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  • Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression Marta Pascual*, Ibane Abasolo†, Ana Mingorance-Le Meur*‡, Albert Martínez*, José A. Del Rio*, Christopher V. E. Wright§, Francisco X. Real†¶, and Eduardo Soriano*¶

    *Institut de Recerca Biomèdica, Parc Cientı́fic de Barcelona, and Department de Biologia Cellular, Universitat de Barcelona, Josep Samitier 1-5, E-08028 Barcelona, Spain; †Institut Municipal d’Investigació Mèdica and Universitat Pompeu Fabra, Dr. Aiguader 88, E-08003 Barcelona, Spain; and §Vanderbilt Developmental Biology Program, Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232-2175

    Edited by Lily Y. Jan, University of California, San Francisco School of Medicine, San Francisco, CA, and approved January 23, 2007 (received for review July 10, 2006)

    We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell pro- genitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1aCre/Cre mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

    cerebellum � GABAergic cells � neural specification

    The cerebellar cortex essentially comprises three major types ofneurons: Purkinje cells (PCs), granule cells, and several types of interneurons that include basket, stellate, and Golgi cells (1). PCs and interneurons are GABA-releasing inhibitory neurons, whereas granule neurons use glutamate as their transmitter. A crucial issue in neural development is the identification of the mechanisms by which distinct types of neurons are specified and settle in their correct layers. In the mouse, PCs arise at the ventricular zone (VZ) at embryonic day (E)11–E13 and migrate radially to form the PC layer (PCL). Granule cell progenitors migrate tangentially from the rhombic lip (RL) to form the external granule cell layer (EGL). At postnatal stages, EGL precursors expand dramatically to ultimately migrate inwards and form the granule cell layer (GCL) (2–5). Cerebellar interneurons stem from the VZ and remain mitotically active in the white matter to finally give rise to diverse types of interneurons at postnatal stages (6, 7). It has been shown that the deep cerebellar nuclei and unipolar brush cells originate in the RL (4, 8, 9).

    In the cerebellum, the transcription factors Math1 and NeuroD are essential for the specification and generation of glutamater- gic granule cells (4, 10, 11), but little is known about the genes that control PC specification. The Ptf1a gene encodes a basic helix–loop–helix transcription factor, which is required for the specification and formation of the pancreas (12, 13). A recent study has shown that a 300-kb deletion in the enhancer of the Ptf1a gene leads to abnormal cerebellar development as well as loss of PCs and interneurons (7). Ptf1a is also required for the generation of dorsal horn GABAergic interneurons in the spinal cord, and, in its absence, Ptf1a-derived cells adopt a glutama- tergic phenotype (14). A recent study in the retina shows that inactivation of Ptf1a leads to a fate switch in horizontal and amacrine cell precursors that causes them to adopt a ganglion cell fate (15). We have examined the role of Ptf1a in the

    development of the cerebellum by analyzing the phenotype of mice lacking the complete Ptf1a coding sequence (Ptf1aCre knockin). We confirm that Ptf1a is required for the generation of PCs and interneurons. Moreover, by using cell lineage analysis employing Ptf1aCre/Cre;R26R mice, we show that, in the absence of Ptf1a, progenitors fated to produce PCs and interneurons become incorporated in the EGL and express typical markers of granule cells. These findings indicate that Ptf1a is not only required for the specification of PCs and interneurons, but also that Ptf1a expression suppresses a granule cell phenotype from the cerebellar VZ.

    Results Ptf1a Is Expressed in PC and Interneuronal Progenitors. To determine the cell lineages arising from cells in which the Ptf1a locus had ever been activated, we examined the cerebella of Ptf1aCre/� mice crossed with R26R reporter mice (Ptf1aCre/�;R26R). X-Gal staining was strong in the brainstem, spinal cord, and developing cerebellar anlage (Fig. 1A). At E12, cells located just above the VZ were labeled in the cerebellum [see supporting information (SI) Fig. 7A]; at E14, labeling was detected throughout the cerebellar plate (data not shown); and at E18, �-Gal activity was detected around the PCL and deeper in the cerebellar parenchyma (SI Fig. 7B). In contrast, no staining was observed in either the RL or the EGL at any stage. Using RT-PCR, we found that cerebellar Ptf1a mRNA levels were high at E12, decreased until postnatal day (P)2, and were unde- tectable in the adult (SI Fig. 7C). By in situ hybridization(ISH), Ptf1a mRNA was detected at E12–E14 in the VZ of the cerebellar anlage containing progenitor cells (Fig. 1B). At later stages (E16– P5), mRNA was detected in single cells embedded in the cerebellar parenchyma, most notably below the PCL. No expression was detected in either the EGL or the PCL at any stage analyzed (Fig. 1C). To substantiate this expression pattern, sections were immu- nolabeled with anti-Ptf1a antibodies (16). At E14–E16, Ptf1a was

    Author contributions: F.X.R. and F.S. contributed equally to this work; M.P., I.A., A.M.-L.M., F.X.R., and E.S. designed research; M.P., I.A., A.M.-L.M., A.M., and J.A.D.R. performed research; J.A.D.R. and C.V.E.W. contributed new reagents/analytic tools; M.P., I.A., A.M.-L.M., A.M., J.A.D.R., C.V.E.W., F.X.R., and E.S. analyzed data; and M.P., I.A., F.X.R., and E.S. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS direct submission.

    Abbreviations: Calb, calbindin; E(n), embryonic day (n); EGL, external granule cell layer; PC, Purkinje cell; PCL, PC layer; P(n), postnatal day (n); RL, rhombic lip; VZ, ventricular zone.

    ‡Present address: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

    ¶To whom correspondence may be addressed. E-mail: esoriano@pcb.ub.es or preal@ imim.es.

    This article contains supporting information online at www.pnas.org/cgi/content/full/ 0605699104/DC1.

    © 2007 by The National Academy of Sciences of the USA

    www.pnas.org�cgi�doi�10.1073�pnas.0605699104 PNAS � March 20, 2007 � vol. 104 � no. 12 � 5193–5198

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  • detected in the nuclei of many cells located in the upper part of the VZ (Fig. 1D). In addition, single cells exhibiting elongated nuclei, typical of migrating neurons, were immunostained just above the VZ at E14–E16 (Fig. 1D Inset and E). At E18–P5, Ptf1a was detected in single cells located in the developing white matter, the GCL, and around PCs (Fig. 1F). The distribution of these cells resembled that of cerebellar interneurons migrating toward the molecular layer (Fig. 1F Inset). Ptf1a was not detected in PCs in the PCL at any developmental stage, nor was it detected in the cerebellum at P15 (Fig. 1G) or in adult mice (data not shown). These results suggested that Ptf1a is expressed in a short temporal window in postmitotic PCs and in interneuronal progenitors.

    The fate of cells that activate the Ptf1a locus was analyzed in Ptf1aCre/�;R26R mice (12). Lineage tracing in these mice confirmed that, at E18, Ptf1a-derived cells (expressing �-Gal) populated the PCL and the cerebellar parenchyma but not the EGL (SI Fig. 8 A–C and Fig. 3A). Double labeling with �-Gal and calbindin (Calb) confirmed that PCs expressed �-Gal (SI Fig. 8 A–C). At postnatal ages (P20) and adult, �-Gal was detected in PCs (labeled by Calb) (SI Fig. 8 D–F) and in parvalbumin-labeled interneurons of the molecular layer, but not in the GCL (SI Fig. 8 H–J) or in glial fibrilary acidic protein (GFAP)-immunoreactive cells (SI Fig. 8G). These lineage analyses indicated that Ptf1a-expressing cells selec- tively give rise to PCs and interneurons.

    Ptf1a Is Required for the Development of PCs and Interneurons. To examine the in vivo role of Ptf1a, we analyzed the phenotype of Ptf1a null embryos (Ptf1aCre/Cre). Mice lacking Ptf1a expression die at birth (12, 13), so only embryos up to E18 were analyzed. Histological examination of coronal and sagittal sections at

    E14–E18 revealed cerebellar hypoplasia in mutant embryos (SI Fig. 9 A and B). At E14, Calb was detected in a broad band of migrating PCs, which were more abundant in the caudal half of cerebella (SI F