genomes of invertebrate species (D. melanogaster and C. elegans). However, analysis of DCX proteins from D. melanogaster revealed a catalytic "protein C kinase-like" domain (IPR000719, PF00069) ( Figure S2) located between residues 477-743. Based on these results, we performed a multiple-sequence alignment of all the retrieved DCX and DCLK sequences. Results revealed more than 50% sequence similarity between DCX and DCLK sequences ( Table 2).
We then extracted the DCX1 conserved domain from DCX and DCLKs proteins, which was used to perform a multiple-sequence alignment. A similar analysis was conducted for DCX2 conserved domain. In both cases, a high conservation degree was observed between DCX and DCLKs proteins ( Table 3). The same approach was applied to the kinase domain found in DCLK proteins from vertebrates, using the "protein C kinase-like" catalytic domain of the D. melanogaster DCX sequence as reference (Accession number: AAM11416) ( Figure S2). Remarkably, results revealed that the DCX2 domain is much more conserved than DCX1 and the kinase domains among species analyzed, and the latter, appears to be not much conserved in the evolutionary scale ( Table 3). Comparison of DCX1 and DCX2 domains of DCLK proteins from zebrafish and of DCX proteins from A. burtoni fish revealed a high similarity of sequence and structure (Table 3).
To verify the absence of DCX in the zebrafish genome, we performed a tblastn analysis using human DCX amino acidic sequence as query. Although no DCX orthologous genes were found on zebrafish, we found three loci putatively encoding different DCLK isoforms. Likewise, the genomes of the two invertebrate species did not show any conserved region corresponding to DCLK sequences. This supports the hypothesis that the DCLK-encoding gene is absent in invertebrates.

Western Blot Analysis Confirms the Presence of DCX in Burton's Mouthbrooder Fish and Mice, but Not Zebrafish
Using a commercially available anti-DCX antibody, western blot assays revealed an intense and specific band of 40 KDa in protein samples from mice and A. burtoni fish (Figures 2 and S3), which corresponded to the weight described for DCX protein (information related to sequence similarity between mice and A. burtoni DCX can be found in Figure 3A). No specific DCX band was found in zebrafish samples ( Figure 2). A positive immunoreactive band for β-actin (loading control) in all samples proved that protein lysates from zebrafish brain were correct. In contrast, DCLK expression was found in protein lysates from the three studied organisms (Figures 2 and S3); a strong immunoreactive band at 82 KDa is observed in lysates from mice brains, as well as a less intense band at 130 KDa. These two isoforms were also detected in samples from the A. burtoni brain, especially when 60 mg and 80 mg proteins were loaded, although the expression of the 82 KDa isoform is significantly lower. In the case of zebrafish, a specific immunoreactive 130 KDa band was found for the three tested protein concentrations (information related to conservation of the immunogen sequence among the studied species can be found in Figure 3B).   Analysis shows the expression of DCX in A. burtoni and mice, but not in zebrafish extracts. β-actin was used as the loading control after the membrane stripping.

Discussion
Doublecortin (DCX) is a microtubule-binding protein expressed in differentiating and migrating neurons of the nervous system during embryonic and postnatal development [1,36]. Various reports have associated DCX to migrating cells, especially during the formation of cortical layers [1,3,37]. Our results indicate that DCX is also expressed in non-migrating neurons such as those located in the transition zone of the retina next to proliferating cells. In contrast, precursor cells giving rise to new rods are known to migrate but were negative for DCX. This is consistent with our previous finding in the fish telencephalon, where DCX is expressed by new neurons but not in migrating cells [38].

Discussion
Doublecortin (DCX) is a microtubule-binding protein expressed in differentiating and migrating neurons of the nervous system during embryonic and postnatal development [1,36]. Various reports have associated DCX to migrating cells, especially during the formation of cortical layers [1,3,37]. Our results indicate that DCX is also expressed in non-migrating neurons such as those located in the transition zone of the retina next to proliferating cells. In contrast, precursor cells giving rise to new rods are known to migrate but were negative for DCX. This is consistent with our previous finding in the fish telencephalon, where DCX is expressed by new neurons but not in migrating cells [38].
DCX gene expression have been described in different species, including invertebrates [29,[39][40][41]. Apart of the MTs association, Friocourt et. al. identified and characterized a DCX-interacting protein in D. melanogaster, which is involved in cleaving ubiquitin from protein-ubiquitin conjugates [42]. In adults of the afrotherian tenrecs, DCX was shown to be expressed in the paleocortex [43,44]. In adult mammals, DCX is highly expressed in newly produced cells in the neurogenic zones; the subventricular zone along the lateral ventricle and the subgranular zone of the dentate gyrus [7,45]. Indeed, DCX has been adopted as a marker for neuronal precursors during adult neurogenesis [46]. In the visual system, DCX positive cells were found in the retinas of rats [47,48], sharks [5], teleost fish [6,23], and lampreys [4]. Here, we support the use of DCX as a marker for maturing neurons in the A. burtoni visual system. However, these results might not be extensive to all species since we were unable to find DCX in zebrafish in any of our experiments. This might suggest that DCX function could be compensated by other proteins.
An important question for understanding DCX function concerns its intracellular localization, since DCX may act similarly to classical MAPs and influence microtubule stability [49]. Although mutations in human DCX produces several defects in neuronal migration, a genetic deletion of DCX in mice causes a milder deformity [11,12,41]. Deuel et al. identified a different locus, doublecortin-like-kinase (DCLK), that encodes a protein with a similar "doublecortin domain" and microtubule stabilization properties that could compensate DCX function in rodents [50][51][52]. DCX and DCLK could directly or indirectly regulate microtubule-based vesicle transport, a process critical to both axon growth and neuronal migration [41,51,53,54].
The high level of conservation found in the evolutionary branches [11,29,41] and the very specific neuronal cell type that express DCX [2,45,46,55] would make this protein an excellent marker to detect neurogenesis events, not only during development but also in adulthood. It could be suggested that the absence of DCX in zebrafish could be compensated by DCLK. However, the exact compensatory mechanism and its implications during neuron migration and microtubule dynamics would require further investigation. The absence of DCX in zebrafish genome in addition to the lack of a corresponding amino acid sequence raises the question of how neurogenesis in zebrafish compensates for the missing DCX protein. This might ultimately help in investigating and better understanding neurogenesis in the vertebrate CNS.

Conclusions
The different techniques applied to detect the DCX protein in the visual system of teleost fish allowed us to determine the expression in A. burtoni but not in D. rerio. This work brings to light the high conservation of DCX sequences between different evolutionary groups, which makes DCX an ideal marker to study neurogenesis in various species. In A. burtoni, DCX is very useful for identifying new neurons generated in the retina and to follow their axons through components of the fish visual system. In addition, in teleosts, DCX apparently is not associated to migrating cells but could be related to axonal growth. Taking together, DCX represents an excellent marker for neurogenesis of most but not all animal models.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biology11020248/s1, Figure S1: Scheme of DCX1 and DCX2 conserved domains found in DCX protein sequences. Figure S2: Scheme of the kinase conserved domain found in DCX protein sequences. Figure S3: Relative intensity quantification of western blot. File S1: Full Western Blot.