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Doublecortin
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This entry is adapted from the peer-reviewed paper 10.3390/biology11020248

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. Immunohistochemical, in silico, and western blot analysis to detect the DCX protein in the visual system of teleost fish are described here.

doublecortin visual system Danio rerio Astatotilapia burtoni neurogenesis

1. Introduction

Doublecortin (DCX; also known as doublin or lissencephalin-X) is a microtubule-associated 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], researchers were interested in testing DCX as a potential marker to study differentiating neurons within the retinal transition zone of the fish retina.

2. 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.
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Figure 1. Sections of retina (A–D) and cleared tissue (E). DCX positive cells (green arrows) are detected in A. burtoni retina (A) but not in zebrafish (C). Magnification of (A,C) are presented in (B,D). The proliferating cell nuclear antigen (PCNA; red arrows) binding antibodies detect cells in the peripheral germinal zone (PGZ; of both species. In addition, DCX (green arrows) and PCNA (red arrows) are detected in the ONL corresponding to cones and rod precursors, respectively. Nuclei are stained with DRAQ5 (blue). No double positive cells for DCX (green arrows) and PCNA (red arrows) are found. By analyzing the whole transparent eyeball, it is possible to follow DCX positive processes in the neural fiber layer (green arrow) into the optic nerve (E; white arrow). Scale bar 20 µm (A,C); 10 µm (B,D). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; NFL: neural fiber layer.

3. DCX Proteins Are Present in All the Genomes Analyzed but Not in Zebrafish

Researchers 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).
Table 1. Database of 159 amino acidic sequences selected to perform protein alignments. Different species are used to compare DCX and DCLK sequence identity. From this database researchers also extract DCX1 and DCX2 conserved domains for further sequence analyses.
Table
Protein and Species Accession Number Protein and Species Accession Number Protein and Species Accession Number
DCLK from Kanglang fish ROJ66296 DCLK from Human NP_004725 DCLK from Zebrafish AAI63926
DCLK from Kanglang fish ROL01462 DCLK from Human XP_016876336 DCLK from Zebrafish AAI68500
DCLK from Kanglang fish ROL42582 DCLK from Human XP_016876337 DCLK from Zebrafish BAF45322
DCLK from Kanglang fish ROL54251 DCLK from Medaka XP_004075345 DCLK from Zebrafish BAF45323
DCLK from Betta fish XP_029027475 DCLK from Medaka XP_011474963 DCLK from Zebrafish BAF45324
DCLK from Betta fish XP_029027480 DCLK from Medaka XP_011474968 DCLK from Zebrafish BAF45325
DCLK from Betta fish XP_029027952 DCLK from Medaka XP_011474974 DCLK from Zebrafish BAF45326
DCLK from Betta fish XP_029027953 DCLK from Medaka XP_011480649 DCLK from Zebrafish NP_001128593
DCLK from Betta fish XP_029027954 DCLK from Medaka XP_011481611 DCLK from Zebrafish NP_001139259
DCLK from Betta fish XP_029027955 DCLK from Medaka XP_011485544 DCLK from Zebrafish NP_001139260
DCLK from Betta fish XP_029030116 DCLK from Medaka XP_020563782 DCLK from Zebrafish NP_001139261
DCLK from A. burtoni fish XP_005926513 DCLK from Medaka XP_020564443 DCLK from Zebrafish XP_005172728
DCLK from A. burtoni fish XP_005926514 DCLK from Medaka XP_024115177 DCLK from Zebrafish XP_009290868
DCLK from A. burtoni fish XP_005928154 DCLK from Medaka XP_024143878 DCLK from Zebrafish XP_009303718
DCLK from A. burtoni fish XP_005935114 DCLK from Mouse AAH21354 DCLK from Zebrafish XP_009303720
DCLK from A. burtoni fish XP_005935115 DCLK from Mouse AAH50903 DCLK from Zebrafish XP_021325742
DCLK from A. burtoni fish XP_005935116 DCLK from Mouse AAH64783 DCLK from Zebrafish XP_021334856
DCLK from A. burtoni fish XP_005951753 DCLK from Mouse AAI33686 DCX from Betta fish XP_029029871
DCLK from A. burtoni fish XP_005951754 DCLK from Mouse AF155819_1 DCX from Chick AF330009
DCLK from A. burtoni fish XP_014186335 DCLK from Mouse NP_001104521 DCX from Chimp PNI40040
DCLK from A. burtoni fish XP_014190393 DCLK from Mouse NP_001104522 DCX from A. burtoni fish XP_005922256
DCLK from A. burtoni fish XP_014190900 DCLK from Mouse NP_001104523 DCX from Cow NP_001193894
DCLK from A. burtoni fish XP_014190901 DCLK from Mouse NP_001182467 DCX from Dog XP_022271525
DCLK from A. burtoni fish XP_014190902 DCLK from Mouse NP_001182468 DCX from Dog XP_022271526
DCLK from A. burtoni fish XP_014190903 DCLK from Mouse NP_001344395 DCX from Dog XP_022271527
DCLK from A. burtoni fish XP_014190904 DCLK from Mouse NP_001344397 DCX from Dog XP_022271528
DCLK from A. burtoni fish XP_014190905 DCLK from Mouse NP_001344398 DCX from Dog XP_022271529
DCLK from A. burtoni fish XP_014190906 DCLK from Mouse NP_001344404 DCX from Dog XP_022271530
DCLK from A. burtoni fish XP_014190907 DCLK from Mouse NP_001344405 DCX from Dog XP_022271531
DCLK from A. burtoni fish XP_014193189 DCLK from Mouse NP_064362 DCX from Dog XP_022271532
DCLK from A. burtoni fish XP_014193190 DCLK from Mouse Q9JLM8 DCX from Fly AAM11416
DCLK from A. burtoni fish XP_014193191 DCLK from Mouse XP_006501044 DCX from Glassy fish XP_028278320
DCLK from Codfish XP_030217849 DCLK from Mouse XP_006501045 DCX from Glassy fish XP_028278321
DCLK from Glassy fish XP_028258882 DCLK from Mouse XP_006501046 DCX from Glassy fish XP_028278322
DCLK from Glassy fish XP_028258890 DCLK from Mouse XP_017174936 DCX from Human AAC31696
DCLK from Glassy fish XP_028258898 DCLK from Mouse XP_030108271 DCX from Human AAC31797
DCLK from Glassy fish XP_028258907 DCLK from Mouse XP_036018776 DCX from Human AAC52037
DCLK from Glassy fish XP_028275059 DCLK from Rat NP_445795 DCX from Human AAH27925
DCLK from Glassy fish XP_028275060 DCLK from Tibetan fish KAA0702164 DCX from Human CAA05867
DCLK from Human AAI52457 DCLK from Tibetan fish KAA0704105 DCX from Human CAA06617
DCLK from Human NP_001317000 DCLK from Tibetan fish KAA0710366 DCX from Human NP_001182482
DCLK from Human NP_001317001 DCLK from Tibetan fish KAA0722200 DCX from Human NP_001356299
DCX from Human NP_001356300 DCX from Mouse AAT58219 DCX from Rat AAG18479
DCX from Human NP_001356301 DCX from Mouse AF155820_1 DCX from Rat NP_445831
DCX from Human NP_835364 DCX from Mouse BAA33387 DCX from Rat Q9ESI7
DCX from Human NP_835365 DCX from Mouse NP_001103692 DCX from Rat XP_006257444
DCX from Human NP_835366 DCX from Mouse NP_001103693 DCX from Rat XP_006257447
DCX from Human O43602 DCX from Mouse NP_001103694 DCX from Rat XP_006257448
DCX from Killifish AEY83972 DCX from Mouse NP_034155 DCX from Rat XP_017457656
DCX from Medaka XP_023818290 DCX from Mouse O88809 DCX from Shark AFP00992
DCX from Mouse AAC31799 DCX from Mouse XP_006528761 DCX from Tibetan fish KAA0710823
DCX from Mouse AAH56391 DCX from Mouse XP_030107072 Synapsin from Zebrafish BAH84839
DCX from Mouse AAH57010 DCX from Mouse XP_030107073    
DCX from Mouse AAH62974 DCX from Mouse XP_030107074
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.
Table 2. Sequence similarities between selected proteins containing DCX1 and DCX2 domains. Comparison of entire DCX and/or DCLK amino acid sequences to assess protein conservation between different species.
Table
Protein Organism Number of Aligned Sequences Similarity
DCX Human + Shark 15 96.7%
Mammals 47 89%
Mammals + A. burtoni fish 48 88.8%
Mammals + Teleost Fish 58 83.3%
Teleost Fish 11 82.5%
Mammals + Teleost Fish + Drosophila + Shark + Chick 61 78.6%
DCLK Mammals 30 55.9%
Teleost Fish 71 53.5%
Mammals + Teleost Fish 101 49.3%
DCLK from zebrafish + DCX from D. melanogaster 16 72.5%
DCLKs + DCX 159 50.1%
Since researchers could not find DCX in zebrafish, researchers also considered the DCLK proteins, which have been previously described in zebrafish, and belong to the DCX superfamily [24]. 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) located between residues 477–743. Based on these results, researchers 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).
Researchers 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). 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).
Table 3. Sequece similarities between DCX1 and DCX2 domains after its extraction from DCX and/or DCLK. Comparison of DCX1 and DCX2 conserved domains to analyze the identity between different species.
Table
Domain Organism Number of Sequences Similarity
DCX1 All 159 73.4%
DCX2 All 159 77.4%
Kinase All 159 47.4%
DCX1 Teleost Fish 79 74.5%
DCX2 Teleost Fish 79 79.1%
Kinase Teleost Fish 79 69.9%
DCX1 DCLK ZF + DCLK A. burtoni + DCX from another organisms 97 79.6%
DCX2 DCLK ZF + DCLK A. burtoni + DCX from another organisms 97 83.7%
To verify the absence of DCX in the zebrafish genome, researchers performed a tblastn analysis using human DCX amino acidic sequence as query. Although no DCX orthologous genes were found on zebrafish, researchers 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.

4. 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 (Figure 2), 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 (Figure 2); 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).
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Figure 2. Western blot analysis of protein extracts derived from zebrafish, A. burtoni, and mice brains. Increasing concentrations of proteins were loaded in each well (40, 60 and 80 μg per well). 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.
Biology 11 00248 g003 550
Figure 3. Alignments of DCX and DCLK amino acid sequences. Conserved residues between mice and A. burtoni DCX proteins (A); Conservation of the DCLK2 immunogen sequence use to detecting DCLK protein among the studied species (B).

References

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