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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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Dcc is required for correct asymmetric outgrowth of ADt neuronal axons.(A) Dcc expression was reduced after injection of dcc translation-blocking morpholinos into zebrafish embryos. Endogenous Dcc protein was detected as a band of approximately 170 kb. Tubulin served as a loading control. M: size marker. (B) ADt neurons project axons dorsally when Dcc function is inhibited by morpholino injection. Images of live animals were acquired as in Fig. 1C. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (C) 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. Asterisks and brackets represent p<0.05 by Mann-Whitney U test.
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pone-0036516-g003: Dcc is required for correct asymmetric outgrowth of ADt neuronal axons.(A) Dcc expression was reduced after injection of dcc translation-blocking morpholinos into zebrafish embryos. Endogenous Dcc protein was detected as a band of approximately 170 kb. Tubulin served as a loading control. M: size marker. (B) ADt neurons project axons dorsally when Dcc function is inhibited by morpholino injection. Images of live animals were acquired as in Fig. 1C. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (C) 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. Asterisks and brackets represent p<0.05 by Mann-Whitney U test.

Mentions: To inhibit Dcc function in zebrafish, we injected a morpholino antisense oligonucleotide (dcc-MO) that blocked Dcc protein synthesis [29]. In embryos injected with dcc-MO (6 ng per embryo), the expression of Dcc protein was reduced to levels below the detection limits of our Western blot analyses (Fig. 3A). Photo-conversion of Kaede in dcc-MO injected lhx5:Kaede animals revealed that ADt neurons sent long processes dorsally, in addition to the normal ventrally projecting axons (Fig. 3B). These dorsally projecting processes sometimes occupied lateral positions and extended posteriorly, or they crossed the midline and extended into the contralateral side of the telencephalon (Fig. 3B). Injection of dcc-MO did not affect ADt neuron migration from the medial region to the lateral region of the telencephalon. The dorsally projecting processes were rarely observed in animals injected with a standard control morpholino. To summarize and compare our results, we analyzed each confocal data file and quantified the pixel intensity values of aberrant dorsal processes using custom written ImageJ macro and MATLAB scripts (Fig. S1). To present the quantified results, we assigned phenotypic scores (Grade 0–3) to each embryo based on the pixel intensity value (Fig. S1). Embryos with more pronounced dorsal processes were assigned higher scores (Fig. 3C). Average phenotypic scores (a.p.s.) were calculated for each treatment group where a higher a.p.s. reflected more severe aberrant dorsal projections. For statistical analyses, we used the non-parametric Mann-Whitney U method to test whether samples of two independent observations had equally severe phenotypic values. The rank based Mann-Whitney U test had the advantage that the statistical conclusions were independent of the specific phenotypic score matrix we employed. Based on this analysis, the dcc-MO injected embryos (a.p.s = 0.983) were significantly different from the standard control morpholino injected animals (a.p.s = 0.063) (Fig. 3C, p = 7.01×10−7). Similar statistical results were obtained using the original quantification data and the ANOVA method (Fig. S1). Injection with a lower dosage of dcc-MO (3 ng per embryo) reduced the severity (a.p.s. = 0.500) of the aberrant dorsal axons. Injection with a higher dosage of dcc-MO (12 ng per embryo) caused early death in about 30% of the injected embryos (52 out of 156). The intensity of the abnormal dorsal axon was increased in the remaining embryos, but the differences were not statistically significant (a.p.s = 1.13, p = 0.425 versus embryos injected with 6 ng of dcc-MO). Embryos were injected with 6 ng of dcc-MO in following experiments.


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)

Dcc is required for correct asymmetric outgrowth of ADt neuronal axons.(A) Dcc expression was reduced after injection of dcc translation-blocking morpholinos into zebrafish embryos. Endogenous Dcc protein was detected as a band of approximately 170 kb. Tubulin served as a loading control. M: size marker. (B) ADt neurons project axons dorsally when Dcc function is inhibited by morpholino injection. Images of live animals were acquired as in Fig. 1C. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (C) 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. Asterisks and brackets represent p<0.05 by Mann-Whitney U test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351449&req=5

pone-0036516-g003: Dcc is required for correct asymmetric outgrowth of ADt neuronal axons.(A) Dcc expression was reduced after injection of dcc translation-blocking morpholinos into zebrafish embryos. Endogenous Dcc protein was detected as a band of approximately 170 kb. Tubulin served as a loading control. M: size marker. (B) ADt neurons project axons dorsally when Dcc function is inhibited by morpholino injection. Images of live animals were acquired as in Fig. 1C. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (C) 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. Asterisks and brackets represent p<0.05 by Mann-Whitney U test.
Mentions: To inhibit Dcc function in zebrafish, we injected a morpholino antisense oligonucleotide (dcc-MO) that blocked Dcc protein synthesis [29]. In embryos injected with dcc-MO (6 ng per embryo), the expression of Dcc protein was reduced to levels below the detection limits of our Western blot analyses (Fig. 3A). Photo-conversion of Kaede in dcc-MO injected lhx5:Kaede animals revealed that ADt neurons sent long processes dorsally, in addition to the normal ventrally projecting axons (Fig. 3B). These dorsally projecting processes sometimes occupied lateral positions and extended posteriorly, or they crossed the midline and extended into the contralateral side of the telencephalon (Fig. 3B). Injection of dcc-MO did not affect ADt neuron migration from the medial region to the lateral region of the telencephalon. The dorsally projecting processes were rarely observed in animals injected with a standard control morpholino. To summarize and compare our results, we analyzed each confocal data file and quantified the pixel intensity values of aberrant dorsal processes using custom written ImageJ macro and MATLAB scripts (Fig. S1). To present the quantified results, we assigned phenotypic scores (Grade 0–3) to each embryo based on the pixel intensity value (Fig. S1). Embryos with more pronounced dorsal processes were assigned higher scores (Fig. 3C). Average phenotypic scores (a.p.s.) were calculated for each treatment group where a higher a.p.s. reflected more severe aberrant dorsal projections. For statistical analyses, we used the non-parametric Mann-Whitney U method to test whether samples of two independent observations had equally severe phenotypic values. The rank based Mann-Whitney U test had the advantage that the statistical conclusions were independent of the specific phenotypic score matrix we employed. Based on this analysis, the dcc-MO injected embryos (a.p.s = 0.983) were significantly different from the standard control morpholino injected animals (a.p.s = 0.063) (Fig. 3C, p = 7.01×10−7). Similar statistical results were obtained using the original quantification data and the ANOVA method (Fig. S1). Injection with a lower dosage of dcc-MO (3 ng per embryo) reduced the severity (a.p.s. = 0.500) of the aberrant dorsal axons. Injection with a higher dosage of dcc-MO (12 ng per embryo) caused early death in about 30% of the injected embryos (52 out of 156). The intensity of the abnormal dorsal axon was increased in the remaining embryos, but the differences were not statistically significant (a.p.s = 1.13, p = 0.425 versus embryos injected with 6 ng of dcc-MO). Embryos were injected with 6 ng of dcc-MO in following experiments.

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