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First published online 27 June 2007
doi: 10.1242/dev.000141

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Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner

Anna L. M. Ferri^1,*, Wei Lin^1,*, Yannis E. Mavromatakis¹, Julie C. Wang¹, Hiroshi Sasaki², Jeffrey A. Whitsett³ and Siew-Lan Ang^1,

¹ Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
² Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
³ Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Foxa1/2 are expressed in mDA progenitors and neurons and the timing of inactivation of Foxa2 in Foxa2cko embryos. (A-F,H-I''') Coronal adjacent sections of mouse embryos. (A-F) Wild-type embryos. Co-localization of Foxa1/2 proteins with Lmx1a (A,B), Nurr1 (C,D) and TH (E,F) demonstrate that Foxa1/2 are expressed in all mDA cells in the ventral midbrain at E10.5 (A,B) and E12.5 (D-F). (G) Schematic of the ventral part of the midbrain showing the expression of markers in mDA cells at different developmental stages. For clarity, expression of Foxa1/2 genes in other neuronal populations have been omitted. (H-I''') Foxa2 protein detected by immunohistochemistry is largely absent in ventral midbrain progenitors from E10.5 onwards. Scale bars: 75 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 2. Nkx2.2 is abnormally expressed ventrally, including in a small number of mDA progenitors, in Foxa1-/-;Foxa2cko but not Foxa2cko or Foxa1-/- embryos. (A-H') Coronal sections of mouse embryos. (A-D,A'-D') Lmx1a and Lmx1b are expressed normally in mDA progenitors of Foxa1-/-, Foxa2cko and Foxa1-/-;Foxa2cko mutants, as in control embryos. (E-H,E'-H') Nkx2.2 expression is expanded ventrally starting at E10.5. (I-L) A small number of Lmx1a+ Nkx2.2+ progenitors are observed in the ventral midbrain of Foxa1-/-;Foxa2cko but not Foxa1-/-, Foxa2cko and control embryos at E12.5. Scale bars: 75 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Normal development of ventral midbrain progenitors and Nurr1+ TH+ mDA neurons in Shhcko embryos at E12.5. (A-H) Coronal sections of mouse embryos. (A,E) Shh expression is missing in Shhcko embyos (E), whereas it is expressed in basal midbrain progenitors in control embryos (A). (B-D,F-H) Similar expression of Foxa1/2 and Nurr1 and TH in the ventral midbrain of Shhcko (F-H) and in control embryos (B-D). Scale bars: 75 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 4. Reduced expression of Ngn2 in mDA progenitors and incomplete specification of immature neurons in Foxa1-/-;Foxa2cko double-mutant embryos at E12.5. (A-F) Coronal sections of mouse embryos. (A,B) Ngn2 expression is reduced in mDA cells in mutant compared with wild-type embryos. (C-F) Lmx1a, Tuj1+ immature neurons are generated (C,D) that do not express Nurr1 and TH (E,F) in mutant as compared with control embryos. Scale bars: 75 µm.

View larger version (104K): [in this window] [in a new window]   Fig. 5. Foxa1/2 are required for early and late differentiation of mDA neurons at E12.5. (A-L) Coronal sections of mouse embryos. (A-H) The number of immature Nurr1+ TH- (A-D) and En1+ TH- (E-H) neurons (green) increases at the expense of mature Nurr1+ TH+ (A-D) and En1+ TH+ mDA (E-H) neurons (yellow) in Foxa1/2 single-mutant embryos compared with the corresponding numbers in control embryos. The total number of neurons (immature plus mature mDA neurons) is reduced only in Foxa2cko;Foxa1-/- embryos when compared with the total number in control embryos. (I-L) TH+ AADC+ mature neuron number decreases in all mutant compared with control embryos (see quantification in Fig. 6). Scale bars: 75 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Dosage-dependent requirement for Foxa1/2 in the differentiation of mDA neurons and a progressive rescue of mDA neuron number in mutant embryos during development. (A-C) Bar charts showing the number of immature (Nurr1+ TH-) and mature (Nurr1+ TH+) neurons, as determined by immunohistochemistry, in embryos carrying 0-4 copies of Foxa1 and/or Foxa2 at three different stages. The x-axis shows the genotype of the mouse embryos, and the y-axis indicates the number of cells.

View larger version (9K): [in this window] [in a new window]   Fig. 7. Schematic summarising the sequential roles of Foxa1/2 during the three different phases of development of mDA neurons from neural stem/progenitor cells. During regional and neuronal specification, Foxa1/2 positively regulates Ngn2 expression in mDA progenitors. Foxa1/2 is subsequently required for Nurr1 and En1 expression in immature mDA neurons and for the expression of aromatic L-amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) in mature mDA neurons during early and late differentiation.