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Adult neural stem cells in distinct microdomains generate previously unknown interneuron types

Nature Neuroscience volume 17, pages 207–214 (2014)Cite this article

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Abstract

Throughout life, neural stem cells (NSCs) in different domains of the ventricular-subventricular zone (V-SVZ) of the adult rodent brain generate several subtypes of interneurons that regulate the function of the olfactory bulb. The full extent of diversity among adult NSCs and their progeny is not known. Here, we report the generation of at least four previously unknown olfactory bulb interneuron subtypes that are produced in finely patterned progenitor domains in the anterior ventral V-SVZ of both the neonatal and adult mouse brain. Progenitors of these interneurons are responsive to sonic hedgehog and are organized into microdomains that correlate with the expression domains of the Nkx6.2 and Zic family of transcription factors. This work reveals an unexpected degree of complexity in the specification and patterning of NSCs in the postnatal mouse brain.

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Figure 1: Production of type 1–4 cells in the OB by specifically labeled NSCs.
Figure 2: Type 1–4 cells continue to be produced by GFAP+ NSCs in the adult anterior ventral V-SVZ.
Figure 3: Molecular and morphological characterization of type 1–4 cells.
Figure 4: Gli1-expressing progenitors produce type 1–4 cells.
Figure 5: Type 1–4 cells are produced by postnatal progenitors expressing Nkx6.2, but not Nkx2.1.
Figure 6: Expression of Zic proteins partitions type 1–4 production domains.
Figure 7: Type 1–4 cells are produced in distinct subregions of the anterior ventral V-SVZ.

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Acknowledgements

We would like to thank T. Nguyen, Z. Mirzadeh and R. Ihrie for comments and discussions that improved this study, and D. Rowitch (University of California, San Francisco) and R. Segal (Harvard Medical School) for generously sharing antibodies. R. Romero provided outstanding technical assistance with histology. M. Grist, U. Dennehy and M. Humphreys generated the Nkx6.2∷CreERT2 transgenic mice, and Gli1∷CreERT2 mice were generously provided by A. Joyner (Sloan-Kettering Institute). F.T.M. was supported by the US National Science Foundation, the Jane Coffin Childs Memorial Fund, the US National Institutes of Health (NIH) and the Harvard Stem Cell Institute. L.C.F. was supported by the Howard Hughes Medical Institute and the Helen Hay Whitney Foundation. This study was supported by grants from the NIH (HD 32116 and NS 28478), the John G. Bowes Research Fund, the European Research Council (207807) and the UK Medical Research Council (86419).

Author information

Author notes

  1. Florian T Merkle and Luis C Fuentealba: These authors contributed equally to this work.

  2. Nicoletta Kessaris and Arturo Alvarez-Buylla: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Neurological Surgery, University of California, San Francisco, San Francisco, California, USA

    Florian T Merkle, Luis C Fuentealba, Timothy A Sanders & Arturo Alvarez-Buylla

  2. Eli and Edythe Broad Center of Regeneration, Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA

    Florian T Merkle, Luis C Fuentealba, Timothy A Sanders & Arturo Alvarez-Buylla

  3. Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA

    Florian T Merkle

  4. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Florian T Merkle

  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Florian T Merkle

  6. Wolfson Institute for Biomedical Research, University College London, Gower Street, London, UK

    Lorenza Magno & Nicoletta Kessaris

  7. Department of Cell and Developmental Biology, University College London, Gower Street, London, UK

    Lorenza Magno & Nicoletta Kessaris

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Contributions

F.T.M., L.C.F. and A.A.-B. conceived and designed the experiments. F.T.M., L.C.F., T.A.S. and L.M. conducted experiments. N.K. developed the Nkx6.2CreERT2 transgenic mice and critically reviewed and edited the manuscript. F.T.M., L.C.F. and A.A.-B. analyzed data and prepared the manuscript. F.T.M. wrote the manuscript.

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Correspondence to Arturo Alvarez-Buylla.

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Integrated supplementary information

Supplementary Figure 1 Type 1–4 cells have unique morphologies.

All cells are shown to scale and were traced using a camera lucida, digitized, and colored. Type 1 cells (red) resemble known granule cell types, but have cell bodies located in the superficial GRL and dendrites restricted to the deep layers of the EPL and below. Type 2 cells (blue) have cell bodies in the MCL and a spatially restricted, superficially directed, and highly branched dendritic arbor. Type 3 cells (magenta) have cell bodies in the MCL and relatively thin but highly branching processes concentrated in the IPL and MCL. Type 4 cells (green) are located in the EPL and have branched dendritic arbors with processes that tend to align vertically. Scale bar is 50 μm.

Supplementary Figure 2 Type 1–4 cells express markers of interneurons.

A subpopulation of Type 1-4 cells strongly expresses the calcium binding protein and interneuron marker calretinin (CalR). Type 1-4 cells were negative for parvalbumin (PV), though rare cells in the EPL are clearly immunopositive for PV (pictured). These cells were larger than Type 4 cells and likely respond instead to Van Gehuchten cells. Scale bar for all photomigrographs is 50 μm.

Supplementary Figure 3 Generation and characterization of Nkx6.2::CreERT2 mice

a,b) Strategy for the generation of mice expressing CreERT2 under control of Nkx6.2. Structure of the unmodified genomic BAC used for generation of the transgene (a) and modification of the genomic BAC containing the Nkx6.2 gene by insertion of iCreERT2-polyA within the first coding exon (b). c) RNA in situ hybridization showing expression of Nkx6.2 at E11.5 and E15.5. d) RNA in situ hybridization showing expression of the CreERT2 transgene at E11.5 and E15.5. The endogenous Nkx6.2 gene and the transgene are both expressed in the interganglionic sulcus at E11.5. At E15.5, the transcripts can still be detected in the sulcus but strong expression can also be observed in the V-SVZ.

Supplementary Figure 4 Zic-immunopositive OB interneurons are generated in a medial and anterior domain.

Neurolucida traces of coronal sections from Z/EG mice brains injected at P0 with Ad:Cre to target radial glia on the medial wall of the anterior ventral V-SVZ. The surface of the brain is colored in gray, the lateral ventricle is shown in light purple, and the domain containing Zic immunopositive cells is shown in light red. Radial glial-derived (GFP+) V-SVZ cells are indicated in bright green. Injections were then classified into two groups (a and b) based on the ratio of periglomerular to granule cells in the OB (PGC/GC). As previously described, high ratios (>2) correlated with the presence of more rostrally located GFP+ cells in the V-SVZ. a) The more posterior labeling group had low PGC/GC ratios and intermediate percentages of Zic immunoreactivity among PGCs. Labeling in the V-SVZ was concentrated near the ventral tip of the lateral ventricle. b) The more anterior labeling group was characterized by high PGC/GC ratios and a high percentage (>90%) of PGCs that expressed Zic. Furthermore, the vast majority of Type 1 and Type 3 cells derived from this domain were Zic+.

Supplementary Figure 5 Zic is expressed in a subset of CalR+ PGCs.

Double immunostaining for Zic and markers of PGC subtypes demonstrates co-labeling among Zic and CalR, but very little overlap with CalB or TH. This result is consistent with the previously identified medial anterior domain of CalR+ PGC generation. The presence of a Zic-/CalR+ population is consistent with the observed origin of CalR+ PGCs from other regions such as the cortical V-SVZ, whereas the presence of Zic+/CalR- cells suggests the presence of additional interneuron subtypes among the Zic+ population.

Supplementary Figure 6 The location and morphology of type 1–4 cells suggests unique key roles in OB function.

Here we speculate as to what roles Type 1-4 cells might play in the OB circuitry, bearing in mind that these hypotheses must be tested in future experiments. Type 1 cells (red) may receive axonal (possibly dendritic) input within the superficial granule cell layer and internal plexiform layer and inhibit the cell bodies and proximal dendrites of mitral (black) and tufted cells above them, thereby mediating columnar inhibition. The highly branched, spatially restricted arbors of Type 2 cells (blue) are positioned to inhibit the cell bodies and proximal dendrites of neighboring mitral and deep tufted cells and could mediate localized lateral inhibition. The varicosities and spines of Type 3 (magenta) and 4 cells (green) may be sites of unidirectional (pre or post-synaptic only) or reciprocal synapses. If they are post-synaptic, Type 3 and 4 cells may detect the output of mitral and tufted cells or local processing in their dendrites and relay this activity to other cells in the column via their radially projecting axons. If they have reciprocal synapses or pre-synaptic structures, Type 3 and 4 cells may inhibit the output of mitral and tufted cells or inhibit their dendrites, respectively.

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Merkle, F., Fuentealba, L., Sanders, T. et al. Adult neural stem cells in distinct microdomains generate previously unknown interneuron types. Nat Neurosci 17, 207–214 (2014). https://doi.org/10.1038/nn.3610

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