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Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish.

Gao J, Zhang C, Yang B, Sun L, Zhang C, Westerfield M, Peng G - PLoS ONE (2012)

Bottom Line: We found that ADt axons normally project ventrally.We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function.We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China.

ABSTRACT
The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.

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ADt neurons project axons ventrally.In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower left corners. Lateral, animals mounted in lateral view, rostral to the left; Frontal, animals mounted in frontal view, dorsal to the top. (A) lhx5 is expressed in the anterior dorsal region of the telencephalon. Dashed line marks the telencephalon-diencephalon border. D: dorsal; A: anterior ventral. Scale bar: 100 µm for lateral view; 60 µm for frontal view. (B) BAC modification via recombination methods. Vertical blue bars represent exons of the lhx5 gene. The Kaede expression cassette replaced the first exon of lhx5 gene. pA: polyadenylation signal sequence; Kan: kanamycin resistant marker; FRT, flippase recognition target. (C) Photo-conversion of Kaede in Tg(lhx5BAC:Kaede) transgenic embryos demonstrates ADt neurons project axons ventrally into the AC and SOT. A live Tg(lhx5BAC:Kaede) transgenic animal was mounted in tilted frontal view to reveal the AC and SOT simultaneously. The region of the left telencephalon was selected for photoconversion. D: dorsal; A: anterior ventral; AC: anterior commissure; SOT: supraoptic tract. Scale bar: 60 µm.
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pone-0036516-g001: ADt neurons project axons ventrally.In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower left corners. Lateral, animals mounted in lateral view, rostral to the left; Frontal, animals mounted in frontal view, dorsal to the top. (A) lhx5 is expressed in the anterior dorsal region of the telencephalon. Dashed line marks the telencephalon-diencephalon border. D: dorsal; A: anterior ventral. Scale bar: 100 µm for lateral view; 60 µm for frontal view. (B) BAC modification via recombination methods. Vertical blue bars represent exons of the lhx5 gene. The Kaede expression cassette replaced the first exon of lhx5 gene. pA: polyadenylation signal sequence; Kan: kanamycin resistant marker; FRT, flippase recognition target. (C) Photo-conversion of Kaede in Tg(lhx5BAC:Kaede) transgenic embryos demonstrates ADt neurons project axons ventrally into the AC and SOT. A live Tg(lhx5BAC:Kaede) transgenic animal was mounted in tilted frontal view to reveal the AC and SOT simultaneously. The region of the left telencephalon was selected for photoconversion. D: dorsal; A: anterior ventral; AC: anterior commissure; SOT: supraoptic tract. Scale bar: 60 µm.

Mentions: In the developing zebrafish forebrain, a cluster of cells are specified as neurons in the dorsal rostral region of the neural tube. By the pharyngula period, these telencephalic neurons have extended axons along a commissural tract (the anterior commissure, AC) and a descending tract (the supraoptic tract, SOT) [20]. In the anterior dorsal telencephalon, ADt neurons express lhx5 (Fig. 1A), which encodes a LIM homeodomain protein that regulates the antagonism of Wnt signaling, thus promoting forebrain development [21]. To visualize the lhx5 expressing ADt neurons and their axons in live intact animals, we generated transgenic zebrafish expressing the photo-convertible fluorescent protein Kaede [22] using a bacteria artificial chromosome (BAC) homologous recombination method [23], [24]. The BAC clone carried approximately 200 kb of zebrafish genomic sequence, of which approximately 110 kb was upstream of the transcriptional start site of the lhx5 gene (Fig. 1B). The modified BAC construct contained Kaede coding sequence that replaced the first exon of the lhx5 gene. Two lines of Tg(lhx5BAC:Kaede) stable transgenic fish were obtained after screening 80 of the modified BAC injected fish. The expression patterns of the Kaede protein were identical between the two lines. We maintained one of the Tg(lhx5BAC:Kaede) lines (referred to as lhx5:Kaede hereafter).


Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish.

Gao J, Zhang C, Yang B, Sun L, Zhang C, Westerfield M, Peng G - PLoS ONE (2012)

ADt neurons project axons ventrally.In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower left corners. Lateral, animals mounted in lateral view, rostral to the left; Frontal, animals mounted in frontal view, dorsal to the top. (A) lhx5 is expressed in the anterior dorsal region of the telencephalon. Dashed line marks the telencephalon-diencephalon border. D: dorsal; A: anterior ventral. Scale bar: 100 µm for lateral view; 60 µm for frontal view. (B) BAC modification via recombination methods. Vertical blue bars represent exons of the lhx5 gene. The Kaede expression cassette replaced the first exon of lhx5 gene. pA: polyadenylation signal sequence; Kan: kanamycin resistant marker; FRT, flippase recognition target. (C) Photo-conversion of Kaede in Tg(lhx5BAC:Kaede) transgenic embryos demonstrates ADt neurons project axons ventrally into the AC and SOT. A live Tg(lhx5BAC:Kaede) transgenic animal was mounted in tilted frontal view to reveal the AC and SOT simultaneously. The region of the left telencephalon was selected for photoconversion. D: dorsal; A: anterior ventral; AC: anterior commissure; SOT: supraoptic tract. Scale bar: 60 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351449&req=5

pone-0036516-g001: ADt neurons project axons ventrally.In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower left corners. Lateral, animals mounted in lateral view, rostral to the left; Frontal, animals mounted in frontal view, dorsal to the top. (A) lhx5 is expressed in the anterior dorsal region of the telencephalon. Dashed line marks the telencephalon-diencephalon border. D: dorsal; A: anterior ventral. Scale bar: 100 µm for lateral view; 60 µm for frontal view. (B) BAC modification via recombination methods. Vertical blue bars represent exons of the lhx5 gene. The Kaede expression cassette replaced the first exon of lhx5 gene. pA: polyadenylation signal sequence; Kan: kanamycin resistant marker; FRT, flippase recognition target. (C) Photo-conversion of Kaede in Tg(lhx5BAC:Kaede) transgenic embryos demonstrates ADt neurons project axons ventrally into the AC and SOT. A live Tg(lhx5BAC:Kaede) transgenic animal was mounted in tilted frontal view to reveal the AC and SOT simultaneously. The region of the left telencephalon was selected for photoconversion. D: dorsal; A: anterior ventral; AC: anterior commissure; SOT: supraoptic tract. Scale bar: 60 µm.
Mentions: In the developing zebrafish forebrain, a cluster of cells are specified as neurons in the dorsal rostral region of the neural tube. By the pharyngula period, these telencephalic neurons have extended axons along a commissural tract (the anterior commissure, AC) and a descending tract (the supraoptic tract, SOT) [20]. In the anterior dorsal telencephalon, ADt neurons express lhx5 (Fig. 1A), which encodes a LIM homeodomain protein that regulates the antagonism of Wnt signaling, thus promoting forebrain development [21]. To visualize the lhx5 expressing ADt neurons and their axons in live intact animals, we generated transgenic zebrafish expressing the photo-convertible fluorescent protein Kaede [22] using a bacteria artificial chromosome (BAC) homologous recombination method [23], [24]. The BAC clone carried approximately 200 kb of zebrafish genomic sequence, of which approximately 110 kb was upstream of the transcriptional start site of the lhx5 gene (Fig. 1B). The modified BAC construct contained Kaede coding sequence that replaced the first exon of the lhx5 gene. Two lines of Tg(lhx5BAC:Kaede) stable transgenic fish were obtained after screening 80 of the modified BAC injected fish. The expression patterns of the Kaede protein were identical between the two lines. We maintained one of the Tg(lhx5BAC:Kaede) lines (referred to as lhx5:Kaede hereafter).

Bottom Line: We found that ADt axons normally project ventrally.We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function.We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China.

ABSTRACT
The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.

Show MeSH
Related in: MedlinePlus