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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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Inhibition of Dcc function causes ADt axons to project dorsally or to form multiple processes.(A) Labeling of individual ADt neurons by mosaic expression of fluorescent protein tdTomato. Image of a live 36 hpf Tg(lhx5BAC:Kaede) animal that was injected with emx3:Gal4FF and UAS:tdTomato plasmids is shown. The tdTomato labeled neuron projected an axon into the AC. Merge panel shows the position of the tdTomato labeled soma (marked by an arrowhead). Scale bar = 50 µm. (B) Injection of dcc morpholino causes ADt neurons to project axons dorsally or to form multiple processes. Labeled ADt neurons are marked by arrowheads in the merged panels. Left panels show an ADt neuron with a normal ventrally projecting axon in a control animal. Middle panels show an ADt neuron with an aberrant dorsally projecting axon in a dcc-MO injected animal. Right panels show an ADt neuron with both ventrally and dorsally projecting processes. Black arrow in the single slice images indicates the origin of the axon on the surface of the cell body. Red bar indicates the middle of the dorsal and the ventral side of the labeled neuron cell body. Scale bar equals to 20 µm in the projected images or 10 µm in the single slice images. (C) Additional examples of ADt neurons with multiple aberrant axons in dcc-MO injected animals. Scale bar = 15 µm.
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pone-0036516-g004: Inhibition of Dcc function causes ADt axons to project dorsally or to form multiple processes.(A) Labeling of individual ADt neurons by mosaic expression of fluorescent protein tdTomato. Image of a live 36 hpf Tg(lhx5BAC:Kaede) animal that was injected with emx3:Gal4FF and UAS:tdTomato plasmids is shown. The tdTomato labeled neuron projected an axon into the AC. Merge panel shows the position of the tdTomato labeled soma (marked by an arrowhead). Scale bar = 50 µm. (B) Injection of dcc morpholino causes ADt neurons to project axons dorsally or to form multiple processes. Labeled ADt neurons are marked by arrowheads in the merged panels. Left panels show an ADt neuron with a normal ventrally projecting axon in a control animal. Middle panels show an ADt neuron with an aberrant dorsally projecting axon in a dcc-MO injected animal. Right panels show an ADt neuron with both ventrally and dorsally projecting processes. Black arrow in the single slice images indicates the origin of the axon on the surface of the cell body. Red bar indicates the middle of the dorsal and the ventral side of the labeled neuron cell body. Scale bar equals to 20 µm in the projected images or 10 µm in the single slice images. (C) Additional examples of ADt neurons with multiple aberrant axons in dcc-MO injected animals. Scale bar = 15 µm.

Mentions: Because our Kaede photo-conversion results could not discriminate whether the ADt neurons extended single aberrant or multiple processes in the dcc-MO injected embryos, we labeled single ADt neurons with a transient transgenic method (Fig. 4A). Plasmids injected into early stage zebrafish embryos are mosaically distributed among the dividing cells, and mosaic expression of transgenes encoded by the injected plasmids may result in random labeling of single or small numbers of cells in the injected embryos [30], [31]. We coinjected a plasmid carrying emx3:Gal4FF and a plasmid carrying UAS:tdTomato, with and without dcc-MO into lhx5:Kaede embryos at the one cell stage. The emx3:Gal4FF plasmid carries a Gal4 transcriptional activator variant [32] under control of a forebrain specific enhancer element from the emx3 gene [33]. The UAS:tdTomato plasmid carries tdTomato, a red fluorescent protein gene [34] under control of 5 copies of the Gal4 recognition element UAS. By screening the injected embryos for axons that can be traced and assigned to single labeled ADt neurons, we obtained ADt neuronal projection patterns resolvable at single cell resolution (Fig. 4B). Among the labeled ADt neurons in control animals, none had dorsally projecting axons (Fig. 4B, n = 80). In contrast, about 40% (19 out of 50) of the labeled ADt neurons in dcc-MO injected animals had either dorsally projecting axons or multiple processes (Fig. 4B). The dorsally projecting axons extended laterally or crossed the midline to extend into the contralateral telencephalon (n = 7). The axons with multiple processes often projected both dorsally and ventrally (n = 12). We further examined all single confocal slices to determine the origins of the axons on the surface of the labeled neurons (Fig. 4B). In control embryos, the origins of the axons were found overwhelmingly on the ventral side of the cell bodies (92%, 74 out of 80). The other 6 labeled neurons either had the axon origins on the dorsal side (4%, n = 3) or close to the middle (4%, n = 3) of the cell bodies. In contrast, for those labeled neurons that had dorsally projecting axons in dcc-MO injected embryos, the origins of the axons were found on the dorsal side of the cell bodies (84%, 16 out of 19). Thus, the single cell labeling results mirrored our results obtained from group labeling, and together these results suggest that Dcc function is required for the asymmetric outgrowth of the ADt axons. Furthermore, because the labeled ADt neurons always extend single axons in control animals, the formation of ADt neuron axons with multiple processes in dcc morpholino injected embryos (Figure 4C) suggest that Dcc function is required for polarized axon initiation of the forebrain neurons in vivo.


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)

Inhibition of Dcc function causes ADt axons to project dorsally or to form multiple processes.(A) Labeling of individual ADt neurons by mosaic expression of fluorescent protein tdTomato. Image of a live 36 hpf Tg(lhx5BAC:Kaede) animal that was injected with emx3:Gal4FF and UAS:tdTomato plasmids is shown. The tdTomato labeled neuron projected an axon into the AC. Merge panel shows the position of the tdTomato labeled soma (marked by an arrowhead). Scale bar = 50 µm. (B) Injection of dcc morpholino causes ADt neurons to project axons dorsally or to form multiple processes. Labeled ADt neurons are marked by arrowheads in the merged panels. Left panels show an ADt neuron with a normal ventrally projecting axon in a control animal. Middle panels show an ADt neuron with an aberrant dorsally projecting axon in a dcc-MO injected animal. Right panels show an ADt neuron with both ventrally and dorsally projecting processes. Black arrow in the single slice images indicates the origin of the axon on the surface of the cell body. Red bar indicates the middle of the dorsal and the ventral side of the labeled neuron cell body. Scale bar equals to 20 µm in the projected images or 10 µm in the single slice images. (C) Additional examples of ADt neurons with multiple aberrant axons in dcc-MO injected animals. Scale bar = 15 µm.
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Related In: Results  -  Collection

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pone-0036516-g004: Inhibition of Dcc function causes ADt axons to project dorsally or to form multiple processes.(A) Labeling of individual ADt neurons by mosaic expression of fluorescent protein tdTomato. Image of a live 36 hpf Tg(lhx5BAC:Kaede) animal that was injected with emx3:Gal4FF and UAS:tdTomato plasmids is shown. The tdTomato labeled neuron projected an axon into the AC. Merge panel shows the position of the tdTomato labeled soma (marked by an arrowhead). Scale bar = 50 µm. (B) Injection of dcc morpholino causes ADt neurons to project axons dorsally or to form multiple processes. Labeled ADt neurons are marked by arrowheads in the merged panels. Left panels show an ADt neuron with a normal ventrally projecting axon in a control animal. Middle panels show an ADt neuron with an aberrant dorsally projecting axon in a dcc-MO injected animal. Right panels show an ADt neuron with both ventrally and dorsally projecting processes. Black arrow in the single slice images indicates the origin of the axon on the surface of the cell body. Red bar indicates the middle of the dorsal and the ventral side of the labeled neuron cell body. Scale bar equals to 20 µm in the projected images or 10 µm in the single slice images. (C) Additional examples of ADt neurons with multiple aberrant axons in dcc-MO injected animals. Scale bar = 15 µm.
Mentions: Because our Kaede photo-conversion results could not discriminate whether the ADt neurons extended single aberrant or multiple processes in the dcc-MO injected embryos, we labeled single ADt neurons with a transient transgenic method (Fig. 4A). Plasmids injected into early stage zebrafish embryos are mosaically distributed among the dividing cells, and mosaic expression of transgenes encoded by the injected plasmids may result in random labeling of single or small numbers of cells in the injected embryos [30], [31]. We coinjected a plasmid carrying emx3:Gal4FF and a plasmid carrying UAS:tdTomato, with and without dcc-MO into lhx5:Kaede embryos at the one cell stage. The emx3:Gal4FF plasmid carries a Gal4 transcriptional activator variant [32] under control of a forebrain specific enhancer element from the emx3 gene [33]. The UAS:tdTomato plasmid carries tdTomato, a red fluorescent protein gene [34] under control of 5 copies of the Gal4 recognition element UAS. By screening the injected embryos for axons that can be traced and assigned to single labeled ADt neurons, we obtained ADt neuronal projection patterns resolvable at single cell resolution (Fig. 4B). Among the labeled ADt neurons in control animals, none had dorsally projecting axons (Fig. 4B, n = 80). In contrast, about 40% (19 out of 50) of the labeled ADt neurons in dcc-MO injected animals had either dorsally projecting axons or multiple processes (Fig. 4B). The dorsally projecting axons extended laterally or crossed the midline to extend into the contralateral telencephalon (n = 7). The axons with multiple processes often projected both dorsally and ventrally (n = 12). We further examined all single confocal slices to determine the origins of the axons on the surface of the labeled neurons (Fig. 4B). In control embryos, the origins of the axons were found overwhelmingly on the ventral side of the cell bodies (92%, 74 out of 80). The other 6 labeled neurons either had the axon origins on the dorsal side (4%, n = 3) or close to the middle (4%, n = 3) of the cell bodies. In contrast, for those labeled neurons that had dorsally projecting axons in dcc-MO injected embryos, the origins of the axons were found on the dorsal side of the cell bodies (84%, 16 out of 19). Thus, the single cell labeling results mirrored our results obtained from group labeling, and together these results suggest that Dcc function is required for the asymmetric outgrowth of the ADt axons. Furthermore, because the labeled ADt neurons always extend single axons in control animals, the formation of ADt neuron axons with multiple processes in dcc morpholino injected embryos (Figure 4C) suggest that Dcc function is required for polarized axon initiation of the forebrain neurons in vivo.

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