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Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum

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Abstract

Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

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Abbreviations

M-L:

Medio-lateral

A-P:

Anterior-posterior

VZ:

Ventricular zone

pWM:

Presumptive white matter tracts

TMX:

Tamoxifen

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Acknowledgments

We are grateful to the staff of USU’s Biomedical Instrumentation Center for technical assistance.

Funding

This work is supported by the National Science Foundation (NSF) Award #1121839.

Coflicts of Interest

The authors disclose that no competing financial interests or personal relationships exist that might bias this work.

Author information

Authors and Affiliations

  1. Department of Anatomy, Physiology and Genetics, Center for Neuroscience and Regenerative Medicine (CNRM), Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA

    Edwin A. Obana, Travis G. Lundell, Kevin J. Yi, Kryslaine L. Radomski, Qiong Zhou & Martin L. Doughty

Authors
  1. Edwin A. Obana
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  2. Travis G. Lundell
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  3. Kevin J. Yi
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  4. Kryslaine L. Radomski
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  5. Qiong Zhou
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  6. Martin L. Doughty
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Corresponding author

Correspondence to Martin L. Doughty.

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Supplementary Figure 1

Patterns of GIFM Purkinje cells following TMX on E12.5 do not coincide with expression patterns of Zebrin II or Hsp25. Coronal sections through the rostro-caudal axis of the cerebellum were immunostained using ABC enhancement. All mice were analyzed at P42. Note there are some detachments from whole cerebellar sections during the IHC process such as the paraflocculus (e’, f), simple lobule and Crus 1 (e’, l, l’), or middle cerebellar peduncle region (e). Missing fragments do not interfere with our expression pattern analysis, which is focused on the vermis. E12.5 GIFM Purkinje cells settle in three parasagittal stripes along the medio-lateral axis of the adult vermis and paravermis (a-l). Consecutive slices were analyzed for Zebrin II and Hsp25 expression patterns. Multiple Zebrin II negative stripes (arrow heads) appear in lobules VIII and IX (a’-b’) and three distinct Zebrin II positive stripes (arrows) appear in lobules I-V (d’-f’). Three Hsp25 positive stripes (arrows) appear in lobules VI, VII, IX and X (g’-j’) and Hsp25 is not expressed in rostral sections (k’-l’). Dual IHC reveals RFP labeled Purkinje cells expressing Zebrin II (m) or Hsp25 (n) indicated with arrows. (o) Although PLCβ4 expression patterns do not compliment GIFM Purkinje cells, dual anti-RFP/anti-PLCβ4 Purkinje cells are present in lobule VIII. (GIF 381 kb)

High resolution image (TIFF 24710 kb)

Supplementary Figure 2

GIFM is specific to Purkinje cell lineages in Neurog1-CreER;R26td-Tomato/+ mice but RFP reporter expression occurs independent of TMX delivery in some basket, stellate and Golgi cells. Neurog1-CreER;R26td-Tomato/+ mice administered with TMX or sunflower oil only on E12.5 were analyzed at P42. Dual IHC for RFP with the basket and stellate cell marker Pvlb or the Golgi cell marker Nrg co-labels RFP interneurons in mice injected with TMX or sunflower oil (arrows). Leaky RFP labeling in sunflower oil controls is more numerous in basket and stellate cell interneurons compared to Golgi cells. In contrast, RFP is not detected in Calb1+ Purkinje cells in the absence of TMX administration. (GIF 110 kb)

High resolution image (TIFF 24692 kb)

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Obana, E.A., Lundell, T.G., Yi, K.J. et al. Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum. Cerebellum 14, 247–263 (2015). https://doi.org/10.1007/s12311-014-0641-9

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