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Astrocyte-encoded positional cues maintain sensorimotor circuit integrity


Astrocytes, the most abundant cells in the central nervous system, promote synapse formation and help to refine neural connectivity. Although they are allocated to spatially distinct regional domains during development, it is unknown whether region-restricted astrocytes are functionally heterogeneous. Here we show that postnatal spinal cord astrocytes express several region-specific genes, and that ventral astrocyte-encoded semaphorin 3a (Sema3a) is required for proper motor neuron and sensory neuron circuit organization. Loss of astrocyte-encoded Sema3a leads to dysregulated α-motor neuron axon initial segment orientation, markedly abnormal synaptic inputs, and selective death of α- but not of adjacent γ-motor neurons. In addition, a subset of TrkA+ sensory afferents projects to ectopic ventral positions. These findings demonstrate that stable maintenance of a positional cue by developing astrocytes influences multiple aspects of sensorimotor circuit formation. More generally, they suggest that regional astrocyte heterogeneity may help to coordinate postnatal neural circuit refinement.

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Figure 1: AS express region-specific genes.
Figure 2: Axon-repulsive effects of AS-encoded Sema3a maintain α-MN AIS orientation.
Figure 3: AS-encoded Sema3a is required for postnatal α-MN survival.
Figure 4: AS-encoded Sema3a regulates MN synaptogenesis and function.
Figure 5: AS-encoded Sema3a regulates DV positioning of sensory axons.

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Gene Expression Omnibus

Data deposits

Microarray data has been deposited to GEO under accession number GSE55054.


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We thank J. Flanagan, T. Jessell, J. de Nooj, N. Balaskas, M. Hancock, S. Ohata and R. Krencik for comments on the manuscript and technical suggestions. We are grateful to K. Sabeur, M. Wong and the UCSF Flow Cytometry and Genomics core facilities for expert technical help, A. Kolodkin for Sema3a probe construct, L. Reichardt for the TrkA antibody, J. Dasen for FoxP1 and Scip antibodies, and N. Heintz and J. Dougherty for Aldh1L1-cre mice. A.V.M. is supported by an NIMH Training Grant (5T32MH089920-04) and an APA/Pfizer MD/PhD Psychiatric Research Fellowship. K.W.K is supported by the California Institute for Regenerative Medicine (TG2-01153). S.A.R. is supported by a Ruth L. Kirschstein NRSA FNS081905A. This work was supported by grants from the NINDS (to D.H.R. (R01 NS059893) and J.R.C.), E.M.U. is supported by NIMH (R01MH099595-01), an NIH New Innovator Award (1DP2OD006507-01) and That Man May See. D.H.R. is a HHMI Investigator.

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Authors and Affiliations



A.V.M. performed most experiments and data analysis. K.W.K performed electrophysiology under supervision of E.M.U. H.-H.T. contributed to data analysis and experimental design. S.A.R. performed MN purification under supervision of J.R.C. S.M.C performed mouse genotyping. L.M. and S.E.B. performed bioinformatics data processing and analysis. A.V.M. and D.H.R. designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to David H. Rowitch.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Flow cytometry gating strategy and microarray.

a, Schematic indicating microdissection of Aldh1l1–GFP-positive P7 spinal cord and isolation by flow cytometry using scatter gates, doublet exclusion (not shown) and sorting for GFP-positive cells with live/dead exclusion by DAPI staining. Percentage of Aldh1l1–GFP cells was not significantly different between dorsal and ventral (not shown). b, Summary of differentially expressed genes in astrocytes (AS), whole cord, or both using the analysis parameters indicated. c, Heatmap of all 39 genes differentially expressed between dorsal and ventral cord, highlighting astrocyte-enriched genes with known roles in neural circuit development (red) or extracellular matrix (blue).

Extended Data Figure 2 Coordinate expression of Sema3a and Nrp1 in astrocytes and neurons.

ac, Sema3a mRNA is expressed in radial glia (RG) and in protoplasmic cells that are NeuN negative throughout the embryonic and early postnatal period. Sema3a was not detected in DRG or in SC white matter (b). d, Sema3a is segregated from Plp-positive oligodendrocytes. e, MN Sema3a expression is detected in α-MN but not γ-MN in cervical SC. f, g, High levels of Nrp1 expression in TrkA+ fibres and cell bodies (white arrowhead) and in MN, but not in PV-positive fibres and cell bodies (yellow arrows). h, Quantification of percentage of Nrp1+ neurons per condition.

Extended Data Figure 3 Fate map of conditional astrocyte deletion lines used in this study.

a, hGFAPcre fate map labels fibrous and a subset of protoplasmic AS but not MN or interneurons in P10 SC. b, Aldh1l1cre fate maps to astrocytes but not to neurons in P10 SC, including α-MN (purple), γ-MN (blue) and interneurons (red).

Extended Data Figure 4 Motor neuron AIS orientation defects in cervical spinal cord.

a, Representative images of cervical SC confocal sections stained to distinguish α− and γ-MN and identify their proximal axon segment (asterisk denotes ventral root). b, Inset shows high-magnification view of representative MN with identifiable AIS and a schematic of their location with respect to the ventral root. c, Overlay of all cervical a-MN angles measured to generate data summarized in Fig. 2c, with positional information preserved, demonstrates that misoriented AIS can be seen at all DV positions.

Extended Data Figure 5 No evidence of abnormal MN cell body positioning with loss of astrocyte-encoded Sema3a.

a, Representative FoxP1 Islet1/2 co-labelling at three rostrocaudal levels in control and mutant animals shows no differences between control and mutant. b, Similar stainings using Scip (a PMC and LMC marker. c, d, No obvious differences in DV or mediolateral boundaries of ChAT+ MN at comparable cervical or lumbar levels at P0 (using Aldh1l1cre to delete Sema3a) and P7 (with hGFAPcre), both time periods where misorientation of AIS is clearly evident.

Extended Data Figure 6 Quantification of ventral interneuron populations after loss of astrocyte-encoded Sema3a.

a, Chx10 staining at E18 and quantification. b, Calbindin staining of Renshaw interneurons at P30 and quantification demonstrates a significant increase at this age. Data are mean ± s.e.m., student’s t-test. Data in a from n = 2 per group, 4 sections per animal; data in b from 4 per group, 4 sections per animal.

Extended Data Figure 7 Additional data and controls for MN electrophysiology.

a, 2 μM strychnine and 20 μM bicuculline block postsynaptic currents (at −55 mV) in a ChAT–GFP+ lumbar MN. b, 20 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 50 μM (2R)-amino-5-phosphonovaleric acid (AP5) block postsynaptic currents (at −75 mV) in a ChAT–GFP+ lumbar MN. c, No difference in input resistance, sIPSC amplitude or sEPSC amplitude between control (Cre-) and hGFAPcre:Sema3afl/fl (fl/fl) MN. n = 5/each; mean ± s.e.m., Student’s t-test.

Extended Data Figure 8 Normal dorsal root ganglia in Aldh1L1cre:Sema3afl/fl mice.

a, No difference in the number of subtype-specific neurons per DRG in control or Aldh1l1cre:Sema3afl/fl mice (n = 3 from 4–5 sections per animal; mean ± s.e.m.; Student’s t-test).

Extended Data Figure 9 Differential expression of regionally heterogeneous astrocyte genes is partly preserved in vitro.

qPCR quantification demonstrates that many regionally heterogeneous microarray genes prospectively identified in vivo remain differentially expressed in vitro after 17 days in culture, including ventral Sema3a. Mean ± s.e.m., n = 3 independent experiments.

Extended Data Table 1 Subgroup analyses of motor neuron data presented in Figs 2 and 3.

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Molofsky, A., Kelley, K., Tsai, HH. et al. Astrocyte-encoded positional cues maintain sensorimotor circuit integrity. Nature 509, 189–194 (2014).

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