ReviewUnraveling inner ear induction by gene manipulation using Pax2-Cre BAC transgenic mice
Introduction
Isolation of embryonic stem (ES) cells and gene-targeting techniques have dramatically improved mouse genetic study in recent decades, and culminated in the awarding of the Nobel Prize for Physiology or Medicine to Evans, Smithies and Capecchi in 2007. With this technology, researchers are able to modify or inactivate genes of interest in a living mammalian organism. Improvements upon the technology using the Cre-Lox and Flp-Frt systems have been developed more recently (Dymecki, 1996, Gu et al., 1993, Rossant and McMahon, 1999, Sauer, 1998) and these techniques have already been applied to the inner ear (Bouchard et al., 2004, Cohen-Salmon et al., 2002, Gao et al., 2004, Hebert and McConnell, 2000, Ohyama and Groves, 2004, Tian et al., 2006). The most commonly used tissue-specific gene manipulation techniques rely on the Cre-loxP recombination system. LoxP is a 34-base pair DNA sequence recognized by the Cre recombinase gene product of bacteriophage P1. CRE excises DNA sequences flanked by loxP sites when they are in the same orientation. Thus, the Cre-loxP system has been extensively used for conditional knockout (CKO) mice. This technology can also be used for conditional gene activation (cAct). A more detailed description of these techniques is available in a previous review issue (Tian et al., 2006). In this system, spatiotemporal regulation of the Cre expression is central to a successful gene manipulation in a tissue of interest. There are several Cre-expressing mouse strains that can be used for inner ear study (Tian et al., 2006). Here, we briefly describe several discoveries in early inner ear development achieved by one of these inner ear-specific Cre lines, Pax2-Cre, developed in our laboratory (Ohyama and Groves, 2004).
Section snippets
Induction of the otic placode
The first morphological sign of inner ear development is a thickening of the ectoderm next to the hindbrain, called the otic placode. In the 1950s and 60s, Jacobson carefully analyzed the timing of induction and the inducing tissues for several cranial sensory placodes by tissue grafting experiments in salamanders (Jacobson, 1963, Jacobson, 1966). His experiments provided evidence for the existence of a common sensory precursor domain, or pre-placodal domain, that is competent to give rise to
Lineage tracing of Pax2+ ectoderm by Pax2-Cre mice
We generated a Pax2-Cre BAC transgenic mouse line in which Cre expression recapitulates the native Pax2 expression in the presumptive otic ectoderm, firstly to create a useful tool to manipulate genes of interest in the entire presumptive otic ectoderm and secondly to trace the descendants of Pax2+ ectoderm by crossing these mice with Cre-loxP reporter strains (Novak et al., 2000, Soriano, 1999). In this experiment, Pax2+ cells are permanently labeled by activation of a reporter gene.
Inner ear-specific gene manipulation of Wnt and Notch signaling
To search for additional signals that induce the otic placode, we first focused on Wnt signals that have been shown to promote otic genes synergistically with FGFs (Ladher et al., 2000). A number of Wnt family members are expressed in the hindbrain at the level of the otic placode such as Wnt8a (Bouillet et al., 1996, Ohyama et al., 2006) or at the hindbrain-placode boundary such as Wnt1, Wnt3a and Wnt6 (Jayasena et al., 2008, Parr et al., 1993, Wilkinson et al., 1987) around the onset of otic
The three-step model
A number of studies suggest that all cranial sensory placodes develop from a common precursor domain or pre-placodal domain in the border region between the neural plate and surface ectoderm which is competent to give rise to different placodes if grafted to the appropriate location (Step 1 in Fig. 2; Groves and Bronner-Fraser, 2000, Martin and Groves, 2006). These pre-placodal cells are capable of responding to otic inducing signals such as FGFs. The initial expression of the early otic genes
Pax2-Cre BAC transgenic strain
The BAC clone 242K18 was obtained by library screening with a fragment of Pax2 exons. It contains about 101 kb upstream of the mouse Pax2 ATG and 20 kb downstream including the first three exons of the Pax2 gene. The IRES-Cre-polyA fragment is inserted by the BAC modification system (Yang et al., 1997). The Pax2-Cre BAC DNA fragment was then used for pronuclear injection (for more detail, see Ohyama and Groves, 2004). The Pax2-Cre transgenic strain is available from the Mutant Mouse Regional
Acknowledgments
I thank Dr. Andrew Groves (Baylor College of Medicine, Houston, TX) for discussion and comments.
References (47)
- et al.
Vertebrate cranial placodes I. Embryonic induction
Dev. Biol.
(2001) - et al.
A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid
Mech. Dev.
(1996) - et al.
Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death
Curr. Biol.
(2002) - et al.
Targeting hearing genes in mice
Brain Res. Mol. Brain Res.
(2004) - et al.
Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting
Cell.
(1993) - et al.
Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures
Dev. Biol.
(2000) - et al.
Ringing in the new ear: resolution of cell interactions in otic development
Dev. Biol.
(2003) Inducible gene targeting in mice using the Cre/lox system
Methods.
(1998)Extensive cell movements accompany formation of the otic placode
Dev. Biol.
(2002)- et al.
Conditional and inducible gene recombineering in the mouse inner ear
Brain Res.
(2006)
Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo
Cell.
Sensory organs: making and breaking the pre-placodal region
Curr. Top. Dev. Biol.
Tissue-specific expression of cre recombinase from the Pax8 locus
Genesis.
Induction and specification of the vertebrate ectodermal placodes: precursors of the cranial sensory organs
Biol. Cell.
Notch1 is required for the coordinate segmentation of somites
Development.
Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice
Proc. Natl. Acad. Sci. U. S. A.
Jagged 1 is a beta-catenin target gene required for ectopic hair follicle formation in adult epidermis
Development
The Induction of the Otic Placode in Development of the Ear
Competence, specification and commitment in otic placode induction
Development.
Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors
Development.
Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b
BMC Dev. Biol.
The determination and positioning of the nose, lens and ear. III. Effects of reversing the antero-posterior axis of epidermis, neural plate and neural fold
J. Exp. Zool.
Inductive processes in embryonic development
Science
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