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doi: 10.1242/10.1242/dev.00358

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Regulation of motor neuron subtype identity by repressor activity of Mnx class homeodomain proteins

Christopher M. William1, Yasuto Tanabe1,2 and Thomas M. Jessell1,*

1 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, 701 West 168th Street, New York, NY 10032, USA
2 Mitsubishi Kagaku Institute of Life Sciences, PREST, Japan Science and Technology, 11 Minami-Ooya, Machida-shi, Tokyo 194 8511, Japan



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  		Fig. 1. Restricted expression of MNR2 and HB9 by postmitotic spinal motor neurons. Transverse sections through stage 29 chick spinal cord reveal the columnar restriction of MNR2 (A,C,and E) and HB9 (B,D,F) expression within motor neurons. (A) At brachial levels of the spinal cord, MNR2 is expressed in the medial MMC and in a lateral population of motor neurons, located within the confines of the LMC. (B) At brachial levels, HB9 is expressed by medial MMC and a subset of lateral LMC neurons. Isl1/2 is expressed in all medial MMC and LMC neurons. A dorsal population of interneurons also expresses Isl1. (C) At thoracic levels, MNR2 is expressed in all medial (m) MMC neurons but by only a few lateral (l) MMC neurons and not by CT neurons. (D) At thoracic levels, HB9 is expressed in all MMC neurons but not in CT neurons. (E) At lumbar levels, MNR2 is expressed in medial MMC neurons but not in LMC neurons. (F) At lumbar levels, HB9 is expressed in medial MMC and lateral LMC neurons. INT, interneuron.

 


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  		Fig. 2. Motor neuron inducing activity of MNR2 in chick spinal cord. (A) Widespread unilateral expression of MNR2 in stage 24 chick spinal cord, obtained by unilateral (right side) in ovo electroporation at stages 10-12. (B-F) Ectopic dorsal expression, after MNR2 electroporation, of Lim3 (B), Isl1 (C), Isl2 (D), HB9 (E) and SC1 (F). Images are representative of more than 40 electroporated embryos.

 


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  		Fig. 3. Molecular markers of CT and LMC motor neuron subtype identity. (A) Selective expression of Lim3 by medial MMC neurons at brachial levels of stage 29 spinal cord. (B) Selective expression of RALDH2 by LMC neurons at brachial levels of stage 29 spinal cord. RALDH2 is also expressed by roof-plate cells. (C) Co-expression of Isl2 and Lim1 by lateral LMC neurons at brachial levels of stage 29 spinal cord. (D) Pattern of Isl1/2 expression in motor neurons and dorsal interneurons at brachial levels of stage 29 spinal cord. (E,F) Absence of expression of BMP5 in motor neurons at brachial levels of the spinal cord. Arrows (F) indicate sites of BMP5 expression in roof-plate (left arrow) and mesenchymal cells surrounding the spinal cord (right arrow). (G) Expression of ephrinA5 in a subset of LMC motor neurons at brachial levels of the spinal cord. Arrow indicates ephrinA5 expression in a small group of cells just ventral to the dorsal root entry zone. (H) Motor neuron columnar organization at brachial levels of the spinal cord. (I,J) Selective expression of BMP5 in CT motor neurons at thoracic levels of the spinal cord. BMP5 is also expressed by roof-plate cells. (K) Expression of ephrinA5 in CT motor neurons at thoracic levels of the spinal cord. (L) Motor neuron columnar organization at thoracic levels of the spinal cord. (M) Widespread expression of MNR2 in dorsal spinal cord after in ovo electroporation of MNR2. Analysis performed at stage 29. (N) Co-expression of Lim3 by Isl1/2+ ectopic motor neurons after MNR2 electroporation. (O) Lack of expression of BMP5 in ectopic motor neurons at thoracic levels after MNR2 electroporation. (P) Lack of expression of RALDH2 in ectopic motor neurons at limb levels after MNR2 electroporation. Images are representative of over 30 electroporated embryos

 


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  		Fig. 7. Evidence that MNR2 can function as a transcriptional repressor. (A) Deletion isoforms of MNR2 tested for Lim3-inducing activity in vivo. Approximate activity of these proteins, assessed by induction of Lim3+ cells, is indicated on the right, by comparison with wild-type MNR2. Electroporation of each of these constructs was performed at stage 10-12, and stage 25 embryos were analyzed for expression of Lim3. +++, more than 20 Lim3+ cells per 12 µM section; ++, 10-12 Lim3+ cells per section; +, 10 cells per section; –, no ectopic Lim3+ cells per section. Six to ten embryos were analyzed for the activity of each construct. (B) Deletion of the C-terminal domain of MNR2 does not abolish Lim3 inductive activity. Expression of the MNR2 HD alone has no inductive activity. (C) The N-terminal domain of MNR2 functions as a potent transcriptional repressor when fused to a Gal4 DNA-binding domain in vitro. The repressive activity of the MNR2 domain is similar to that of a Gal4-Engrailed repressor domain fusion. A MyoD-Gal4 DNA-binding domain fusion acts as a potent transcriptional activator. COS-1 cells were co-transfected with Gal4-fusion constructs and a Gal4-E1b-luciferase reporter plasmid. In controls, a Gal4-MyoD construct activated E1b-luciferase activity 19.2±3.6-fold (s.e.m.) overexpression of Gal4 alone. A Gal4-EnR construct repressed E1b-luciferase activity 7.7±4.5-fold (s.e.m.). A fusion of Gal4 to protein sequence N-terminal to the MNR2 HD repressed E1b-luciferase activity 8.3±1.3-fold (s.e.m.). (D) Detection of an eh1 domain in all Mnx class HD proteins. MNR2 protein sequence is represented as a black bar. The HD is depicted as a white box and an N-terminal eh1 motif is depicted with a gray box. The sequence containing the eh1 motif in MNR2 has been aligned with other Mnx class HD proteins to illustrate the high degree of conservation of this motif. An alignment with the eh1 motif in Engrailed reveals several identical residues, highlighted by black circles. Eh1 motifs characterized in Nkx proteins are aligned below Engrailed. Black circles below the Nkx6.1 sequence highlight residues in either Nkx2.2 or Nkx6.1 that are identical to Engrailed. Black diamonds above MNR2 and below Nkx6.1 highlight acidic residues conserved between Mnx class and Nkx HD proteins. cMNR2, chick MNR2; hHB9, human HB9; AmphiHB9, Amphioxus homolog of HB9; dHB9, Drosophila homolog of HB9; dEn, Drosophila Engrailed; mNkx2.2, mouse Nkx2.2; rNkx6.1, rat Nkx6.1. (E) Expression of an MNR2 isoform lacking the 14 N-terminal residues retains wild-type Lim3 inducing activity. (F) Negligible Lim3 inductive activity of an MNR2 HD-Engrailed repressor domain fusion protein. Images are representative of over 10 electroporated embryos.

 


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  		Fig. 4. Suppression of CT neuron generation by MNR2, HB9 and Lim3. (A) Widespread ectopic expression of MNR2 protein on the right side of spinal cord after MNR2 electroporation. (B-D) Loss of dorsomedially located Isl1+ motor neurons (B), and absence of expression of BMP5 (C) and ephrinA5 (D) after unilateral MNR2 electroporation. (E) Widespread ectopic expression of HB9 protein on the right side of spinal cord after HB9 electroporation. (F-H) Loss of dorsomedially located Isl1+ motor neurons (F) and absence of expression of BMP5 (G) and ephrinA5 (H) after unilateral Hb9 electroporation. (I) Widespread ectopic expression of Lim3 protein on the right side of spinal cord after Lim3 electroporation. (J-K) Loss of dorsomedially located Isl1+ motor neurons (J) and absence of BMP5 expression (K), after unilateral Lim3 electroporation. (L) Ectopic dorsal Chx10+ (V2) interneurons induced by Lim3 electroporation. Brackets indicate area of expression on the untreated side. Images are representative of 10-30 electoporated embryos.

 


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  		Fig. 5. Hierarchical interactions between MNR2, HB9 and Lim3 in the suppression of CT neuron generation. (A-E) Electroporation of MNR2 within the normal domain of motor neuron generation at thoracic levels (A) represses BMP5 expression (B), but does not result in maintained Lim3 (C,D) or HB9 (E) expression in motor neurons. (F-J) Electroporation of Lim3 within the normal domain of motor neuron generation at thoracic levels (F) represses BMP5 expression (G), and results in maintained MNR2 (H) and HB9 (I,J) expression in postmitotic motor neurons. Images are representative of 20 electroporated embryos. Arrows in C-E,H-J indicate the approximate position of newly generated CT neurons, prior to their dorsal migration.

 


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  		Fig. 6. Repression of LMC neuron specification by Lim3 but not by MNR2 or HB9. (A-C) Electroporation of MNR2 within the normal domain of LMC neuron generation at brachial levels of the spinal cord does not maintain Lim3 expression in postmitotic LMC neurons. B and C, enlarged view of boxed area in A. (D) Electroporation of HB9 within the normal domain of LMC neuron generation at brachial levels of the spinal cord does not maintain Lim3 expression in postmitotic LMC neurons. (E-H) Electroporation of MNR2 within the normal domain of LMC neuron generation at brachial levels of the spinal cord does not repress expression of RALDH2 or the generation of motor neurons that co-express Isl2 and Lim1. G and H, enlarged view of boxed area in E. (I-L) Electroporation of Lim3 within the normal domain of LMC neuron generation at brachial levels of the spinal cord represses expression of RALDH2 and the generation of motor neurons that co-express Isl2 and Lim1. K and L, enlarged view of boxed area in J. Images are representative of 10-30 electroporated embryos.

 


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  		Fig. 8. Inductive activity of a MNR2-E1a C-terminal domain fusion protein. (A) Position of PxxL motifs in MNR2 and related Mnx class HD proteins. (B) Diagram of MNR2HD-E1a C-terminal fusion proteins. (C) Widespread ectopic expression of MNR2 HD-E1a C-terminal fusion protein. (D) Lim3 inductive activity of MNR2 HD-E1a C-terminal fusion protein in vivo. (E) Widespread expression of the mutated MNR2 HD-E1a C-terminal fusion protein. (F) Lack of Lim3 inductive activity of MNR2 HD-E1a C-terminal fusion protein mutated in its three C-terminal residues. (G) Widespread expression of MNR2 HD-E1a C-terminal fusion protein in spinal cord at stage 29 after in ovo electroporation. (H) Repression of CT neuron generation (arrow) by MNR2 HD-E1a C-terminal fusion protein. The MNR2 HD fused to a VP-16 activation domain was also inactive in inducing Lim3 expression (data not shown). Images are representative of over 20 electroporated embryos per construct.

 


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  		Fig. 9. Progressive restrictions in homeodomain protein expression and the assignment of motor neuron columnar identity. (A) Profiles of HD and Olig2 protein expression in motor neuron progenitors and postmitotic motor neurons. Proteins shown in black designate expression in columnar neuronal subsets where expression is required for progression to a specific motor neuron columnar fate. Proteins shown in gray indicate expression in a particular subset of motor neurons under conditions in which these proteins do not influence motor neuron subtype determination, as assessed by gain- and loss-of-function studies. Late-stage motor neuron progenitors express Nkx6.1, MNR2, Lim3 and Olig2. Cell-cycle exit is accompanied by the extinction of Olig2 expression, the loss of Nkx6.1 from most motor neurons, and by the onset of expression of Isl1, Isl2 (not shown) and HB9. The emergence of medial MMC [MMC(m)] neuronal fate is accompanied by the persistence of expression of Isl1, HB9, MNR2 and Lim3, whereas lateral MMC [MMC(l)] and LMC fates are associated with the extinction of expression of Lim3 and MNR2. CT neuronal fate is associated with the extinction of MNR2, Lim3 and HB9 expression. Within the lateral MMC and LMC lineages, Lim3 represses motor neuron columnar fates independent of its ability to induce MNR2 expression, whereas within the CT lineage, the Lim3-mediated repression of CT identity is likely to be mediated by induction of Mnx class HD protein expression. (B) In dorsal neural progenitors, the repressor activity of MNR2 appears to induce motor neuron differentiation by repression of intermediate repressors (Xn) that function to repress expression of Lim3 and Isl1, proteins that when co-expressed have the capacity to direct motor neuron generation.

 

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