The Journal of Neuroscience, August 31, 2011 • 31(35):12413–12425 • 12413 Development/Plasticity/Repair Lmx1a and Lmx1b Function Cooperatively to Regulate Proliferation, Specification, and Differentiation of Midbrain Dopaminergic Progenitors Carol H. Yan,1* Martin Levesque,1* Suzanne Claxton,1 Randy L. Johnson,2 and Siew-Lan Ang1 1Department of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom, and 2Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Centre, Houston, Texas 77030 LIM homeodomain transcription factors, Lmx1a and Lmx1b, are required for the development of midbrain dopaminergic (mDA) neurons. Lmx1b is required for the specification and maintenance of mDA neurons, primarily due to its role in isthmic organizer development that is essential for the induction of mDA neurons. Here, we conditionally deleted Lmx1b in the ventral neural tube using ShhCre and found that Lmx1b conditional mutant mouse embryos show no defect in the development and maintenance of mDA neurons. In addition, Dreher (Lmx1a mutant) embryos display only a moderate reduction in the number of mDA neurons, suggesting that the related family member Lmx1b might compensate for Lmx1a function. We therefore generated Lmx1a and Lmx1b double mutants. Severe loss of mDA neurons occurred in Lmx1adr/dr;ShhCre/ϩ;Lmx1bf/f double mutants due to essential roles for Lmx1a and Lmx1b in regulating the proliferation and neuronal commitment of mDA progenitors through the expression of Wnt1 and Ngn2, respectively. Lmx1a and Lmx1b also negatively regulate Hes1 expression and consequently cell cycle exit through activation of p27 Kip1 expression. In addition, Lmx1a and Lmx1b also regulate the expression of floor plate genes such as Corin and Slit2 and specification of postmitotic mDA neurons. These defects were more severe with decreasing gene dosage of Lmx1a and Lmx1b or observed only when all four copies of Lmx1a and Lmx1bgeneswereinactivated.Together,ourresultsdemonstratethatLmx1aandLmx1bfunctioncooperativelytoregulateproliferation, specification, and differentiation of mDA progenitors, including their floor plate-like properties. Introduction code, including expression of Otx2, Foxa1, Foxa2, Lmx1a, and Midbrain dopaminergic (mDA) neurons have diverse roles in Lmx1b. regulating motor and cognitive functions (Bjorklund and Lind- Otx2 is a homeodomain transcription factor required for pat- vall, 1984). Degeneration or dysfunctions of these neurons can terning the mid-hindbrain region (Puelles et al., 2003) and spec- lead to severe neurological disorders such as Parkinson’s disease ification of mDA progenitors (Omodei et al., 2008). Foxa1 and (Lang and Lozano, 1998; Olanow et al., 2003). These neurons are Foxa2 are members of the forkhead/winged helix family of tran- thought to arise from progenitors in the floor plate region of the scription factors required for development and maintenance of caudal diencephalon and the mesencephalon (Marin et al., 2005; mDA neurons in adults (Ferri et al., 2007; Kittappa et al., 2007; Andersson et al., 2006a; Ono et al., 2007). Extrinsic signals, such Lin et al., 2009). Lmx1a and Lmx1b belong to the Lmx group of as sonic hedgehog (Shh), fibroblast growth factor-8 (Fgf8), LIM homeodomain transcription factors and share 64% homol- Wnt1, and transforming growth factor-␤2 (Tgf-␤2) and Tgf␤3 ogy in their overall amino acid composition (Hobert and West- specify mDA progenitor identity (Hynes et al., 1997; Ye et al., phal, 2000). Lmx1a spontaneous mutants dreher (Lmx1adr/dr) 1998; Farkas et al., 2003; Prakash et al., 2006). Consequently, exhibit a complex phenotype, including circling behavior, steril- mDA progenitors are assigned a combined transcription factor ity, pigmentation, and tail abnormalities (Lyons and Wahlsten, 1988; Chizhikov et al., 2006a). Loss- and gain-of-function studies in chick embryos also demonstrate that Lmx1a is an essential Received March 1, 2011; revised June 20, 2011; accepted July 7, 2011. Authorcontributions:C.H.Y.,M.L.,andS.-L.A.designedresearch;C.H.Y.,M.L.,andS.C.performedresearch;R.L.J. determinant of mDA neuron development (Andersson et al., contributed unpublished reagents/analytic tools; C.H.Y., M.L., and S.-L.A. analyzed data; C.H.Y., M.L., and S.-L.A. 2006a). However, only 46% of mDA neurons are lost in wrote the paper. Lmx1adr/dr mutant mouse embryos, suggesting that the related This work was supported by Medical Research Council United Kingdom and by a research grant from Parkinson’s family member Lmx1b may compensate for Lmx1a function United Kingdom (S.-L.A.). We thank members of the laboratory for critical reading of this manuscript, Drs. T. Perlmann and J. Ericson for sharing data prior to publication, and Dr. B. Harfe for generously providing ShhCre/ϩ (Ono et al., 2007). mice. Loss-of-function studies demonstrate that Lmx1b regulates *C.H.Y. and M.L. contributed equally to this work. mid-hindbrain patterning, and consequently severe reduction of Correspondence should be addressed to Siew-Lan Ang, Division of Developmental Neurobiology, National Insti- mDA neuron number is primarily due to early loss of most of the tute for Medical Research, The Ridgeway, London NW7 1AA, UK. E-mail: [email protected]. Ϫ/ Ϫ C. H. Yan’s present address: Department of Biochemistry, University of Hong Kong, Hong Kong. midbrain in Lmx1b mouse embryos (Guo et al., 2007). DOI:10.1523/JNEUROSCI.1077-11.2011 Lmx1b also failed to rescue the loss of mDA neurons in chick Copyright © 2011 the authors 0270-6474/11/3112413-13$15.00/0 embryos with specific downregulation of Lmx1a in the ventral 12414 • J. Neurosci., August 31, 2011 • 31(35):12413–12425 Yan et al. • Lmx1a and Lmx1b Regulate Dopamine Neuron Development midbrain using siRNAs (Andersson et al., 2006a), suggesting that number of cells of the mDA region was obtained by multiplying raw Lmx1b cannot compensate for Lmx1a function in the develop- counts by 12. For E18.5 data, we counted all THϩ neurons from five ment of these neurons. In contrast, Lmx1a and Lmx1b show matching sections of 12 ␮m (one section every six) from wild-type, dr/dr dr/dr cre/ϩ f/f ϭ similar roles in mDA neuron development in gain-of-function Lmx1a , and Lmx1a Shh Lmx1b embryos (n 3 for each studies in mouse embryos (Lin et al., 2009; Nakatani et al., 2010) genotype). The rostral-most level corresponds to mouse brain atlas at bregma Ϫ2.78 mm (Allen Institute for Brain Science). At this level, part and in ES cells (Chung et al., 2009); however, in these experi- of the posterior hypothalamus and the fasciculus retroflexus are located ments, Lmx1b induces Lmx1a expression in mDA cells and a dorsally and medially to the VTA. The posterior-most section corre- specific role of Lmx1b in mDA neuron development remains sponds to bregma Ϫ3.08, where the fasciculus retroflexus forms a round unclear. bundle of fiber located ventrally to the VTA. The total number of em- We therefore investigated whether Lmx1a and Lmx1b func- bryos counted was 74 that were harvested from 24 litters. Quantitative tion cooperatively to regulate mDA neuron development using data represent mean Ϯ SEM for cell counts of the entire midbrain. Two- genetic studies in mice. ShhCre/ϩ;Lmx1bflox/flox embryos (referred tailed Student’s t tests or ANOVA were used to determine statistical to as ShhCre/ϩ;Lmx1bf/f) showed no obvious phenotype in the significance. *p Ͻ 0.05; **p Ͻ 0.01; ***p Ͻ 0.001; N.S., not significant. generation of mDA neurons. In contrast, severe loss of mDA In vivo proliferation assays. Pregnant females received intraperitoneal neurons occurred in Lmx1adr/dr;ShhCre/ϩ;Lmx1bf/f double mu- injection of BrdU (100 mg/kg; Sigma-Aldrich) 1 h before embryos being harvested and fixed with 4% paraformaldehyde. Sections were immuno- tants. Together, our results demonstrate cooperative roles for stained with rat anti-BrdU in combination with mDA cell marker (anti- Lmx1a and Lmxb in regulating proliferation, specification, and Lmx1a or anti-Lmx1b) and/or with anti-Ki67. differentiation of mDA progenitors. Quitting fraction. Twenty-four hours before harvesting embryos, preg- nant females received intraperitoneal injection of BrdU (100 mg/kg; Materials and Methods Sigma-Aldrich). Sections from E11.5 embryos were then triple stained Animals. Lmx1adr/dr mutant mice (B6C3Fe-a/a-Lmx1adr-J; The Jackson for Ki67, BrdU, and Lmx1b. The quitting fraction is obtained by Laboratory), ShhCre/ϩ (Harfe et al., 2004), and Lmx1b floxed (Lmx1bf/f) dividing the number of BrdU-labeled cells that had left the cell cycle Ϫ ϩ ϩ mice (Zhao et al., 2006) were genotyped as previously described (Ki67 BrdU Lmx1b ) by the number of BrdU-labeled cells that ϩ ϩ ϩ (Mishima et al., 2009). All the mice were maintained in a mixed back- were still cycling (Ki67 BrdU Lmx1b ). ground. Conditional Lmx1b mice were generated by intercrossing ShhCre/ϩ and Lmx1bf/f mouse lines. Timed mating was set up for 3 h between dr/ϩ Cre/ϩ f/ϩ dr/ϩ f/f Results Lmx1a ;Shh ;Lmx1b males and Lmx1a ;Lmx1b females for Expression of Lmx1b in mDA progenitors and neurons is not Lmx1a/b double-mutant studies, after which the mice were separated. required for their generation and maintenance The embryos were staged as E0 when a vaginal plug was detected. Tissue analysis. The procedures for whole-mount and section in situ To study the specific effect of Lmx1b on mDA neuron develop- hybridization have been described previously (Conlon and Herrmann, ment without disrupting the isthmic organizer, the Lmx1b floxed