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First published online 4 December 2008
doi: 10.1242/dev.028043

Development 136, 231-240 (2009)
Published by The Company of Biologists 2009
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Nolz1 is induced by retinoid signals and controls motoneuron subtype identity through distinct repressor activities

Sheng-Jian Ji*, Goran Periz and Shanthini Sockanathan{dagger}

The Solomon Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, MD 21205, USA.


Figure 1
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  		Fig. 1. Nolz1 expression is induced by RA signals in spinal cord explants. (A) Conserved domains between chick (c), mouse (m), zebrafish (z) and human (h) predicted Nolz1 ORFs. Sp, Sp1-related domain; FKPY, putative Groucho consensus binding site; Btd, Drosophila Buttonhead domain; ZF, zinc-finger domain. Numbers indicate percentage identity in the regions between these domains. (B) Reverse northern of two different chick Nolz1 EST clones (EST1, EST2) probed with pools of cDNAs generated from St 19 ventral chick embryonic spinal cord explants grown in the presence (+) or absence (-) of retinol (Rol). c29.1 is a cDNA fragment from an unrelated gene known to be transcriptionally unresponsive to RA signals (Rao and Sockanathan, 2005Go). (C,D) Confocal images of chick spinal cords electroporated with RAR403 showing decreased dorsal Nolz1 expression (asterisk). Motoneurons are reduced as their generation is dependent on RA signaling.

 

Figure 2
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  		Fig. 2. Nolz1 is expressed in subsets of postmitotic spinal MNs. (A-H) In situ hybridization of Nolz1 mRNA expression on transverse sections of embryonic chick spinal cords. (I-R) Confocal micrographs showing analysis of Nolz1 protein expression as compared with molecular markers of specific spinal motor columns. Arrows mark Nolz1 and Lim3 coexpressing MNs. (S) Summary of Nolz1 expression in relation to motor column development. The dashed orange line indicates transient Nolz1 expression. MMC, median motor column; MMCm, medial division of the MMC; MMCl, lateral division of the MMC; LMC, lateral motor column; LMCm, medial division of the LMC; LMCl, lateral division of the LMC; CT, column of Terni.

 

Figure 3
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  		Fig. 3. Nolz1 ablation causes a decrease of Lim3- motor columns. (A-D,F-I) Confocal micrographs of sectioned St 23/24 chick spinal cords electroporated with Nolz1 siRNAs and examined for Nolz1 expression and motor column markers by immunohistochemistry at forelimb (FL) and thoracic (T) levels of the spinal cord. (E,J) Bar charts showing the percentage of MNs corresponding to different motor columns in electroporated Nolz1 siRNAs (black bars), control siRNAs (gray bars) and non-electroporated contralateral (white bars, set as 100%) sides of the spinal cord at forelimb (E) and thoracic (J) regions. n=4-5 embryos, mean ± s.e.m.

 

Figure 4
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  		Fig. 4. Nolz1 and Grg5 interact and function as transcriptional repressors. (A) In situ hybridization of Grg5 mRNA expression in transverse sections of chick embryonic spinal cords. (B) Co-immunoprecipitation (IP) analyses of Grg5 protein interactions with Nolz1. (C) Schematic of the Nolz1 coding region. FKPY, putative Grg consensus binding site; Btd, Buttonhead domain; ZF, atypical zinc-finger domain. Lines beneath indicate the regions deleted within the Nolz1 coding sequence; the numbers indicate the corresponding amino acids that are deleted. Whether Nolz1 retains (+) or loses (-) its ability to interact with Grg5 in co-IP is indicated alongside. (D) Luciferase-based transcriptional assays using extracts from transfected COS-7 cells. Data are from three replicate experiments, mean ± s.e.m. Student's t-test compared with Nolz1: Nolz1+Grg5, P=0.0055; Nolz1{Delta}C22, P=0.0016. (E) Luciferase-based transcriptional assays using extracts from electroporated chick spinal cords. n=6-8 embryos, mean ± s.e.m. Student's t-test compared with Nolz1: Nolz1+Grg5, P<0.000001; Nolz1{Delta}C22, P=0.0029. Compared with GAL4-only: Nolz1+Grg5, P=0.0003.

 

Figure 5
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  		Fig. 5. Nolz1 represses Lim3 expression in postmitotic MNs. (A-F) Confocal micrographs of St 21 chick spinal cord sections electroporated on the right. The high levels of Nolz1 elicited by overexpression preclude the detection of endogenous Nolz1 in the spinal cord under these imaging conditions. (G) Bar chart quantifying the percentage of electroporated cells expressing Lim3. n=10 embryos, mean ± s.e.m.

 

Figure 6
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  		Fig. 6. Nolz1-Grg5 complexes induce postmitotic MNs. Transverse sections of St 21 embryonic chick spinal cords electroporated on the right. White brackets denote ectopic MNs. (A-H,J-M) Immunohistochemical analyses using antibodies against MN and ventral interneuron markers. Arrows indicate ectopic MNs expressing the MN surface marker SC1 (J) or Chx10 (K). (I) Arrows mark ectopic MNs expressing transcripts for choline acetyltransferase (ChAT). Dashed lines mark the lateral extent of the spinal cord.

 

Figure 7
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  		Fig. 7. Grg5 knockdown decreases the number of Lim3- motor columns. (A-D) St 24 chick embryos electroporated with Grg5 siRNAs. (A) The right-hand half of the spinal cord is electroporated, as indicated by GFP expression. (B) In situ hybridization of Grg5 mRNA showing efficient ablation of Grg5 mRNA (asterisk) on the Grg5 siRNA-electroporated side. (C,D) Confocal micrographs of sectioned chick spinal cords showing immunohistochemical analysis of motor column markers. (E) Bar chart showing the percentage of motor columns in electroporated Grg5 siRNAs (black bars), control siRNAs (gray bars) and contralateral non-electroporated (white bars, set as 100%) sides of the spinal cord at forelimb regions. n=4-5 embryos, mean ± s.e.m.

 

Figure 8
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  		Fig. 8. Coexpression of Nolz1 and Grg5 induces Hoxc6 expression and partial lateral LMC specification. (A-C) Confocal micrographs of ventral chick embryonic spinal cords showing that the onset of Nolz1 expression occurs prior to that of Hoxc6. The dashed line outlines the spinal cord. (D-G) Confocal micrographs of electroporated dorsal right halves of St 23 chick spinal cords. Grg5 expression is not shown but is ~95-98% coincident with exogenous Nolz1. The dashed line marks the lateral boundary of the spinal cord. (H,I) Confocal micrographs of the ventral MN domain of St 23/24 chick embryonic spinal cords electroporated on the right. Yellow cells in panel I mark thoracic MNs that express Hoxc6. (J-M) Confocal micrographs of electroporated dorsal right halves of St 23 chick spinal cords. The dashed line marks the lateral boundary of the spinal cord. The right-hand side is electroporated in all cases. The high levels of Nolz1 elicited by overexpression preclude the detection of endogenous Nolz1 under these imaging conditions. (N) Model for Nolz1-dependent specification of MN subtype identity. Orange lines mark cells expressing Nolz1. Broken orange line refers to transient Nolz1 expression. Nolz1 downregulates Lim3 expression in prospective Lim3-negative MNs and, in a complex with Grg5, maintains the expression of key MN determinants. At limb levels, Nolz1-Grg5 complexes confer forelimb LMC identity through induction of Hoxc6, and induce Lim1 expression in prospective LMCl MNs.

 

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