Elsevier

Brain Research

Volume 1206, 24 April 2008, Pages 33-43
Brain Research

Research Report
Differential and dose-dependent regulation of gene expression at the mid-hindbrain boundary by Ras–MAP kinase signaling

https://doi.org/10.1016/j.brainres.2008.01.100Get rights and content

Abstract

Recent experiments suggest that activation of the Ras–MAP kinase pathway at the mid-hindbrain boundary (MHB) induces cerebellar development, whereas tectal development occurs in the absence of Ras–MAP kinase activity. To test this model we have stimulated or inhibited Ras–MAP kinase signaling in chick embryos through targeted misexpression of a constitutive active (RasV12) or dominant negative (RasN17) form of Ras. The consequence of these manipulations on the expression of several genes that are expressed in distinct patterns at or around the MHB organizer, including En1, Pax2, Pax3, Pax5, Wnt1, Meis2, and ephrin-A2, -A5, and -B1, was assessed. Extending previous findings we show that inhibition of Ras–MAP kinase signaling differently affects Pax3 expression in different regions of the mid-hindbrain territory, inhibiting its expression in the midbrain but inducing it in the MHB region. Expression of the midbrain specific marker gene Meis2 was not affected by RasN17 at first but later upregulated concomitantly with the morphological transformation of hindbrain to midbrain. In addition, we show that different dosages of Ras–MAP kinase activity are required for transcriptional activation of Wnt1 and En1 at the MHB. Collectively, these results validate and extend previous findings on the molecular changes associated with Fgf8 loss-of-function or gain-of-function phenotypes at the MHB, demonstrate that gene expression at the MHB is regulated by Ras–MAP kinase signaling in a spatially and temporally distinct manner and provide evidence for a dosage dependent function of Fgf8 signaling at the MHB.

Introduction

Early development of the mid-hindbrain region is largely governed by the isthmic organizer (or mid-hindbrain boundary (MHB) organizer), a signaling center located at the boundary between the mes- and metencephalic vesicles. Development of the MHB organizer initiates already during gastrulation and requires graded activity of the Wnt and Fgf8 signaling pathways (Rhinn et al., 2005, Olander et al., 2006). Later during embryogenesis, expression of an interdependent network of nuclear factors and signaling proteins, which includes the transcription factors En1/En2, Pax2/Pax5, Lmx1b, Hes1/Hes3, iro1/iro7 and members of the Fgf- and Wnt-families of secreted proteins, maintains isthmic organizer activity and controls the development of the mid-hindbrain territory (Adams et al., 2000, Araki and Nakamura, 1999, Bally-Cuif et al., 1992, Davis and Joyner, 1988, Hidalgo-Sanchez et al., 1999, Hirata et al., 2001, Itoh et al., 2002, Ristoratore et al., 1999, Rowitch and McMahon, 1995, Shamim et al., 1999; Buckles et al., 2004).

Fibroblast growth factor 8 (Fgf8) is expressed in rhombomere1 (r1) adjacent to the mid-hindbrain boundary and is of critical importance for mid- and hindbrain development. Beads soaked in recombinant Fgf8 and implanted into the chick diencephalon, midbrain or rostral hindbrain can mimic the inductive capacity of isthmic transplants and induce organizer characteristic gene expression and cell fate changes (Crossley et al., 1996, Shamim et al., 1999). Conversely, Fgf8 mutant zebrafish (acerebellar (ace) mutants), mice hypomorphic for Fgf8 or mice mutant for Fgf receptor 1 fail to maintain isthmus specific gene expression and exhibit defects in midbrain- and cerebellar development (Meyers et al., 1998, Reifers et al., 1998, Trokovic et al., 2003). Two splicing isoforms of Fgf8, Fgf8a and Fgf8b, are expressed at the organizer of which Fgf8b can elicit a mes- to metencephalic cell fate change when ectopically delivered to the mid-hindbrain region (Lee et al., 1997, Liu et al., 1999, Liu et al., 2003, Sato et al., 2001). Fgf signals are transmitted into the recipient cells via tyrosine kinase type receptors. Four Fgf receptor genes (Fgfr1–4) exist in the mammalian genome of which Fgfr1 is the most relevant receptor for isthmic organizer function and maintenance (Blak et al., 2005, Trokovic et al., 2003, Blak et al., 2007, Walshe and Mason, 2000). Fgf binding to its receptor at the MHB induces activation of the Ras–MAP kinase pathway, which is important for cerebellar development (Corson et al., 2003, Sato and Nakamura, 2004). A central component of the Ras–MAP kinase pathway is the GTPase Ras. Activated Ras initiates signal transduction through the mitogen-activated protein kinase (MAP kinase) pathway, a phosphorylation cascade that comprises of the protein kinases Raf, Mek and Erk. A mutated form of Ras in which a serine at position 17 is changed to an asparagine (RasN17) has previously been shown to block Ras–MAP kinase signal transduction, whereas a glycine to valine mutation at residue 12 (RasV12) impairs the intrinsic GTPase activity of Ras, allowing the protein to accumulate in the active, GTP-bound state and thereby leading to continuous stimulation of the Ras–MAP kinase pathway (Feig and Cooper, 1988, Trahey and McCormick, 1987).

Recent studies have examined the consequence of activation or inhibition of the Ras–MAP kinase pathway on mid- and hindbrain development in chick embryos (Sato et al., 2001, Sato and Nakamura, 2004, Suzuki-Hirano et al., 2005). Strong Ras–MAP kinase pathway activation, caused by misexpression of the potent splicing isoform Fgf8b in the MHB region, induced ectopic expression of hindbrain associated gene products, such as Gbx2 or Irx2, in the midbrain and repressed the expression of midbrain marker genes like Otx2. This was accompanied by a morphological mes- to metencephalic transformation (Sato et al., 2001). Conversely, inhibition of the Ras–MAP kinase pathway through overexpression of the mutated RasN17 or the pathway antagonist Spry2, led to the appearance of an ectopic swelling in the r1 region that exhibited a cellular lamination reminiscent of the normal optic tectum (Sato and Nakamura, 2004, Suzuki-Hirano et al., 2005). These results together with the phenotypes of Fgf8 loss-of-function models have led to the model that strong activation of the Ras–MAP kinase pathway forces cells to adopt a metencephalic fate, whereas absence of Ras–MAP kinase signaling favors the development of midbrain specific characteristics. However, most of the molecules that were shown to be altered when Ras–MAP kinase signal transduction at the MHB was blocked in these studies, such as Otx2, Gbx2, En1, Pax2/5, or Wnt1, are themselves part of the interdependent regulatory loop that maintains the MHB organizer (Wurst and Bally-Cuif, 2001; and references therein). To gain further insight into the molecular events that occur at the MHB in absence of Ras–MAP kinase activity or following excessive stimulation of this pathway, we have assessed the expression profile of a number of genes, which do or do not participate in this regulatory maintenance loop at the MHB, following ectopic activation or inhibition of the Ras–MAP kinase pathway respectively. We describe spatial and temporal differences in the dependency of different MHB marker genes on Ras–MAP kinase pathway activity. In addition, we provide evidence that transcriptional activation of Wnt1 and En1, two targets of Fgf-signaling at the MHB, requires different threshold levels of Ras–MAP kinase activity. These findings support the idea that Fgf8 functions in a dose-dependent manner at the MHB.

Section snippets

Forced activation or inhibition of Ras–MAP kinase signaling at the MHB alters expression of En1, Pax2, Pax5, and ephrin-A2 and perturbs mid-hindbrain development

Activation of the Ras–MAP kinase pathway can be visualized with a monoclonal antibody specific for the di-phosphorylated forms of Erk1 and Erk2 (dpErk), two signaling intermediates of this pathway (Gabay et al., 1997). When this antibody is applied to mouse or chick embryos, strong Erk-phosphorylation and, thus, strong activity of the Ras–MAP kinase pathway can be detected around the mid-hindbrain boundary (Corson et al., 2003, Sato et al., 2004; arrowhead in Fig. 1A). Ectopic expression of the

Discussion

Ras–MAP kinase pathway activation plays an important role in the specification of cerebellar structures during early embryonic development (Sato et al., 2001, Reifers et al., 1998, Jaszai et al., 2003, Meyers et al., 1998). Here we have investigated the consequence of ectopic activation or inhibition of Ras–MAP kinase signaling at the MHB on the expression of several genes that are known to participate or to not participate in isthmic organizer function and maintenance. We find that these genes

Expression constructs and in ovo electroporation

For constitutive activation of the Ras–MAP kinase pathway a mutated form of Ras, in which a glycine to valine mutation at residue 12 results in constitutive activation of the Ras–MAP kinase pathway (RasV12; Clontech, Mountain View, CA, USA), was cloned into the expression vector pMES, which includes an IRES–GFP cassette (Swartz et al., 2001; pMES-RasV12). To block Ras–MAP kinase signal transduction a dominant negative form of Ras (RasN17), in which a serine to asparagine mutation at position 17

Acknowledgments

We thank Dalit Sela-Donenfeld for the gift of pCAGGS-dnFgfr1, Christa Ziegler for excellent technical assistance and members of the Schulte lab for helpful discussions.

References (60)

  • OlanderS. et al.

    Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the isthmic organizer

    Mech. Dev.

    (2006)
  • PruittS.C. et al.

    Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo

    Gene Expr. Patterns

    (2004)
  • RowitchD.H. et al.

    Pax-2 expression in the murine neural plate precedes and encompasses the expression domains of Wnt-1 and En-1

    Mech. Dev.

    (1995)
  • SchulteD. et al.

    Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map

    Neuron

    (1999)
  • WalsheJ. et al.

    Expression of FGFR1, FGFR2 and FGFR3 during early neural development in the chick embryo

    Mech. Dev.

    (2000)
  • WilkinsonD.G. et al.

    Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo

    Cell

    (1987)
  • AdamsK.A. et al.

    The transcription factor Lmx1b maintains Wnt1 expression within the isthmic organizer

    Development

    (2000)
  • AlexandreP. et al.

    The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures

    Development

    (2003)
  • AlexandreP. et al.

    Positive and negative regulations by FGF8 contribute to midbrain roof plate developmental plasticity

    Development

    (2006)
  • ArakiI. et al.

    Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate

    Development

    (1999)
  • Bally-CuifL. et al.

    Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras

    Development

    (1994)
  • Bally-CuifL. et al.

    Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon

    Development

    (1992)
  • BlakA.A. et al.

    Expression of Fgf receptors 1, 2, and 3 in the developing mid- and hindbrain of the mouse

    Dev. Dyn.

    (2005)
  • CorsonL.B. et al.

    Spatial and temporal patterns of ERK signaling during mouse embryogenesis

    Development

    (2003)
  • CrossleyP.H. et al.

    Midbrain development induced by FGF8 in the chick embryo

    Nature

    (1996)
  • DavisC.A. et al.

    Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development

    Genes Dev.

    (1988)
  • FeigL.A. et al.

    Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP

    Mol. Cell. Biol.

    (1988)
  • FunahashiJ. et al.

    Role of Pax-5 in the regulation of a mid-hindbrain organizer's activity

    Dev. Growth Differ.

    (1999)
  • GabayL. et al.

    In situ activation pattern of Drosophila EGF receptor pathway during development

    Science

    (1997)
  • HamburgerV. et al.

    A series of normal stages in the development of the chick embryo

    J. Morph.

    (1951)
  • Cited by (11)

    • MEIS2 gene is responsible for intellectual disability, cardiac defects and a distinct facial phenotype

      2020, European Journal of Medical Genetics
      Citation Excerpt :

      Depletion of MEIS2 in neuroblastoma cells leads indeed to mitotic aberrations, while ectopic MEIS2 expression leads to the proliferation of tumor cells (Zha et al., 2014). Thus, MEIS2 can be counted among the genes implicated both in neurodevelopmental disorders and in cancers, such as RAS Pathway Genes or BAF-complex genes (Agoston et al., 2012; Vennemann et al., 2008). Overall, our results reinforce the involvement of MEIS2 in patients with cardiac and nervous system involvement and delineate a distinct facial phenotype making the MEIS2-related condition a distinct and recognizable neurodevelopmental disorder through the application of “reverse phenotyping”.

    View all citing articles on Scopus
    1

    Present address: Pharmazentrum Frankfurt, Institut für Klinische Pharmakologie, Klinikum der J.-W. Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, D-60590 Frankfurt.

    View full text