Doublecortin | Encyclopedia MDPI

Doublecortin: Comparison

Please note this is a comparison between Version 1 by Laura DeOliveira-Mello and Version 6 by Vivi Li.

Doublecortin (DCX) is an microtubule associatedessential protein, essential for correctin in the development of the central nervous system development andand in lamination inof the mammalian cortex. It has been demonstrated to bis known that the expressed in developing but not in matureion of DCX is restricted to newborn neurons. The visual system of teleost visual system ifish has been postulated as an ideal model to study mechanisms of adult neurogenesis due to its continuous life-long growth. Immunohistochemical, in silico,since it continuously grows throughout the animal’s life. Here, we report a comparative expression analysis of DCX between two teleost fish species ands western blotll as a bioinformatic analysis to detect the DCX proteinwith other animal groups. Our results demonstrate that DCX is very useful for identifying new neurons in the visual systems of Astatotilapia burtoni, buteleost fish are described is absent in hereDanio rerio.

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][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][7,11]. In newly formed neurons, DCX are involved in the growth of neuronal processes ^[12][13][12,13].

The continuous life-long growth of the visual system of anamniotes (such as teleost fish) has been an intriguing phenomenon ^[14][15][14,15], especially since such extensive growth does not occur in mammals ^[16][17][16,17]. Several studies have used different fish species to understand the mechanism of adult neurogenesis in vertebrates ^[2][18][19][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][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][6,23]. Given that commercially available anti-DCX-antibodies efficiently label processes from maturing neurons [12], rwesearchers 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.

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

ResearchWers 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 rwesearchers also extract DCX1 and DCX2 conserved domains for further sequence analyses.

Protein and Species	Accession Number	Protein and Species	OrganismAccession Number	Protein and Species	Accession Number
Number of Aligned Sequences	Similarity
DCLK from Kanglang fish	ROJ66296	DCLK from Human	NP_004725
DCX	Human + Shark	15	DCLK from Zebrafish	AAI63926
	96.7%	DCLK from Kanglang fish	ROL01462	DCLK from Human	XP_016876336	DCLK from Zebrafish	AAI68500
	Mammals	47	89%	DCLK from Kanglang fish	ROL42582
	Mammals + A. burtoni fish	DCLK from Human	XP_016876337	DCLK from Zebrafish	BAF45322
	48	88.8%

). 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.

Domain	Organism	Number of Sequences	Similarity
DCX1	All	159	73.4%
DCX2	All	159	77.4%
DCLK from Kanglang fish
Kinase	All	159	ROL54251	DCLK from Medaka	XP_004075345	DCLK from Zebrafish	BAF45323
DCLK from Betta fish
47.4%	Mammals + Teleost Fish	58	83.3%	XP_029027475
DCX1	Teleost Fish	79	74.5%	DCLK from Medaka	XP_011474963
Teleost Fish	11	DCLK from Zebrafish	BAF45324
DCLK from Betta fish	XP_029027480	DCLK from Medaka	XP_011474968	DCLK from Zebrafish	BAF45325
82.5%
Mammals + Teleost Fish + Drosophila + Shark + Chick	61	78.6%	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
DCX2	Teleost Fish	79	79.1%
Kinase	Teleost Fish	79	69.9%	DCLK
DCX1	Mammals	30	DCLK ZF + DCLK A. burtoni + DCX from another organisms		97	79.6%	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
DCX2	DCLK ZF + DCLK A. burtoni + DCX from another organisms	97	83.7%	DCLK from Zebrafish	XP_005172728
DCLK from A. burtoni fish	XP_005926514	DCLK from Medaka	XP_024115177	DCLK from Zebrafish	XP_009290868
55.9%
Teleost Fish	71	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
53.5%	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 fishA. 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
DCX from Rat
Mammals + Teleost Fish	101	49.3%	DCLK from Human	NP_445831
DCLK from zebrafish + DCX from D. melanogaster		16	72.5%
DCLKs + DCX		159	50.1%	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	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 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.

Protein

Since rwesearchers could not find DCX in zebrafish, researcherswe also considered the DCLK proteins, which have been previously described in zebrafish, and belong to the DCX superfamily ^[24][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, rwesearchers 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).

RWesearchers 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

To verify the absence of DCX in the zebrafish genome, reswearchers performed a tblastn analysis using human DCX amino acidic sequence as query. Although no DCX orthologous genes were found on zebrafish, researcherswe 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 and Figure 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 (Figure 2 and Figure 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).

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.

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).