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NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord.

Leggere JC, Saito Y, Darnell RB, Tessier-Lavigne M, Junge HJ, Chen Z - Elife (2016)

Bottom Line: RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system.We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance.Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, United States.

ABSTRACT
RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

No MeSH data available.


Related in: MedlinePlus

Fluorescent in situ hybridization of Dcc in cultured embryos electroporated with Actb-gfp.Nova dKO was used as there are more GFP+ neurons in the VZ. Dcc expression is seen in GFP+ neuroprogenitors and interneurons. Scale bars, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.14264.009
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fig2s1: Fluorescent in situ hybridization of Dcc in cultured embryos electroporated with Actb-gfp.Nova dKO was used as there are more GFP+ neurons in the VZ. Dcc expression is seen in GFP+ neuroprogenitors and interneurons. Scale bars, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.14264.009

Mentions: To determine the identity of the ACTB-GFP+ neurons and axons, we immunostained the cultured embryos with neuronal and axonal markers. We used PAX3/7 antibodies to label the dorsal interneuron progenitors that give rise to different populations of commissural and ipsilateral neurons (Caspary and Anderson, 2003; Helms and Johnson, 2003). Pax3 and Pax7 are required to specify the majority of commissural neurons, as their double knockout greatly reduces the ventral commissure formed by commissural axons crossing the midline (Mansouri and Gruss, 1998). We labeled differentiated interneurons with antibodies against BARHL2 and LHX5, two transcription factors among many others that are expressed by post-mitotic interneurons. Combinations of these transcription factors define the different lineages of commissural and ipsilateral neurons (Caspary and Anderson, 2003; Helms and Johnson, 2003). We also labeled commissural axons specifically with anti-ROBO3 (Sabatier et al., 2004). Our studies show that the GFP+ neurons within the VZ are dorsal interneuron progenitors and the ones at the lateral spinal cord are differentiated interneurons. In addition, the GFP+ axons that fail to project to the midline are commissural axons (Figure 2). Furthermore, using fluorescent in situ hybridization, we found that the GFP+ neuroprogenitors and interneurons also express Dcc (Figure 2—figure supplement 1), consistent with previous reports that Dcc is expressed in these neuronal populations (Keino-Masu et al., 1996; Phan et al., 2011). Taken together, Nova deficiency appears to disturb commissural axon projection and also interferes with earlier stages of commissural neuron development in the progenitors.10.7554/eLife.14264.008Figure 2.Expression of neuronal and axonal markers in cultured Nova WT and dKO embryos electroporated with Actb-gfp.


NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord.

Leggere JC, Saito Y, Darnell RB, Tessier-Lavigne M, Junge HJ, Chen Z - Elife (2016)

Fluorescent in situ hybridization of Dcc in cultured embryos electroporated with Actb-gfp.Nova dKO was used as there are more GFP+ neurons in the VZ. Dcc expression is seen in GFP+ neuroprogenitors and interneurons. Scale bars, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.14264.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930329&req=5

fig2s1: Fluorescent in situ hybridization of Dcc in cultured embryos electroporated with Actb-gfp.Nova dKO was used as there are more GFP+ neurons in the VZ. Dcc expression is seen in GFP+ neuroprogenitors and interneurons. Scale bars, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.14264.009
Mentions: To determine the identity of the ACTB-GFP+ neurons and axons, we immunostained the cultured embryos with neuronal and axonal markers. We used PAX3/7 antibodies to label the dorsal interneuron progenitors that give rise to different populations of commissural and ipsilateral neurons (Caspary and Anderson, 2003; Helms and Johnson, 2003). Pax3 and Pax7 are required to specify the majority of commissural neurons, as their double knockout greatly reduces the ventral commissure formed by commissural axons crossing the midline (Mansouri and Gruss, 1998). We labeled differentiated interneurons with antibodies against BARHL2 and LHX5, two transcription factors among many others that are expressed by post-mitotic interneurons. Combinations of these transcription factors define the different lineages of commissural and ipsilateral neurons (Caspary and Anderson, 2003; Helms and Johnson, 2003). We also labeled commissural axons specifically with anti-ROBO3 (Sabatier et al., 2004). Our studies show that the GFP+ neurons within the VZ are dorsal interneuron progenitors and the ones at the lateral spinal cord are differentiated interneurons. In addition, the GFP+ axons that fail to project to the midline are commissural axons (Figure 2). Furthermore, using fluorescent in situ hybridization, we found that the GFP+ neuroprogenitors and interneurons also express Dcc (Figure 2—figure supplement 1), consistent with previous reports that Dcc is expressed in these neuronal populations (Keino-Masu et al., 1996; Phan et al., 2011). Taken together, Nova deficiency appears to disturb commissural axon projection and also interferes with earlier stages of commissural neuron development in the progenitors.10.7554/eLife.14264.008Figure 2.Expression of neuronal and axonal markers in cultured Nova WT and dKO embryos electroporated with Actb-gfp.

Bottom Line: RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system.We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance.Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, United States.

ABSTRACT
RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

No MeSH data available.


Related in: MedlinePlus