Cre fate mapping reveals lineage specific defects in neuronal migration with loss of Pitx2 function in the developing mouse hypothalamus and subthalamic nucleus

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Abstract

Establishment of neuronal diversity is a central topic in developmental neurobiology. Prior studies implicated Pitx2, a paired-like homeodomain transcription factor, in mouse subthalamic nucleus neuronal development, but precise stages of neuronal differentiation affected (migration, axon outgrowth, fate specification) and underlying mechanisms were unknown. Here we report lineage tracing experiments using Pitx2cre/+, Pitx2cre/null, and conditional nuclear lacZ reporter mice to track embryonic Pitx2 expressing neurons. Migration of subthalamic nucleus and hypothalamic neurons was severely arrested in Pitx2cre/null embryos, and subclasses of subthalamic nucleus neurons identified by Lmx1b, Foxp1, and Foxp2-gene expression revealed differing sensitivities to Pitx2 dosage. Interestingly, embryonic subthalamic nucleus development was unaffected in Lmx1b null mice, suggesting that Pitx2 and Lmx1b act via independent genetic pathways. These data provide the first direct evidence for Pitx2-dependent neuronal migration in the developing hypothalamus, and demonstrate that complex transcriptional networks regulate regional specialization of distinct hypothalamic and subthalamic nucleus neurons.

Introduction

Generation of neuronal diversity in the mammalian brain requires coordinated expression of transcription factors and signaling molecules (Puelles and Rubenstein, 2003, Sur and Rubenstein, 2005, Lim and Golden, 2007). Regional specialization of these complex neuronal populations also requires that differentiating neurons travel long, sometimes circuitous routes to their final destinations in the brain. Central questions in developmental neurobiology include how these distinct neuronal populations are formed and which molecular signals are used to guide their terminal differentiation. Recent advances in genetic fate mapping of cells with restricted gene expression have made it possible to explore cellular fates and phenotypes of specific neuronal lineages in the developing mouse brain (Branda and Dymecki, 2004, Joyner and Zervas, 2006).

Restricted expression of Pitx2, a paired-like homeodomain transcription factor, in the developing mouse hypothalamus occurs concomitant with or soon after terminal mitosis of neural progenitors around E9.5–E10.5 (Martin et al., 2002), and is necessary for normal development of neurons in the subthalamic nucleus (Martin et al., 2004). Due to complex central nervous system phenotypes and a lack of reporter alleles, earlier studies could not distinguish the effects of Pitx2 deficiency on neural cell migration, axon outgrowth, or cell fate specification. Moreover, it was not possible to exclude altered Pitx2 gene expression as an explanation for defects seen in Pitx2 null embryos (Martin et al., 2004).

Pitx2 exhibits pleiotropic, tissue specific effects during development, with a short (30 min) mRNA half-life that is regulated by Wnt/Dvl/β-catenin signaling (Kioussi et al., 2002). Pitx2 promotes migration of cultured HeLa cells (Wei and Adelstein, 2002) and cells that give rise to craniofacial and cardiac structures (Liu et al., 2002, Liu et al., 2003), and Pitx2 function is critical for survival of pituitary hormone-producing cells (Charles et al., 2005). Based on these observations, we hypothesized that Pitx2 may be required for one or more aspects of neuronal differentiation including maintenance of gene expression, cell identity, neuronal migration, and axonal outgrowth. To explore the fates of Pitx2 deficient neurons during mid-gestation, we designed a lineage tracing strategy using previously characterized Pitx2null (Gage et al., 1999) and Pitx2cre knock-in (Liu et al., 2003) alleles and transgenic mice containing a Cre conditional nuclear localized lacZ reporter under the control of the chicken β-actin promoter (N-lacZ) (Zinyk et al., 1998). Our analysis revealed that Pitx2 is essential for lineage specific neuronal migration in the developing hypothalamus and subthalamic nucleus.

Section snippets

Nuclear localized β-galactosidase labels Pitx2 mutant neurons

PITX2 protein and mRNA are expressed in differentiating neurons of the E9.5–E14.5 mouse brain around the time of terminal mitosis (Martin et al., 2002 and unpublished data). Prior studies showed that homozygous Pitx2 null embryos require Pitx2 for normal gene expression and formation of neuronal projections in the developing subthalamic nucleus, but the lack of a permanent reporter made it impossible to track the fates of Pitx2 mutant neurons (Martin et al., 2004). To evaluate for region

Discussion

We demonstrate, through the use of a Cre lineage tracing Pitx2 deficiency allele, neuronal migration defects in the developing mouse hypothalamus with loss of Pitx2 function. Our studies provide direct evidence of a genetic requirement for Pitx2 in normal neuronal migration in the developing hypothalamus, which, unlike the cortex and cerebellum, has not been extensively explored (Ayala et al., 2007, Lim and Golden, 2007).

Generation of mutant mice

We used a previously characterized Pitx2 null allele, Pitx2creneo, that exhibits Cre activity in areas of known Pitx2 expression in the heart and craniofacial structures (Liu et al., 2002, Liu et al., 2003). Pitx2creneo/+ mice are heterozygous for a Pitx2 deficiency allele in which the homeodomain is replaced with an IRES-Cre-neomycin cassette (Liu et al., 2003). Since brain Cre activity patterns in these mice had not been explored in detail, we mated Pitx2creneo/+ mice with FLPe recombinase

Acknowledgments

We thank Ben Novitch and Yasushi Nakagawa for their critical comments on the manuscript. The following people provided reagents: Iain McIntosh (Lmx1b mice), Tord Hjalt (PITX2 antibody), Tom Glaser (β-galactosidase antibody), Tom Jessell (LMX1B antibody), Chris Walsh (CAMKL1 antibody), and Edward Morrissey (FOXP1 antibody). Margaret Lomax and Bob Lyons assisted with Q-PCR for genotyping N-lacZ mice. These data were presented in part at the 2006 annual meetings of the Society for Developmental

References (48)

  • MucchielliM.L. et al.

    Otlx2, an Otx-related homeobox gene expressed in the pituitary gland and in a restricted pattern in the forebrain

    Mol. Cell Neurosci.

    (1996)
  • PuellesL. et al.

    Forebrain gene expression domains and the evolving prosomeric model

    Trends Neurosci.

    (2003)
  • TamuraS. et al.

    Foxp1 gene expression in projection neurons of the mouse striatum

    Neuroscience

    (2004)
  • WinterC.G. et al.

    Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

    Cell

    (2001)
  • ZervasM. et al.

    Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1

    Neuron

    (2004)
  • AltmanJ. et al.

    The development of the rat hypothalamus

    Adv. Anat. Embryol. Cell Biol.

    (1986)
  • Alvarez-BoladoG. et al.

    Winged helix transcription factor Foxb1 is essential for access of mammillothalamic axons to the thalamus

    Development

    (2000)
  • BalterM.

    Genetics: first gene linked to speech identified

    Science

    (2001)
  • BerryF.B. et al.

    Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld–Rieger syndrome and anterior segment dysgenesis

    Hum. Mol. Genet.

    (2006)
  • CharlesM.A. et al.

    PITX genes are required for cell survival and Lhx3 activation

    Mol. Endocrinol.

    (2005)
  • ChenH. et al.

    Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome

    Nat. Genet.

    (1998)
  • DiehlA.G. et al.

    Extraocular muscle morphogenesis and gene expression are regulated by Pitx2 gene dose

    Invest. Ophthalmol. Vis. Sci.

    (2006)
  • DingY.Q. et al.

    Lmx1b is essential for the development of serotonergic neurons

    Nat. Neurosci.

    (2003)
  • FerlandR.J. et al.

    Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain

    J. Comp. Neurol.

    (2003)
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