Lmx1a and Lmx1b Function Cooperatively to Regulate Proliferation, Specification, and Differentiation of Midbrain Dopaminergic Progenitors

Abstract

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 Lmx1a dr/dr ;Shh Cre/ϩ ;Lmx1b f/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 Lmx1b genes were inactivated. Together, our results demonstrate that Lmx1a and Lmx1b function cooperatively to regulate proliferation, specification, and differentiation of mDA progenitors, including their floor plate-like properties.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

“…Tissue samples were cryosectioned in 20-μm coronal sections and mounted on Superfrost Plus slides (Fisher Scientific). The procedures for in situ hybridization have been described previously (8). The mouse anti-sense RNA probes for Lmx1a and Lmx1b were generated from cDNA templates using RT-PCR and were used as described previously (49).…”

“…Our knowledge of the functional roles of these TFs in mature neurons remains rudimentary. Accumulating evidence shows that transcription factors including the nuclear receptor related 1 protein (Nurr1), En1, Pitx3, Otx2, and Foxa2, which are recognized for their role in the early development of mDA neurons, are also required for the maintenance of phenotypic neuronal identity in the adult (5).The LIM homeodomain genes Lmx1a/b are early determinants of the fate of mDA progenitors (6), and their actions are essential at each step of DA neuronal generation (7,8). The murine Lmx1a and Lmx1b proteins are closely related and share an overall amino acid identity of 64%, with 100% identity in their homeodomain and 67% and 83% identity in each LIM domain (9).…”

“…The LIM homeodomain genes Lmx1a/b are early determinants of the fate of mDA progenitors (6), and their actions are essential at each step of DA neuronal generation (7,8). The murine Lmx1a and Lmx1b proteins are closely related and share an overall amino acid identity of 64%, with 100% identity in their homeodomain and 67% and 83% identity in each LIM domain (9).…”

How to make a midbrain dopaminergic neuron

“…2, red arrows) required for mDA specification: on one hand β-catenin directly upregulates Lmx1a and Otx2, and, on the other hand, Lmx1a/b directly upregulate each other as well as Wnt1, Msx1 and two key genes involved in mDA neuronal differentiation and survival, Nurr1 (Nr4a2, nuclear receptor 4a2) and Pitx3 (pituitary homeobox 3, or pairedlike homeodomain transcription factor 3). Accordingly, deletion of both Lmx1a and Lmx1b results in a near-complete loss of mDA neurons (Yan et al, 2011). Similarly, deletion of Wnt1 results in the loss of Lmx1a, Nurr1 and Pitx3 in the mFP and a complete loss of mDA neurons (Andersson et al, 2013;Prakash et al, 2006).…”

A novel floor plate boundary defined by adjacentEn1andDbx1microdomains distinguishes midbrain dopamine and hypothalamic neurons

“…The Lmx1a/b domain is the origin of most DA neurons, whereas the Nkx6-1/Sim1/Neurog1 domain is the source of Pou4f1 + red nucleus (RN) neurons and other smaller populations. Knockouts of Lmx1a/b results in fewer DA neurons and ectopic Pou4f1 + neurons from the DA domain (Deng et al, 2011;Yan et al, 2011), whereas forced ectopic expression of Lmx1a/b result in DA neuron production from the Nkx6-1/Sim1/Neurog1 domain at the expense of RN neurons (Anderegg et al, 2013;Andersson et al, 2006;Lin et al, 2009;Nakatani et al, 2010). Conversely, forced ectopic expression of Sim1 results in the production of Pou4f1 + neurons from the Lmx1a/b domain, at the expense of DA neurons (Nakatani et al, 2010).…”