and fig. S9I). Thus, some but not all biophysical
properties driven by Dlk1 are mediated by the
secondary actor Kcng4.
The Dlk1 isoforms expressed in the mouse
spinal cord can give rise to membrane-tethered
or cleaved extracellular proteins (fig. S10, A and
B) (11, 12). We therefore forcedly expressed a
noncleavable form of Dlk1 (Dlk1NC) or the extracellular segment of Dlk1 (Dlk1ES) (fig. S10B)
in chick motor neurons. We observed that Dlk1NC,
but not Dlk1ES, promoted fast properties (Fig. 4C).
We further observed that only motor neurons
forcedly expressing Dlk1, but not adjacent nontransfected motor neurons (fig. S10C), exhibited
altered properties (Fig. 4C), together suggesting
that Dlk1 operates cell-autonomously to promote
a fast biophysical signature.
In preadipocytes, Dlk1 actions involve the inhibition of Notch signaling (17). Indeed, our expression of Dlk1 completely abolished the induction
of a reporter for Notch activation in Xenopus embryos (Fig. 4D and fig. S10D). Moreover, forced
expression of the canonical Notch activator Delta-like 1 (18) did not recapitulate the effects of excess Dlk1 on chick motor neuron properties (Fig.
4C). Furthermore, cotransfection of constitutively
active Notch1 abolished the ability of excess Dlk1
to alter motor neuron properties (Fig. 4B), suggesting that Dlk1 action in motor neurons relies
on Notch inhibition. Because Notch signaling is
generally involved in cell fate decisions (18), it is
likely that Dlk1 action involves additional pathways to promote fast motor neuron identity.
Here we have shown that Dlk1 is both neces-
sary and sufficient for determining fast motor
neurons and their corresponding biophysical
signature in the mouse and chick (fig. S10E).
Dlk1 implements expression of motor neuron
type–specific genes such as Kcng4, which mod-
ulates a subset of neural activity parameters. The
result is a biophysical signature in motor neurons
that supports peak neuromuscular outputs. The
strategy by which expression of a neural activity
modulator is confined to a subset of neurons may
similarly drive functional diversity elsewhere in
the developing nervous system.
The overall lack of topographic organization
for slow or fast motor neurons suggests that
motor neuron type is acquired independently of
the mechanisms that, before muscle innervation,
determine motor neuron positional (column or
pool) identities (19, 20). We still do not know
when subsets of motor neurons acquire type-specific biophysical signatures, to what extent
motor neuron functional diversification involves
signals from muscle (21), how motor neuron and
muscle fiber types are matched (22–24), or what
causes the differential vulnerability of motor neuron types to disease or aging (25). However,
57 years after the characterization of fast and
slow motor neurons (1), we can now have insight
into the molecular mechanisms that control their
development and function.
References and Notes
1. J. C. Eccles, R. M. Eccles, A. Lundberg, Nature 179,
2. R. E. Burke, D. N. Levine, F. E. Zajac 3rd, P. Tsairis,
W. K. Engel, Science 174, 709–712 (1971).
3. D. Kernell, The Motoneuron and Its Muscle Fibers (Oxford
Univ. Press, New York, ed. 1, 2006).
4. J. E. Zengel, S. A. Reid, G. W. Sypert, J. B. Munson,
J. Neurophysiol. 53, 1323–1344 (1985).
5. R. Bakels, D. Kernell, J. Physiol. 463, 307–324
6. M. Manuel, C. J. Heckman, J. Neurosci. 31,
7. E. Henneman, Science 126, 1345–1347 (1957).
8. D. Kernell, Science 152, 1637–1639 (1966).
9. M. Gustafsson, M. J. Pinter, Trends Neurosci. 8, 431–433
10. E. Davis et al., Curr. Biol. 14, 1858–1862 (2004).
11. H. S. Sul, Mol. Endocrinol. 23, 1717–1725 (2009).
12. S. R. Ferrón et al., Nature 475, 381–385 (2011).
13. S. Grillner, in Handbook of Physiology: The Nervous
System, Motor Control, Vol. 2, V. B. Brooks, Ed.
(American Physiological Society, Bethesda, MD 1981),
14. N. Ottschytsch, A. Raes, D. Van Hoorick, D. J. Snyders,
Proc. Natl. Acad. Sci. U.S.A. 99, 7986–7991
15. H. Murakoshi, J. S. Trimmer, J. Neurosci. 19, 1728–1735
16. J. M. Wilson, J. Rempel, R. M. Brownstone, J. Comp.
Neurol. 474, 13–23 (2004).
17. M. L. Nueda, V. Baladrón, B. Sánchez-Solana,
M. A. Ballesteros, J. Laborda, J. Mol. Biol. 367,
18. K. Hori, A. Sen, S. Artavanis-Tsakonas, J. Cell Sci. 126,
19. W. A. Alaynick, T. M. Jessell, S. L. Pfaff, Cell 146, 178, e1
20. J. S. Dasen, T. M. Jessell, Curr. Top. Dev. Biol. 88,
21. J. V. Chakkalakal, H. Nishimune, J. L. Ruas,
B. M. Spiegelman, J. R. Sanes, Development 137,
22. W. J. Thompson, L. A. Sutton, D. A. Riley, Nature 309,
23. T. Fladby, J. K. Jansen, Development 109, 723–732
24. V. F. Rafuse, L. D. Milner, L. T. Landmesser, J. Neurosci.
16, 6864–6877 (1996).
25. K. C. Kanning, A. Kaplan, C. E. Henderson, Annu. Rev.
Neurosci. 33, 409–440 (2010).
Acknowledgments: We thank A. Klusowski, B. Veith, T. V. Bui,
and T. G. Hampton for technical support; S. Schiaffino and
K. Kawakami for reagents; P. D'Adamo for phenotyping service;
and O. Shomroni and S. Bonn for bioinformatics advice.
This research received funding from the European Research
Council under the European Union's Seventh Framework
Programme (FP/2007-2013)/ERC Grant Agreement 311710-MU
TUNING and the Emmy-Noether Program of the Deutsche
Forschungsgemeinschaft (MA4278/1-1). S.R.B. was supported
by the Center for Biologics Evaluation and Research, Food
and Drug Administration, operating funds; O.S. by the
Emmy-Noether Program (SCHL592-4); R.M.B. by the Canadian
Institutes of Health Research, Canada Research Chairs Program,
Canada Foundation for Innovation, and Nova Scotia Research and
Innovation Trust; and J.L. by the Ministerio de Educación y
Ciencia, Spain, from whom Dlk1ko mice are available through a
materials transfer agreement. Dlk1fx mice are deposited at
jaxmice.jax.org (no. 019074). Notch1:Gal4 is available from
R. Kopan (Cincinnati Children's Hospital Medical Center).
ENI-G is a cooperation of University Medical Center Göttingen
and the Max-Planck Society.
Materials and Methods
Figs. S1 to S10
Tables S1 to S9
25 September 2013; accepted 14 February 2014