Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of inhibition of Netrin1 or Neogenin1 function on the ADt axons.(A) ADt neurons project axons dorsally when Netrin1 function is inhibited (ntn-MO). Knockdown of Neogenin1 function doesn’t cause ADt neuron to project axon dorsally (neo-MO). Images were processed as in Fig. 3B. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (B) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. (C) Synergistic effects between sub-threshold Dcc-Netrin1 and Dcc-Neogenin morpholino knockdowns. dcc-sub_MO and ntn-sub_MO: sub-threshold concentration morpholino. At least three independent injections were performed for each treatment group. Numbers inside parentheses denote numbers of animals analyzed. Asterisks represent p<0.001 by ANOVA test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351449&req=5

pone-0036516-g005: Effects of inhibition of Netrin1 or Neogenin1 function on the ADt axons.(A) ADt neurons project axons dorsally when Netrin1 function is inhibited (ntn-MO). Knockdown of Neogenin1 function doesn’t cause ADt neuron to project axon dorsally (neo-MO). Images were processed as in Fig. 3B. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (B) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. (C) Synergistic effects between sub-threshold Dcc-Netrin1 and Dcc-Neogenin morpholino knockdowns. dcc-sub_MO and ntn-sub_MO: sub-threshold concentration morpholino. At least three independent injections were performed for each treatment group. Numbers inside parentheses denote numbers of animals analyzed. Asterisks represent p<0.001 by ANOVA test.

Mentions: To provide further evidence for the role of Dcc in the asymmetric growth of ADt axons, we examined the effect of knocking down Netrin1, a Dcc ligand. In lhx5:Kaede embryos co-injected with ntn1a and ntn1b morpholinos (referred to as ntn1-MO, 6 ng each per embryo), ADt neurons developed aberrant dorsal projections similar to neurons in dcc-MO injected embryos (Fig. 5A, a.p.s. = 0.857, p = 0.820 versus dcc-MO injected embryos). Injection of either ntn1a-MO or ntn1b-MO alone didn’t cause ADt neurons to project axons dorsally. Co-injection of ntn1a and ntn1b mismatch control morpholinos didn’t cause ADt neurons to develop aberrant projections (data not shown). Knocking down both Dcc and Netrin1 by co-injecting dcc-MO (6 ng per embryo) and ntn1-MO (3 ng each per embryos) caused the ADt neurons to develop aberrant projections in a manner similar to knock down of Dcc alone (a.p.s. = 1.09, Fig. 5B and Fig. S2). A lower dose of ntn1-MO (3 ng each versus 6 ng each per embryo) was used in the Dcc-Netrin1 double knockdown experiment due to concerns over potential toxicity of high dose morpholino injections.


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)

Effects of inhibition of Netrin1 or Neogenin1 function on the ADt axons.(A) ADt neurons project axons dorsally when Netrin1 function is inhibited (ntn-MO). Knockdown of Neogenin1 function doesn’t cause ADt neuron to project axon dorsally (neo-MO). Images were processed as in Fig. 3B. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (B) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. (C) Synergistic effects between sub-threshold Dcc-Netrin1 and Dcc-Neogenin morpholino knockdowns. dcc-sub_MO and ntn-sub_MO: sub-threshold concentration morpholino. At least three independent injections were performed for each treatment group. Numbers inside parentheses denote numbers of animals analyzed. Asterisks represent p<0.001 by ANOVA test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036516-g005: Effects of inhibition of Netrin1 or Neogenin1 function on the ADt axons.(A) ADt neurons project axons dorsally when Netrin1 function is inhibited (ntn-MO). Knockdown of Neogenin1 function doesn’t cause ADt neuron to project axon dorsally (neo-MO). Images were processed as in Fig. 3B. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (B) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. (C) Synergistic effects between sub-threshold Dcc-Netrin1 and Dcc-Neogenin morpholino knockdowns. dcc-sub_MO and ntn-sub_MO: sub-threshold concentration morpholino. At least three independent injections were performed for each treatment group. Numbers inside parentheses denote numbers of animals analyzed. Asterisks represent p<0.001 by ANOVA test.
Mentions: To provide further evidence for the role of Dcc in the asymmetric growth of ADt axons, we examined the effect of knocking down Netrin1, a Dcc ligand. In lhx5:Kaede embryos co-injected with ntn1a and ntn1b morpholinos (referred to as ntn1-MO, 6 ng each per embryo), ADt neurons developed aberrant dorsal projections similar to neurons in dcc-MO injected embryos (Fig. 5A, a.p.s. = 0.857, p = 0.820 versus dcc-MO injected embryos). Injection of either ntn1a-MO or ntn1b-MO alone didn’t cause ADt neurons to project axons dorsally. Co-injection of ntn1a and ntn1b mismatch control morpholinos didn’t cause ADt neurons to develop aberrant projections (data not shown). Knocking down both Dcc and Netrin1 by co-injecting dcc-MO (6 ng per embryo) and ntn1-MO (3 ng each per embryos) caused the ADt neurons to develop aberrant projections in a manner similar to knock down of Dcc alone (a.p.s. = 1.09, Fig. 5B and Fig. S2). A lower dose of ntn1-MO (3 ng each versus 6 ng each per embryo) was used in the Dcc-Netrin1 double knockdown experiment due to concerns over potential toxicity of high dose morpholino injections.

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