The Cerebellum

, Volume 10, Issue 3, pp 356–372

Spatially Restricted and Developmentally Dynamic Expression of Engrailed Genes in Multiple Cerebellar Cell Types

  • Sandra L. Wilson
  • Anna Kalinovsky
  • Grant D. Orvis
  • Alexandra L. Joyner


The cerebellum is a highly organized structure partitioned into lobules along the anterior–posterior (A-P) axis and into striped molecular domains along the medial–lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P) 21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCß4 and EPHA4.


Engrailed Purkinje cell Cerebellum Compartment Development Stripe 

Supplementary material

12311_2011_254_Fig10_ESM.gif (194 kb)
Figure 1

Purkinje cells co-express LHX 1/5 and RORα. Representative sagittal (A-A″) and coronal (B-B″) sections of E17.5 cerebella show overlapping expression of LHX 1/5 (A and B) and RORα (A′ and B′). High magnification optical section taken from the dorsal Purkinje cell band displaying near complete overlap between LHX 1/5 (C and C″) and RORα (C′ and C″). Asterisk indicates a blood vessel fluorescing in the red channel (C and C″). (GIF 193 KB)

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High resolution image file (TIF 15.2 kb)
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Figure 2

EN1/2 is robustly expressed in ML interneurons, IGL and DCN cells, and at relatively lover levels in Purkinje cells at P21. (A) Nuclear labeling with pan-EN antibody (left panel, red in color composite) highlights most ML interneurons identified by immunodetection with parvalbumin (green), but not calbindin (blue) antibody. (B) Higher magnification of boxed region in (A). (C) All DCN subdivisions express EN1/2 at P21. (GIF 617 KB)

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High resolution image file (TIF 29.5 kb)
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Figure 3

Expression levels of EN1/2 in IGL at P21 differ between cerebellar subregions. Representative images of coronal series through P21 cerebellum demonstrating regional differences in EN expression levels in the IGL, with the highest expression in IGL concentrated in the nodular zone. (GIF 109 KB)

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High resolution image file (TIF 14.5 kb)


  1. 1.
    Larsell O. The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat. J Comp Neurol. 1952;97(2):281–356.PubMedCrossRefGoogle Scholar
  2. 2.
    Ozol K et al. Transverse zones in the vermis of the mouse cerebellum. J Comp Neurol. 1999;412(1):95–111.PubMedCrossRefGoogle Scholar
  3. 3.
    Sillitoe RV, Joyner AL. Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum. Annu Rev Cell Dev Biol. 2007;23:549–77.PubMedCrossRefGoogle Scholar
  4. 4.
    Joyner AL et al. Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. Science. 1991;251(4998):1239–43.PubMedCrossRefGoogle Scholar
  5. 5.
    Cheng Y et al. The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum. Development. 2010;137(3):519–29.PubMedCrossRefGoogle Scholar
  6. 6.
    Sillitoe RV et al. Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum. J Neurosci. 2008;28(47):12150–62.PubMedCrossRefGoogle Scholar
  7. 7.
    Sillitoe RV, Vogel MW, Joyner AL. Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map. J Neurosci. 2010;30(30):10015–24.PubMedCrossRefGoogle Scholar
  8. 8.
    Wurst W, Auerbach AB, Joyner AL. Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development. 1994;120(7):2065–75.PubMedGoogle Scholar
  9. 9.
    Sgaier SK et al. Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins. Development. 2007;134(12):2325–35.PubMedCrossRefGoogle Scholar
  10. 10.
    Millen KJ, Hui CC, Joyner AL. A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum. Development. 1995;121(12):3935–45.PubMedGoogle Scholar
  11. 11.
    Davis CA, Joyner AL. Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. Genes Dev. 1988;2(12B):1736–44.PubMedCrossRefGoogle Scholar
  12. 12.
    Larouche M, Hawkes R. From clusters to stripes: the developmental origins of adult cerebellar compartmentation. Cerebellum. 2006;5(2):77–88.PubMedCrossRefGoogle Scholar
  13. 13.
    Vandaele S et al. Purkinje cell protein-2 regulatory regions and transgene expression in cerebellar compartments. Genes Dev. 1991;5(7):1136–48.PubMedCrossRefGoogle Scholar
  14. 14.
    Oberdick J et al. Control of segment-like patterns of gene expression in the mouse cerebellum. Neuron. 1993;10(6):1007–18.PubMedCrossRefGoogle Scholar
  15. 15.
    Wassef M et al. Transient biochemical compartmentalization of Purkinje cells during early cerebellar development. Dev Biol. 1985;111(1):129–37.PubMedCrossRefGoogle Scholar
  16. 16.
    Karam SD et al. EphA4 is not required for Purkinje cell compartmentation. Brain Res Dev Brain Res. 2002;135(1–2):29–38.PubMedCrossRefGoogle Scholar
  17. 17.
    Apps R, Hawkes R. Cerebellar cortical organization: a one-map hypothesis. Nat Rev Neurosci. 2009;10(9):670–81.PubMedCrossRefGoogle Scholar
  18. 18.
    Ahn AH et al. The cloning of zebrin II reveals its identity with aldolase C. Development. 1994;120(8):2081–90.PubMedGoogle Scholar
  19. 19.
    Brochu G, Maler L, Hawkes R. Zebrin II: a polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J Comp Neurol. 1990;291(4):538–52.PubMedCrossRefGoogle Scholar
  20. 20.
    Armstrong CL et al. Expression of heat-shock protein Hsp25 in mouse Purkinje cells during development reveals novel features of cerebellar compartmentation. J Comp Neurol. 2001;429(1):7–21.PubMedCrossRefGoogle Scholar
  21. 21.
    Larouche M, Che PM, Hawkes R. Neurogranin expression identifies a novel array of Purkinje cell parasagittal stripes during mouse cerebellar development. J Comp Neurol. 2006;494(2):215–27.PubMedCrossRefGoogle Scholar
  22. 22.
    Marzban H et al. Phospholipase Cbeta4 expression reveals the continuity of cerebellar topography through development. J Comp Neurol. 2007;502(5):857–71.PubMedCrossRefGoogle Scholar
  23. 23.
    Sillitoe RV, Gopal N, Joyner AL. Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections. Neuroscience. 2009;162(3):574–88.PubMedCrossRefGoogle Scholar
  24. 24.
    Hashimoto M, Mikoshiba K. Mediolateral compartmentalization of the cerebellum is determined on the “birth date” of Purkinje cells. J Neurosci. 2003;23(36):11342–51.PubMedGoogle Scholar
  25. 25.
    Chedotal A, Bloch-Gallego E, Sotelo C. The embryonic cerebellum contains topographic cues that guide developing inferior olivary axons. Development. 1997;124(4):861–70.PubMedGoogle Scholar
  26. 26.
    Chedotal A, Sotelo C. The ‘creeper stage’ in cerebellar climbing fiber synaptogenesis precedes the ‘pericellular nest’—ultrastructural evidence with parvalbumin immunocytochemistry. Brain Res Dev Brain Res. 1993;76(2):207–20.PubMedCrossRefGoogle Scholar
  27. 27.
    Wassef M et al. Development of the olivocerebellar projection in the rat: II. Matching of the developmental compartmentations of the cerebellum and inferior olive through the projection map. J Comp Neurol. 1992;323(4):537–50.PubMedCrossRefGoogle Scholar
  28. 28.
    Sudarov A, Joyner AL. Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers. Neural Dev. 2007;2:26.PubMedCrossRefGoogle Scholar
  29. 29.
    Baader SL et al. Selective disruption of “late onset” sagittal banding patterns by ectopic expression of engrailed-2 in cerebellar Purkinje cells. J Neurosci. 1999;19(13):5370–9.PubMedGoogle Scholar
  30. 30.
    Vogel MW et al. The Engrailed-2 homeobox gene and patterning of spinocerebellar mossy fiber afferents. Brain Res Dev Brain Res. 1996;96(1–2):210–8.PubMedCrossRefGoogle Scholar
  31. 31.
    Hanks M et al. Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science. 1995;269(5224):679–82.PubMedCrossRefGoogle Scholar
  32. 32.
    Matei V et al. Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit. Dev Dyn. 2005;234(3):633–50.PubMedCrossRefGoogle Scholar
  33. 33.
    Sgaier SK et al. Morphogenetic and cellular movements that shape the mouse cerebellum: insights from genetic fate mapping. Neuron. 2005;45(1):27–40.PubMedGoogle Scholar
  34. 34.
    Kimmel RA et al. Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation. Genes Dev. 2000;14(11):1377–89.PubMedGoogle Scholar
  35. 35.
    Nagy A, Getsenstein M, Vintersten K, Behringer RR. Manipulating the mouse embryo: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2003.Google Scholar
  36. 36.
    Davis CA et al. Examining pattern-formation in mouse. Chicken and frog embryos with an en-specific antiserum. Development. 1991;111(2):287–98.PubMedGoogle Scholar
  37. 37.
    Palay SL, Chan-Palay V. Cerebellar cortex: cytology and organization XII. Heidelberg: Springer; 1974. p. 348.Google Scholar
  38. 38.
    Engelkamp D et al. Role of Pax6 in development of the cerebellar system. Development. 1999;126(16):3585–96.PubMedGoogle Scholar
  39. 39.
    Englund C et al. Unipolar brush cells of the cerebellum are produced in the rhombic lip and migrate through developing white matter. J Neurosci. 2006;26(36):9184–95.PubMedCrossRefGoogle Scholar
  40. 40.
    Fink AJ et al. Development of the deep cerebellar nuclei: transcription factors and cell migration from the rhombic lip. J Neurosci. 2006;26(11):3066–76.PubMedCrossRefGoogle Scholar
  41. 41.
    Graham V et al. SOX2 functions to maintain neural progenitor identity. Neuron. 2003;39(5):749–65.PubMedCrossRefGoogle Scholar
  42. 42.
    Zhao Y et al. LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum. Proc Natl Acad Sci USA. 2007;104(32):13182–6.PubMedCrossRefGoogle Scholar
  43. 43.
    Glassmann A et al. Basic molecular fingerprinting of immature cerebellar cortical inhibitory interneurons and their precursors. Neuroscience. 2009;159(1):69–82.PubMedCrossRefGoogle Scholar
  44. 44.
    Minaki Y et al. Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker. Gene Expr Patterns. 2008;8(6):418–23.PubMedCrossRefGoogle Scholar
  45. 45.
    Nakagawa S, Watanabe M, Inoue Y. Prominent expression of nuclear hormone receptor ROR alpha in Purkinje cells from early development. Neurosci Res. 1997;28(2):177–84.PubMedCrossRefGoogle Scholar
  46. 46.
    Maricich SM, Herrup K. Pax-2 expression defines a subset of GABAergic interneurons and their precursors in the developing murine cerebellum. J Neurobiol. 1999;41(2):281–94.PubMedCrossRefGoogle Scholar
  47. 47.
    Sugitani Y et al. Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons. Genes Dev. 2002;16(14):1760–5.PubMedCrossRefGoogle Scholar
  48. 48.
    Wassef M, Sotelo C. Asynchrony in the expression of guanosine 3′:5′-phosphate-dependent protein kinase by clusters of Purkinje cells during the perinatal development of rat cerebellum. Neuroscience. 1984;13(4):1217–41.PubMedCrossRefGoogle Scholar
  49. 49.
    Smeyne RJ et al. Dynamic organization of developing Purkinje cells revealed by transgene expression. Science. 1991;254(5032):719–21.PubMedCrossRefGoogle Scholar
  50. 50.
    Karam SD et al. Eph receptors and ephrins in the developing chick cerebellum: relationship to sagittal patterning and granule cell migration. J Neurosci. 2000;20(17):6488–500.PubMedGoogle Scholar
  51. 51.
    Sarna JR et al. Complementary stripes of phospholipase Cbeta3 and Cbeta4 expression by Purkinje cell subsets in the mouse cerebellum. J Comp Neurol. 2006;496(3):303–13.PubMedCrossRefGoogle Scholar
  52. 52.
    Baader SL et al. Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions. J Neurosci. 1998;18(5):1763–73.PubMedGoogle Scholar
  53. 53.
    Kuemerle B et al. Pattern deformities and cell loss in Engrailed-2 mutant mice suggest two separate patterning events during cerebellar development. J Neurosci. 1997;17(20):7881–9.PubMedGoogle Scholar
  54. 54.
    Lundell MJ et al. The engrailed and huckebein genes are essential for development of serotonin neurons in the Drosophila CNS. Mol Cell Neurosci. 1996;7(1):46–61.PubMedCrossRefGoogle Scholar
  55. 55.
    Saueressig H, Burrill J, Goulding M. Engrailed-1 and netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons. Development. 1999;126(19):4201–12.PubMedGoogle Scholar
  56. 56.
    Simon HH et al. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci. 2001;21(9):3126–34.PubMedGoogle Scholar
  57. 57.
    Louvi A, Wassef M. Ectopic engrailed 1 expression in the dorsal midline causes cell death, abnormal differentiation of circumventricular organs and errors in axonal pathfinding. Development. 2000;127(18):4061–71.PubMedGoogle Scholar
  58. 58.
    Joly W, Mugat B, Maschat F. Engrailed controls the organization of the ventral nerve cord through frazzled regulation. Dev Biol. 2007;301(2):542–54.PubMedCrossRefGoogle Scholar
  59. 59.
    Solano PJ et al. Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes. Development. 2003;130(7):1243–54.PubMedCrossRefGoogle Scholar
  60. 60.
    Serrano N, Brock HW, Maschat F. beta3-tubulin is directly repressed by the engrailed protein in Drosophila. Development. 1997;124(13):2527–36.PubMedGoogle Scholar
  61. 61.
    Wizenmann A et al. Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo. Neuron. 2009;64(3):355–66.PubMedCrossRefGoogle Scholar
  62. 62.
    Brunet I et al. The transcription factor Engrailed-2 guides retinal axons. Nature. 2005;438(7064):94–8.PubMedCrossRefGoogle Scholar
  63. 63.
    Ashwell KWS, Zhang LL. Ontogeny of afferents to the fetal-rat cerebellum. Acta Anat. 1992;145(1):17–23.PubMedCrossRefGoogle Scholar
  64. 64.
    Grishkat HL, Eisenman LM. Development of the spinocerebellar projection in the prenatal mouse. J Comp Neurol. 1995;363(1):93–108.PubMedCrossRefGoogle Scholar
  65. 65.
    Bourrat F, Sotelo C. Migratory pathways and neuritic differentiation of inferior olivary neurons in the rat embryo—axonal tracing study using the in vitro slab technique. Dev Brain Res. 1988;39(1):19–37.CrossRefGoogle Scholar
  66. 66.
    Chedotal A, Sotelo C. Early development of olivocerebellar projections in the fetal rat using CGRP immunocytochemistry. Eur J Neurosci. 1992;4(11):1159–79.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sandra L. Wilson
    • 1
  • Anna Kalinovsky
    • 1
  • Grant D. Orvis
    • 1
  • Alexandra L. Joyner
    • 1
  1. 1.Developmental Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA

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