Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
Abstract
During development, different classes of neurons and glia are generated
from proliferative progenitor cells lining the ventricles of the brain
and the lumen of the spinal cord. A central issue in developmental
neuroscience is to understand the mechanisms by which these cells are
generated in space and over time. In the ventral spinal cord, the
expression profile of homeodomain (HD) proteins defines five progenitor
domains that each will give rise to a distinct type of neuron. Two
closely related HD transcriptional repressors, Nkx6.1 and Nkx6.2 (Nkx6),
are expressed by progenitors of the ventral spinal cord. We provide
evidence that different levels of Nkx6 repressor activity in progenitor
cells are a critical determinant of ventral neuronal fate, assigning both
redundant and specific roles for these proteins in neuronal
specification. A reduction in Nkx6 activity further permits V0
interneurons to be generated from progenitors that lack HD proteins
normally required for their generation, providing direct evidence for a
model where HD proteins direct specific cell fates by actively repressing
the expression of transcription factors that direct alternative fates.
In the ventral spinal cord, sMNs and oligodendrocyte precursors (OLPs)
are sequentially generated from a domain defined by the expression of
Olig2. We show that the generation of sMNs and OLPs in the ventral spinal
cord is essentially missing in mice lacking Nkx6 function. In contrast,
the same HD proteins instead act to suppress OLP specification in the
ventral hindbrain. The divergent roles for Nkx6 proteins seem to reflect
that OLPs in the spinal cord and hindbrain are produced by distinct
ventral progenitor domains. While a ventral specification of OLPs is well
established, it has remained unclear whether also more dorsal progenitor
cells give rise to oligodendrocytes. We provide in vivo and in vitro
evidence that oligodendrocytes are produced also by dorsal progenitors in
the spinal cord and hindbrain and that the specification of these cells
may result from the progressive evasion of dorsal BUT signalling over
time. Together, our data suggest that oligodendrocytes are generated from
multiple dorsoventral origins in the spinal cord and hindbrain, and
indicate that the activation of Olig2 at different positions is
controlled by distinct genetic programs.
The observation that the loss of sMNs in the spinal cord of Nkx6 mutant
mice correlates with an extinguished expression of the sMN determinant
Olig2 has led to a model where Nkx6 proteins act strictly upstream of
Olig2. However, in the hindbrain of Nkx6 mutant mice the initial
expression of Olig2 is intact and despite this all sMNs are missing,
indicating a parallel requirement for Nkx6 and Olig2 proteins in the
generation of sMNs. Visceral motor neurons (vMNs) are generated
immediately ventral to sMNs. The transcription factor Phox2b has been
found to be an important determinant of these cells, but other factors
involved have not been identified. We show that the HD protein Nkx2.2 is
sufficient to mediate the expression of Phox2b. Furthermore, while the
activities of Nkx6.1 and Nkx6.2 are dispensable for the initial
generation of vMNs, they are required to prevent a parallel program of
more dorsal interneuronal differentiation and to ensure a proper
migration and axonal projection formation of vMNs. Thus, Nkx2 and Nkx6
proteins appear to have complementary roles in the establishment of vMN
identity in the hindbrain. Taken together, our data suggest that both
visceral and somatic motor neuron differentiation rely on the combined
activity of cell intrinsic determinants, rather than on a singe key
determinant of neuronal cell fate.
Neuronal diversity is established by mechanisms that operate in space and
over time. Advances have been made in regard to the mechanisms that
restrict and direct neuronal generation in space, but less is known about
the mechanisms that underlie how neural progenitors produce distinct
types of neurons in a specific temporal order. We addressed this issue by
studying a population of progenitor cells in the ventral hindbrain that
gives rise to vMNs and serotonergic (S) neurons. Each hindbrain segment,
or rhombomere (r), initially generates vMNs, but all the rhombomeres
except for r4 switch to producing S neurons at a defined time point. We
found that the temporal and spatial generation of vMNs and S neurons
critically relies on the integrated activity of Nkx- and Hox-class HD
proteins. A primary function of these proteins is to coordinate the
activation of Phox2b in space and time. Phox2b, in turn, functions as a
binary switch in deciding whether progenitors differentiate into vMNs or
serotonergic neurons. Taken together, these data indicate that
determinants that control spatial patterning may be associated also with
temporal patterning and require that expression patterns are dynamic and
modulated over time