Differentiation of ES cells into cerebellar neurons Enrique Salero* and Mary E. Hatten† Laboratory of Developmental Neurobiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399 Communicated by Torsten N. Wiesel, The Rockefeller University, New York, NY, December 15, 2006 (received for review September 15, 2006) The neuronal circuits of the cerebellar cortex are essential for induces mouse ES cells to differentiate into spinal progenitor cells motor and sensory learning, associative memory formation, and and subsequently into motor neurons. In the present study we have the vestibular ocular reflex. In children and young adults, tumors tested the signals that induce formation of the cerebellar territory of the granule cell, the medulloblastomas, represent 40% of (8–10) from rhombomere 1 (11), signals that dorsalize the neural brain tumors. We report the differentiation of E14 ES cells into tube specify (12) granule neurons (13), and mitogens that expand mature granule neurons by sequential treatment with secreted the pool of granule cell progenitors (GCPs) in the neonatal factors (WNT1, FGF8, and RA) that initiate patterning in the cerebellar cortex (14, 15). To monitor ES cell differentiation we cerebellar region of the neural tube, bone morphogenic proteins measured expression of genes essential for granule neuron devel- (BMP6/7 and GDF7) that induce early granule cell progenitor opment, unique markers for GCPs and differentiated granule markers (MATH1, MEIS1, ZIC1), mitogens (SHH, JAG1) that con- neurons, and markers for differentiating granule neurons. We also trol proliferation and induce additional granule cell markers monitored the expression-specific markers for cerebellar Purkinje (Cyclin D2, PAX2/6), and culture in glial-conditioned medium to neurons (16) and markers for cerebellar astroglia. induce markers of mature granule neurons (GABA␣6r), including During cerebellar development, granule neuron progenitors ZIC2, a unique marker for granule neurons. Differentiated ES arise from the boundary of the mesencephalon and metenceph- cells formed classic “T-shaped” granule cell axons in vitro, and alon in an area known as the rhombic lip. Recent studies reveal implantation of differentiated Pde1c-Egfp-BAC transgenic ES that the anterior rhombic lip, once thought to exclusively gen- cells into the external granule cell layer of neonatal mice erate granule neurons of the cerebellar cortex (17), generates resulted in the extension of parallel fibers, migration across the precursors of the cerebellar and precerebellar nuclei, which molecular layer, incorporation into the internal granule cell project afferent fibers to the cerebellar cortex or receive efferent layer, and extension of short dendrites, typical of young granule fibers from the cerebellum. Progenitors of the Purkinje neuron cells forming synaptic connections with afferent mossy fibers. arise from the ventricular zone of the cerebellar territory and These results underscore the utility of treating ES cells with express specific transcription factors (20). local, inductive signals that regulate CNS neuronal development Over the past 5 years, the Gene Expression Neuronal Database in vivo as a strategy for cell replacement therapy of defined (GENSAT) Project has used Egfp-BAC transgenic mice to define neuronal populations. patterns of CNS gene expression in the developing and adult mouse brain (28). The GENSAT Project has generated hundreds of cerebellum ͉ CNS development ͉ granule neuron ͉ stem cell Egfp-BAC transgenic lines and identified markers for cerebellar granule cells, including Pde1c for granule cells. Pde1c is expressed NEUROSCIENCE he cerebellar cortex is a remarkably simple laminar structure, by GCPs very early in the program of development, commencing at Twith two principal neurons, the granule cell and the Purkinje E10, when the earliest GCP markers, including Math1 (18), Zic1,2 cell, and a diverse set of interneurons, which modulate the output (19), and Meis1 (20), are expressed. Expression continues through- of the Purkinje cell to the cerebellar nuclei (1). The cerebellar out the lifetime of the animal, making Pde1c a granule neuron circuitry coordinates movement and balance and functions in marker that is expressed at all stages of life. To provide markers for sensory discrimination (2) and cognitive processing (3). Long-term ES differentiation into granule cells, we generated ES cells from depression of parallel fiber synapses onto Purkinje has been as- Pde1c-Egfp-BAC transgenic mice. To test whether the local signals sumed to control the simple vestibular ocular reflex. Recent studies and transcription factors that establish the cerebellar primordium in vivo in vitro on both simple functions and adaptive control of the cerebellum and specify neural fates can be harnessed to direct the differentiation of mouse ES cells into granule neurons, Purkinje suggest that multiple plasticity mechanisms may contribute to cells, and cerebellar glial cells, we studied the influence of cerebellar cerebellum-dependent learning. Multiple plasticity mechanisms are ‘‘organizer molecules,’’ dorsal signals and proteins that expand the probably important to encode memories over different time scales, GCP cell population on ES cell differentiation. to regulate the dynamics of movement, and to allow bidirectional changes in the amplitude of movements (4). Although the role of Results the cerebellum in learning and memory is becoming more complex, As discussed, inductive signals and transcription factors involved in the remarkably simple architectonics of the cerebellum make it an cerebellar neuron generation have been identified, raising the attractive model system for developing cell replacement strategies. question of whether these developmental insights can be used to Replacement therapy in the cerebellum, like that in other brain regions, depends on the ability to induce progenitor cells to differentiate into cells with specific cell fates. The pluripotent Author contributions: E.S. and M.E.H. designed research; E.S. performed research; E.S. and nature of mouse ES cells was formally demonstrated by their ability M.E.H. analyzed data; and E.S. and M.E.H. wrote the paper. to contribute to all tissues of adult mice, including the germ line, The authors declare no conflict of interest. after their injection into host blastocysts (5). In addition to their Freely available online through the PNAS open access option. developmental potential in vivo, ES cells display a remarkable Abbreviations: EB, embryoid bodies; EGL, external granule cell layer; GCP, granule cell capacity to form differentiated cell types in culture. Recently, Sato progenitor; Pn, postnatal day n; ML, molecular layer; IGL, internal granule cell layer; DIV, et al. (6) demonstrated that activation of the canonical Wnt pathway days in vivo; RA, retinoic acid; SHH, sonic hedgehog. could replace the requirement of mouse embryonic fibroblast- *Present address: Albany Medical College, Albany, NY 12208. conditioned media in the maintenance of undifferentiated hES cells †To whom correspondence should be addressed. E-mail: [email protected]. for short periods of time (5–7 days). In addition, Wichterle et al. (7) This article contains supporting information online at www.pnas.org/cgi/content/full/ demonstrated that treating ES cells with the series of signals that 0610879104/DC1. induce specific cell populations during normal, in vivo development © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610879104 PNAS ͉ February 20, 2007 ͉ vol. 104 ͉ no. 8 ͉ 2997–3002 Downloaded by guest on September 26, 2021 En1/Tuj1 Math1/Tuj1 Zic1/Tuj1 NeuroD/Tuj1 Meis1/Tuj1 Fig. 1. Differentiation of ES cells into proliferating A B C D E EGL cells, Purkinje neurons, and interneurons. (A–D) After 5 days (12 DIV) of differentiation with FGF8b, WNT1 and WNT3a ES cells expressed a marker of the cerebellar territory (green), EN1 (A), and of proliferat- ing EGL cells MATH1 (B), ZIC1 (C), and NEUROD (D). (E–H) After 4 days (16 DIV) of culture with FGF8b, WNT1, WNT3A, and BMP7, GDF7 and BMP6 ES cells Pax6/Tuj1 Pax2/Tuj1 Calbn/Tuj1 Math1/Pax6 Zic2/Pax6 F G H IJ expressed the granule neuron markers MEIS1 (E), PAX6 (F), and PAX2 (G) and the Purkinje cell marker CALB1 (H). Differentiated ES cells also expressed neuronal marker class III ␤-tubulin (TUJ1; A–H) (red). (I and J)ES cells coexpressed PAX6 (red), MATH1, and ZIC2 (green). (Scale bar: 40 ␮m for A, B, F, and G and 20 ␮m for C–E and H–J.) direct stem cells to a cerebellar fate. We show that developmentally Fig. 8) Double labeling experiments (J) showed that the ES cells relevant signaling factors can induce mouse ES cells to differentiate expressed multiple markers of GCPs. RT-PCR analysis confirmed into midbrain/hindbrain progenitor cells, and subsequently into these results and demonstrated that the cells did not express cerebellar neurons, through a pathway recapitulating that used in markers of spinal cord neurons (26). Thus, treatment of ES cells vivo. To examine the capacity of ES cells to generate cerebella cells, with dorsalizing signals and with the specific combination of BMPs we cultured mouse ES cells for 2 days to form EBs (1,000 cells) and shown previously to induce GCP fates in vivo appeared to recapit- maintained the EB suspension culture (1–7 days) in medium ulate the developmental program that generates cerebellar neu- supplemented with FGF8b and retinoic acid (RA). We observed rons, especially the cerebellar