Suppression of interneuron programs and maintenance of selected spinal motor neuron fates by the transcription factor AML1/Runx1 Nicolas Stifani*, Adriana R. O. Freitas*, Anna Liakhovitskaia†, Alexander Medvinsky†, Artur Kania‡§¶, and Stefano Stifani*ʈ *Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, QC, Canada H3A 2B4; †Institute for Stem Cell Research/Medical Research Council Centre for Stem Cell Biology, University of Edinburgh, Edinburgh EH9 3JQ, United Kingdom; ‡Department of Anatomy and Cell Biology, Division of Experimental Medicine, McGill University, Montreal, QC, Canada H3A 2B2; §De´partement de Me´decine, Universite´de Montreal, Montreal, QC, Canada H3C 3JY; and ¶Neural Circuit Development Laboratory, Institut de recherches cliniques de Montreal, Montreal, QC, Canada H2W 1R7 Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved February 29, 2008 (received for review November 29, 2007) Individual spinal motor neuron identities are specified in large part pattern by examining a previously characterized Runx1lacZ/ϩ by the intrinsic repertoire of transcription factors expressed by knockin mouse line in which ␤-galactosidase (␤-gal) expression undifferentiated progenitors and maturing neurons. It is shown recapitulates the expression of endogenous Runx1 (13, 18, 19). In here that the transcription factor AML1/Runx1 (Runx1) is expressed embryo day (E)9.5 Runx1lacZ/ϩ embryos, ␤-gal expression coin- in selected spinal motor neuron subtypes after the onset of cided with Runx1 protein, detected by using a previously de- differentiation and is both necessary and sufficient to suppress scribed anti-Runx1 antibody (17), in the second branchial arch, interneuron-specific developmental programs and promote main- where Runx1 mRNA expression has been demonstrated (18) tenance of motor neuron characteristics. These findings show an [supporting information (SI) Fig. S1 A–C]. The specificity of the important role for Runx1 during the consolidation of selected spinal motor neuron identities. Moreover, they suggest a require- anti-Runx1 antibody was demonstrated by the absence of im- ment for a persistent suppression of interneuron genes within munoreactivity in Runx1-deficient embryos (Fig. S1 E and F). lacZ/ϩ ␤ maturing motor neurons. In the spinal cord of Runx1 embryos, -gal expression began at ϷE9.5 in a small number of ventrolateral cells at levels ϩ lateral motor column ͉ median motor column ͉ runt ͉ spinal cord C1–C4 (Fig. 1A, arrowheads). ␤-gal cells did not express spinal accessory column neuronal progenitor markers such as Pax6 or Nkx2.2, nor the general cell proliferation marker Ki67 (Fig. 1 A–C). Moreover, we observed no detectable overlap between the expression of pecific transcription factor codes within exclusive ventral ␤ Sprogenitor domains regulate motor neuron and interneuron -gal and the interneuron markers Evx1 (V0 interneurons), En1 differentiation in the developing spinal cord (1, 2). Some deter- (V1), and Chx10 (V2) (Fig. 1 D–F), although some occasional minants of both lineages are coexpressed in mitotic progenitors overlap with Chx10 was observed at later stages (see Fig. 3 NEUROSCIENCE ϩ (3), raising the questions of what molecules control the diver- below). Instead, ␤-gal cells expressed the postmitotic motor gence and maintenance of motor neuron and interneuron dif- neuron markers Isl1 and choline acetyltransferase (ChAT) (3, 4, ferentiation programs. Genetic studies suggest that the gene Hb9 20) (Fig. 1 G and H, arrows). Only a subset of the Isl1ϩ cells is required to suppress interneuron programs actively in matur- expressed ␤-gal, suggesting that Runx1 is expressed in a restricted ing motor neurons (3, 4), but other effectors of the mechanisms number of spinal motor neurons. The expression of ␤-gal that promote the divergence of motor and interneuron fates faithfully recapitulated the Runx1 expression pattern in the remain to be determined. spinal cord (Fig. S1 H–J). These results indicate that Runx1 In both invertebrates and vertebrates, the runt/Runx gene expression in the spinal cord is first activated in a subpopulation family encodes DNA-binding transcription factors that mediate of postmitotic motor neurons, but not their progenitors, at transactivation or repression depending on specific contexts (5). cervical levels C1–C4. Members of this transcription factor family regulate neuron subtype specification and axon target connectivity in Drosophila (6–8), chick (9, 10), and mice (11–16). The runt/Runx family Runx1 Expression Is Activated in Ventrally Exiting Motor Neurons member AML1/Runx1 (Runx1) is expressed in selected popula- After the Onset of Differentiation. To determine the identity of the cervical Isl1ϩ neurons in which ␤-gal is first expressed in tions of motor neurons in the murine and avian spinal cord, ϩ suggesting that it is involved in motor neuron development (13, Runx1lacZ/ embryos, we compared the expression of ␤-gal with 17). Here, we show that mouse Runx1 is expressed in restricted that of the Hb9 protein, which is expressed in virtually all motor groups of ventrally exiting cervical motor neurons during their neurons whose axons exit via the ventral root and innervate postmitotic development. Loss of Runx1 function does not affect skeletal muscles (3, 4). No visible overlap of ␤-gal and Hb9 the survival of those motor neurons but results in a loss of expression was observed at E9.5 (data not shown) and E10.5, the expression of motor neuron-specific genes and a concomitant peak of motor neuron generation (Fig. 1 J–L). At E9.5, we also activation of expression of interneuron-specific genes. Con- versely, ectopic expression of Runx1 in the spinal cord of developing chick embryos suppresses interneuron gene expres- Author contributions: N.S. and A.R.O.F. contributed equally to this work; N.S., A.K., and S.S. sion and promotes motor neuron differentiation programs. designed research; N.S., A.R.O.F., and A.L. performed research; A.L. and A.M. contributed These results identify a role for Runx1 in the establishment of new reagents/analytic tools; N.S., A.R.O.F., A.K., and S.S. analyzed data; and N.S., A.K., and selected motor neuron identities and suggest that maturing S.S. wrote the paper. motor neurons must continually suppress interneuron-specific The authors declare no conflict of interest. developmental programs. This article is a PNAS Direct Submission. ʈTo whom correspondence should be addressed at: Montreal Neurological Institute, 3801 Results rue University, Montreal, QC H3A 2B4, Canada. E-mail: [email protected]. Runx1 Is Expressed in Specific Postmitotic Motor Neurons in the Mouse This article contains supporting information online at www.pnas.org/cgi/content/full/ Cervical Spinal Cord. To determine the role of Runx1 in spinal 0711299105/DCSupplemental. motor neuron development, we first characterized its expression © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711299105 PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 ͉ 6451–6456 Downloaded by guest on October 1, 2021 Fig. 1. Runx1 expression in spinal motor neurons. (A–H) Expression of ␤-gal and the indicated proteins in the spinal cord of E9.5 (A–C and G) or E10.5 (D–F and H) Runx1lacZ/ϩ embryos. ␤-gal expression does not overlap with proliferation (A–C) or ventral interneuron (D–F) markers, but it overlaps with the motor neuron markers ChAT and Isl1 (G and H, arrows). Arrowheads in (A–C) point to ␤-galϩ cells. (I) Summary of Runx1 expression (green) in the cervical spinal cord (SC) at E9.5; the location of the progenitor domain, ventral interneuron 0, 1, and 2 domains, and vMN domain are indicated. (J–O) Expression of ␤-gal and Hb9 in the cervical spinal cord of Runx1lacZ/ϩ embryos. No overlap is observed at E10.5 (J–L, arrowheads), whereas ␤-galϩ/Hb9ϩ cells are visible in a ventromedial domain at E11.5 (M–O, arrows; shown at higher magnification in the Inset). (P–R) Coexpression of ␤-gal and Phox2b in the cervical spinal cord of E9.5 Runx1lacZ/ϩ embryos. Virtually all ␤-galϩ cells coexpress Phox2b (arrows). (S) Analysis of axonal projections in E15.5 Runx1lacZ/ϩ embryos by using retrograde labeling by rhodamine- dextran (Rho) placed in the anterior portion of the trapezius muscle. Arrows point to examples of ␤-galϩ cells that were retrogradely labeled. (T) Schematic displaying the location of the ventrolateral domain of the spinal cord analyzed in S.(U) Summary of Runx1 expression in the cervical SC at E11.5; the lateral domain containing Runx1ϩ cells that express Isl1 and Phox2b and project to the anterior trapezius muscle is shown in yellow, and the ventromedial domain containing Runx1ϩ/Hb9ϩ cells is shown in green; LEP, lateral exit point. When shown, dotted lines depict outline of the spinal cord. (D–S) Dorsal is to the Top and lateral to the Right. failed to detect an overlap between the expression of ␤-gal and and Hb9 expression (E9.5–E10.5). These results suggest that that of Lhx3, a protein whose expression at this stage transiently Runx1 becomes activated in specific vMNs after their initial marks the majority of ventrally exiting motor neurons (vMNs) as differentiation and during their developmental maturation. well as their precursors (20) (data not shown). Coexpression of ␤-gal and Hb9 was first observed in the ventromedial spinal cord Runx1 Is Expressed in Selected Types of Ventrally Exiting Motor at ϷE11 (Fig. 1 M–O, arrows, and data not shown). At this stage, Neurons and Is Not Required for Their Generation or Survival. In Runx1 expression expands ventrally and can be detected at more E13.5 Runx1lacZ/ϩ embryos, when distinct motor columns are caudal levels of the spinal cord (C5–T1) (13). This analysis of discernable, two groups of ␤-galϩ motor neurons were observed Runx1lacZ/ϩ embryos suggests that the onset of Runx1 expression at the forelimb level (C5–C8) (Fig.