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FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains

Nature Neuroscience volume 9, pages 443–452 (2006)Cite this article

Abstract

A major challenge in systems neuroscience is to perform precise molecular genetic analyses of a single neuronal population in the context of the complex mammalian brain. Existing technologies for profiling cell type–specific gene expression are largely limited to immature or morphologically identifiable neurons. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify genetically labeled neurons from juvenile and adult mouse brains for gene expression profiling. We identify and verify a new set of differentially expressed genes in the striatonigral and striatopallidal neurons, two functionally and clinically important projection neuron subtypes in the basal ganglia. We further demonstrate that Ebf1 is a lineage-specific transcription factor essential to the differentiation of striatonigral neurons. Our study provides a general approach for profiling cell type–specific gene expression in the mature mammalian brain and identifies a set of genes critical to the function and dysfunction of the striatal projection neuron circuit.

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Figure 1: EGFP expression in subtypes of striatal projection neurons in three BAC transgenic mice.
Figure 2: FACS-array procedure and validation of known striatal genes.
Figure 3: Microarray comparison schematic.
Figure 4: Independent validation of MSN subtype–specific genes.
Figure 5: BAC transgenic validation of Slc35d3 expression in the striatonigral neurons.
Figure 6: Ebf1 is involved in lineage-specific differentiation of striatonigral neurons.
Figure 7: Striatonigral neuron axonal differentiation deficits in P0 Ebf1−/− mice.

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Acknowledgements

We would like to thank N. Heintz (Rockefeller University) and the National Institute of Neurological Disorders and Stroke (NINDS) Gene Expression Nervous System Atlas (GENSAT) program for providing the BAC transgenic mice; R. Grosschedl (Max-Planck-Institute of Immunobiology, Freiburg, Germany) for the Ebf1 knockout mice; M.S. Levine for equipment use; C. Evans for the met-Enkephalin antibody; D. Anderson (California Institute of Technology) for advice on cell dissociation; and S.L. Zipursky, A. Silva, D.E. Krantz, Y.E. Sun, M.S. Levine and members of the Yang lab for discussions. We would like to thank the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility, and UCLA NINDS/National Institute of Mental Health (NIMH) DNA Microarray Core Facility for their services. X.W.Y. is supported by Semel Institute for Neuroscience and Human Behaviors, NINDS (NS049501 and NS047391) and the Hereditary Disease Foundation, and by pilot grants from UCLA Hatos Center for Neuropharmacology (National Institute of Drug Abuse P50DA05010) and UCLA Brain Research Institute (supported by the Employees Charity Organization of Northrup Grumman). M.K.L. and M.G. are supported by UCLA Mental Retardation Research Center training grants (5T32HD007032 from National Institute of Child Health and Human Development). M.G. is also supported by UCLA Center for Neurobehavioral Genetics (5T32NS048004 from NINDS). D.H.G. is supported by NINDS/NIMH U24 NS43562. S.L.K. is supported by the French Foundation for Alzheimer's Research.

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Authors and Affiliations

  1. Semel Institute for Neuroscience and Human Behavior; Department of Psychiatry and Biobehavioral Sciences, Center for Neurobehavioral Genetics, University of California at Los Angeles (UCLA),

    Mary Kay Lobo, Stanislav L Karsten, Michelle Gray, Daniel H Geschwind & X William Yang

  2. Department of Psychiatry and Biobehavioral Sciences, University of California at Los Angeles (UCLA), 695 Charles Young Drive South, Gonda 3506B, Los Angeles, 90095, California, USA

    Mary Kay Lobo, Stanislav L Karsten, Michelle Gray, Daniel H Geschwind & X William Yang

  3. Brain Research Institute, University of California at Los Angeles (UCLA), 695 Charles Young Drive South, Gonda 3506B, Los Angeles, 90095, California, USA

    Mary Kay Lobo, Stanislav L Karsten, Michelle Gray, Daniel H Geschwind & X William Yang

  4. Department of Neurology, Progam in Neurogenetics, David Geffen School of Medicine, University of California at Los Angeles (UCLA), 695 Charles Young Drive South, Gonda 3506B, Los Angeles, 90095, California, USA

    Stanislav L Karsten & Daniel H Geschwind

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Correspondence to X William Yang.

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Supplementary information

Supplementary Fig. 1

Analysis of cell death and stress related genes in sorted neurons. (PDF 1606 kb)

Supplementary Fig. 2

Gene Ontology analysis identifies enrichment in Signal Transduction (EASE score; p < 0.05). (PDF 398 kb)

Supplementary Fig. 3

A subset of MSN-subtype-specific genes has striatal-enriched expression. (PDF 2956 kb)

Supplementary Fig. 4

Striatonigral neurons are more affected in the dorsal striatum compared to the ventral striatum (nuclear accumbens) in Ebf1−/− mice. (PDF 2856 kb)

Supplementary Fig. 5

Low amounts of starting RNA template yield reproducible and consistent results. (PDF 2031 kb)

Supplementary Table 1

Experimental chart of performed microarray hybridizations. (PDF 53 kb)

Supplementary Table 2

qRT-PCR using Taqman Gene Expression Assays show enrichment of Evi3/Zfp521 and Ebf1 in the striatonigral MSNs. (PDF 64 kb)

Supplementary Table 3

PCR primers used for RT-PCR validation of the striatal MSN-subtype specific genes and to make in situ hybridization probes. (PDF 97 kb)

Supplementary Methods (PDF 125 kb)

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Lobo, M., Karsten, S., Gray, M. et al. FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains. Nat Neurosci 9, 443–452 (2006). https://doi.org/10.1038/nn1654

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