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First published online 1 August 2007
doi: 10.1242/dev.02879

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Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells

Yuichi Ono^1,,*, Tomoya Nakatani^1,, Yoshimasa Sakamoto^1,, Eri Mizuhara¹, Yasuko Minaki¹, Minoru Kumai¹, Akiko Hamaguchi¹, Miyuki Nishimura¹, Yoko Inoue¹, Hideki Hayashi^2,, Jun Takahashi^2, and Toshio Imai¹

¹ KAN Research Institute Inc., KobeMI R&D Center 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
² Department of Neurosurgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.

View larger version (117K): [in this window] [in a new window]   Fig. 1. Lmx1a is selectively expressed in mesDA neurons and their proliferative progenitors. (A) Expression of Lmx1a in the mouse mesencephalon at E12.5. (B-F) Lmx1a is selectively expressed in the mesDA neurons. Lmx1a is co-expressed with Th (B), Lmx1b (C), Nurr1 (D) and Pitx3 (E) but not with Nkx6.1 (F) in the mouse ventral mesencephalon at E12.5. Only Lmx1a is expressed in the VZ region of the mesDA domain. (G-J) Lmx1a is expressed in the proliferative progenitors. Lmx1a was co-expressed with MPM2 (I) and Ngn2 (J), and Lmx1a+ VZ cells incorporate acutely injected BrdU (H) at E11.5.

View larger version (79K): [in this window] [in a new window]   Fig. 2. Lmx1a regulates mesDA neurogenesis by inducing proneural factor expression and cell growth in their progenitors. (A) The mesDA number (indicated by Th and Lmx1b expressions) is reduced in dreher mutant embryos compared with wild-type embryos at E13.5, but the fate of Lmx1a+ neurons is not affected. The intensities of anti-Th and anti-Lmx1b staining were lower in dreher mutants. However, almost all Lmx1a+ neurons and neurons generated from ventral midline regions of the mesencephalon, in mutants and wild-type controls, expressed Lmx1b. (B) Neurogenesis in Lmx1a+ progenitors is affected by the dreher mutation. The Th+ neuron number is severely reduced in dreher mutants at E11.5. Ngn2 and Mash1 expression levels in the Lmx1a+ VZ region are reduced and, consistently, the generation of Lmx1a+ TuJ1+ neurons is less efficient in mutants. Note that reductions in the expression levels of proneural factors are apparent in the medial part of the Lmx1a+ domain. (C) Quantification of Lmx1b+ neurons in the ventral mesencephalon of wild-type, heterozygous and homozygous dreher mutant embryos at E13.5. (D) Quantification of BrdU-incorporating, Ngn2+ and Mash1+ cells in Lmx1a+ VZ region.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Mis-specification of mesDA neurons in dreher embryos. Lim1/2 is expressed in RN neurons and is not co-expressed with mesDA markers in wild-type embryos (A,C,E). By contrast, Lim1/2 is ectopically expressed in Lmx1b+ (B,B'), Lmx1a+ (D) and Nurr1+ (F) neurons (arrowheads). Note that these Lim1/2+ mesDA precursors do not express Th. (G) Model of Lmx1a function in postmitotic mesDA specification.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Mesencephalic ventral midline cells with FP-like marker expression have mesDA progenitor characteristics. (A-C) Co-expression of FP4 and Lmx1a in the rat ventral mesencephalon at E14.5. (D) Virtually all of the neural progenitor cells at the ventral midline of the mesencephalon express Lmx1a. NG2 marks blood vessels. (E,F) Ngn2 and Mash1 are expressed in FP4+ cells in the rat mesencephalon. (G,H) FP cells of the E11.5 rat mesencephalon without proneural gene expression have mesDA characteristics. (I-Q) Ngn2, Mash1, Lmx1a and Dll1 are selectively expressed in mesencephalic FP cells, but not in caudal FP cells of E11.5 mouse embryos. Lmx1b is expressed in midline cells at all AP locations. Mes, mesencephalon; Met, metencephalon; Nuc, nuclear stain; SC, spinal cord.

View larger version (62K): [in this window] [in a new window]   Fig. 5. FACS isolation of mesencephalic ventral midline cells with FP-like characteristics. (A) Corin is selectively expressed in ventral midline cells from the mesencephalon to the spinal cord of E12.5 mouse embryos. (B) Co-expression of Corin and FP4 in E13.5 rat FP cells. Note that Corin is expressed only in medial FP cells in the spinal cord. (C) FACS analysis of E13.5 rat ventral mesencephalon and metencephalon cells. Corin+ cells can be sorted with more than 95% purity. (D) Rat mesencephalic Corin+ cells express Lmx1a, FP4, nestin but not TuJ1. Virtually all the Corin+ cells express Lmx1a and nestin. FP4 expression is detected in more than 95% of the Corin+ cells. (E) Mesencephalic Corin+ cells express Shh, Hnf3ß and Ntn1 transcripts. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Sorted mesencephalic ventral midline cells generate mesDA neurons, but caudal FP cells do not differentiate into neurons in vitro. (A) HuC/D+ neurons are generated from E13.5 rat mesencephalic Corin+ cells, but not from metencephalic Corin+ cells at 6 DIV. (B) Neurons generated in mesencephalic Corin+ cell cultures express Nurr1. (C,D)Th+ and Pitx3+ mesDA neurons are efficiently generated from mesencephalic Corin+ cells, but not from mesencephalic Corin-negative cells. (E) Co-culture of Corin+ cells and DiI-labeled Corin- cells. At 6 DIV, DiI+ cells do not account for the majority of HuC/D+ and Th+ neurons, suggesting that mesDA neurons generated in Corin+ cell cultures are derived from Corin+ cells, but not from contaminated Corin- cells.

View larger version (61K): [in this window] [in a new window]   Fig. 7. mesDA neurons originate from FP cells. (A) Mouse early mesencephalic FP cells express Corin and Lmx1a but not proneural genes. (B) Mesencephalic FP cells sorted from E9.75 mouse embryos generate Nurr1+ mesDA neuron precursors in vitro. Note that Corin+ cells at the sorting period do not express proneural genes (A) and acquire neurogenic activity during culturing, suggesting that mesencephalic FP cells are intrinsically fated to acquire neural progenitor characteristics.

View larger version (86K): [in this window] [in a new window]   Fig. 8. Anterior identity confers neurogenic potential in the FP cells. Otx2 (A) or Mash1 (B) are ectopically expressed under the control of the FP-specific enhancer of the Shh gene. Images show the ventral midline region of the metencephalon at E11.5 (A) or at E12.5 (B). The transgene is selectively expressed in the cFP region. In Otx2-transgenic embryos, cFP cells acquire mesFP-like characteristics and mesDA neurogenesis occurs in the cFP region. By contrast, in Mash1-transgenic embryos, cFP cells differentiate into neurons positive for Th and Lmx1b, but negative for other mesDA markers.

View larger version (57K): [in this window] [in a new window]   Fig. 9. Isolation of mesDA progenitors from ES cell-derived neural populations. (A) Surface expression of Corin in undifferentiated ES cells and SDIA-induced populations. (B) RT-PCR analysis of unsorted SDIA-induced cells (all) and sorted Corin+ and Corin- populations. Expression of undifferentiated ES cell-specific Nanog is not detected in the Corin+ population. (C) mesDA progenitors are enriched in the Corin+ population. Neurons generated from Corin+ cells show correct mesDA neuron identity. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.