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. 2012 Sep;139(17):3109-19.
doi: 10.1242/dev.078501. Epub 2012 Jul 25.

Onecut Transcription Factors Act Upstream of Isl1 to Regulate Spinal Motoneuron Diversification

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Free PMC article

Onecut Transcription Factors Act Upstream of Isl1 to Regulate Spinal Motoneuron Diversification

Agnès Roy et al. Development. .
Free PMC article

Abstract

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.

Figures

Fig. 1.
Fig. 1.
OC factors are required to maintain Isl1 expression during MN subtype diversification. (A-H) Triple label immunofluorescence analysis of Olig2, Hb9 and Isl1 on a single spinal cord section of control mouse embryos or embryos double mutant for Hnf6 and Oc2 (Hnf6/Oc2–/–) at E9.5. At this developmental stage, the spinal cord is not yet divided into distinct brachial, thoracic and lumbar regions, and newly born MNs form a homogeneous column along the anteroposterior axis of the spinal cord. In the absence of OC factors, Isl1 is present in the ‘intermediate’ population (II) but is almost completely absent from newly born MNs (III). I, pMN domain. (I) Quantification of Olig2+ progenitors and Hb9+ or Isl1+ newly born MNs in E9.5 Hnf6/Oc2–/– embryos and control littermates. (J-M) In Hnf6/Oc2–/– embryos at E10.5, the number of thoracic Hb9+ MNs is normal but Isl1 is lost from the newly born MNs. (N) Quantification of Isl1+ or Hb9+ MNs in control and Hnf6/Oc2–/– spinal cord at E10.5. *, P<0.05; error bars indicate s.e.m. Scale bar: 100 μm.
Fig. 2.
Fig. 2.
OC factors directly stimulate the expression of Isl1. (A-C) Overexpression of Hnf6 or Oc2 in chick embryonic spinal cord at HH14 results in ectopic Isl1 expression 6 hours after electroporation. (D) Binding of endogenous OC factors to potential binding sites in Isl1 enhancers was assessed by ChIP (left, gel; right, quantitative real-time PCR) on chick embryonic spinal cord extracts with an anti-Hnf6 antibody or species-matched IgG. The noggin promoter, which contains no potential OC binding site, served as negative control. OC proteins are bound to sites in the CREST2 enhancer, but not to the CREST1 or CREST3 regions. *, P<0.05; error bars indicate s.e.m. (E-G) A reporter construct in which GFP expression is regulated by the CREST2 enhancer is exclusively activated in MNs expressing OC factors (E). This reporter construct is strongly stimulated by Hnf6 co-expression (F,G). Scale bars: 100 μm.
Fig. 3.
Fig. 3.
OC factors control the ratio between somatic and visceral MNs. (A-F) Distribution of visceral MN markers at thoracic levels in control and Hnf6/Oc2–/– mouse embryos at E10.5 unveils a loss of Foxp1 and Sip1 in the absence of OC factors. (G) Quantitative analysis of prospective visceral MN markers at E10.5 on thoracic spinal cord sections of control and Hnf6/Oc2–/– embryos. (H) Comparison of Isl1 protein levels in prospective somatic or visceral MNs in wild-type embryos at E10.5 demonstrates that Isl1 content in prospective visceral MNs is half that in prospective somatic MNs. (I-T) Distribution of MN columnar subtype markers at thoracic levels in control and Hnf6/Oc2–/– spinal cord at E12.5 shows an expansion of visceral MNs at the expense of somatic MNs. (U) Quantitative analysis of MN columnar subtype markers on thoracic spinal cord sections of control and Hnf6/Oc2–/– embryos at E12.5. (V) Quantitative analysis of Isl1 and Foxp1 in visceral MNs on thoracic sections of control and Hnf6/Oc2–/– spinal cord at E12.5. (W) Although the somatic MN population is reduced in Hnf6/Oc2–/– (DKO) spinal cord at E12.5, the ratio between MMC and HMC neurons is unchanged. (X) In Hnf6/Oc2–/– embryos, the total number of MNs at E12.5 is normal but the ratio between somatic and visceral MNs is modified. (Y,Z) Open-book view of control and Hnf6/Oc2–/– spinal cord summarizes the observations obtained at thoracic levels of the spinal cord, i.e. the modification of the ratio between somatic and visceral MNs, the absence of Isl1 and Foxp1 in PGC neurons and the context-dependent regulation of Isl1 by OC factors. (AA-CC) Overexpression of Hnf6 in chick embryonic spinal cord at HH14 results, 96 hours after electroporation (Σ), in a reduction in visceral MNs of the column of Terni and in an expansion of somatic MNs. *, P<0.05; **, P<0.01; ***, P<0.001; error bars indicate s.e.m. PGC, preganglionic column; MMC, median motor column; HMC, hypaxial motor column. Scale bars: 100 μm in A-F; 125 μm in I-L,O-R; 170 μm in M,S; 200 μm in AA,BB.
Fig. 4.
Fig. 4.
OC factors are dispensable for axonal projections of thoracic MNs. (A-F) Whole-mount neurofilament labeling indicates that axonal projections at thoracic levels of the mouse spinal cord are preserved in Hnf6/Oc2–/– embryos. (G-K) MN projections to the paravertebral ganglionic chain were studied by retrograde transport of Rhodamine-dextran in E12.5 control and Hnf6/Oc2–/– embryos. Accuracy of the injection was confirmed by the presence of Rhodamine-dextran in nNOS+ PGC neurons but not in Hb9+ somatic MNs. Supernumerary visceral MNs in Hnf6/Oc2–/– embryos innervate paravertebral ganglia. *, P<0.05; error bars indicate s.e.m. SC, spinal cord; DRG, dorsal root ganglion; PGC, preganglionic column; MMC, median motor column; HMC, hypaxial motor column. Scale bar: 85 μm.
Fig. 5.
Fig. 5.
OC factors are required for LMC subdivisions. (A-L) Colabeling analyses of LMC markers unveil production of supernumerary LMCl neurons at the expense of the LMCm population at E12.5 in the brachial spinal cord of Hnf6/Oc2–/– mouse embryos. (M) Quantification of LMC neurons at E12.5 demonstrates that total MN numbers at brachial levels are not significantly changed in Hnf6/Oc2–/– embryos. (N) Quantitative analysis of LMC subtype markers indicates a conversion of LMCm to LMCl identity in E12.5 Hnf6/Oc2–/– embryos. (O-R) In control embryos at E12.5, Ephb1 mRNA is enriched in the LMCm, whereas high Epha4 mRNA levels are detected in the LMCl. In Hnf6/Oc2–/– embryos, Ephb1 mRNA in the LMC is lost, whereas Epha4 expression is high throughout the LMC. (S,T) Axonal projections of LMC neurons in the limbs of control and Hnf6/Oc2–/– embryos at E12.5. In Hnf6/Oc2–/– embryos, the motor nerve branch that innervates the ventral mesenchyme of the limb is absent (marked by three asterisks). (U,V) LMC neuron projections were studied by retrograde transport of Rhodamine-dextran injected into the dorsal part of the limbs of control and Hnf6/Oc2–/– embryos at E12.5. Foxp1 was used to identify LMC neurons. In the absence of OC factors, most of the LMC neurons innervate dorsal limb mesenchyme. (W-Y) Quantification of LMC neurons labeled after retrograde Rhodamine-dextran transport in control and Hnf6/Oc2–/– embryos demonstrates that the vast majority of LMC axons are redirected towards the dorsal part of the limbs in the absence of OC factors (Y). *, P<0.05; ***, P<0.001; error bars indicate s.e.m. LMCm, medial portion of the lateral motor column; LMCl, lateral portion of the lateral motor column. Scale bars: 150 μm in A-E,G-K; 170 μm in O-R,U-V.
Fig. 6.
Fig. 6.
Re-evaluated model for the control of MN differentiation. (A) Isl1 ensures the survival of newly born MNs, whereas Hb9 and Isl cooperate to consolidate MN identity and simultaneously repress the V2a differentiation program. (B) At thoracic levels of the spinal cord, newly born MNs segregate into visceral or somatic MN populations. The distinction between these two classes is likely to depend on the level of Isl proteins. Sip1 also promotes visceral MN differentiation and is regulated by the OC factors. (C) At brachial or lumbar levels of the spinal cord, retinoic acid (RA) signals provided by early-born LMCm neurons direct the acquisition of LMCl divisional identity. The crossrepressive interaction between Isl1 and Lhx1 reinforces the segregation between LMCm and LMCl. Because they control Isl1 expression in LMCm cells, the OC factors are required for LMC columnar subdivision and LMCm identity. See text for further details.

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