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Unraveling inner ear induction by gene manipulation using Pax2-Cre BAC transgenic mice
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Review
Unraveling inner ear induction by gene manipulation using Pax2-Cre BAC transgenic mice Takahiro Ohyama⁎ Division of Cell Biology and Genetics, House Ear Institute, 2100 West Third Street, Los Angeles, CA 90057, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
One of the biggest drawbacks of conventional mouse knockout techniques in the study of the
Accepted 10 February 2009
inner ear is that loss of a gene of interest may cause embryonic lethality before the inner ear
Available online 2 March 2009
develops. Thus, there is a need for an inner ear-specific gene manipulation system for lossand gain-of-function analysis in the mouse inner ear. We generated a Pax2-Cre BAC transgenic
Keywords:
line in which Cre recombinase expression recapitulates Pax2 expression in the presumptive
Otic placode
otic ectoderm. Here, we present a brief summary of a recent model of inner ear induction
Inner ear
suggested by the results of inner ear-specific gene modification using Pax2-Cre mice.
Induction
© 2009 Elsevier B.V. All rights reserved.
FGF Wnt Notch Conditional knockout mouse
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of the otic placode . . . . . . . . . . . . . . . . . . . . . Lineage tracing of Pax2+ ectoderm by Pax2-Cre mice . . . . . . . . Inner ear-specific gene manipulation of Wnt and Notch signaling The three-step model . . . . . . . . . . . . . . . . . . . . . . . . . Experimental procedures . . . . . . . . . . . . . . . . . . . . . . . 6.1 Pax2-Cre BAC transgenic strain . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
Isolation of embryonic stem (ES) cells and gene-targeting techniques have dramatically improved mouse genetic study
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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
⁎ Fax: +1 213 273 8088. E-mail address: [email protected]. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.036
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organism. Improvements upon the technology using the CreLox 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 tissuespecific 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).
thickening such as Pax2/8 and zebrafish foxi1 (Hans et al., 2004, 2007; Mackereth et al., 2005; Riley et al., 1999; Solomon et al., 2003, 2004; Torres et al., 1996). These genes are necessary for otic placode formation and thus are considered as early otic markers of placode induction. With these markers in hand, the classic grafting experiments were reexamined in the chick to reveal the timing of otic specification and commitment (Groves and Bronner-Fraser, 2000; Martin and Groves, 2006; Ohyama et al., 2007). Four decades ago, Jacobson wrote in his 1966 review: But the concept of induction, once separated out and named, has suffered reification. A number of papers imply or refer to “the moment of induction,” and attention has prematurely shifted from study of the process of embryonic induction to a search for “the inductor substance” (Jacobson, 1966). Indeed, we often refer to the expression of early otic marker genes as “the moment of otic induction.” In order to test this assertion, we determined whether the expression of one of the early otic genes, Pax2, represents the moment of otic induction.
2.
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. Surprisingly, we found that reporter-positive cells give rise to both otic placode and epidermis (Ohyama and Groves, 2004; Ohyama et al., 2007). In chick, similar results have been observed by lineage analysis of dye-labeled cells in the presumptive otic ectoderm (Streit, 2002). These results strongly suggest that the induction of the early otic marker genes induced by FGFs is not equivalent to the induction of the inner ear and thus, we described the domain marked by the early otic marker genes such as Pax2 and Pax8 as a ‘pre-otic field’, distinct from the ‘otic placode’ (Ohyama et al., 2007).
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, 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 all cranial sensory placodes. A number of later experiments support this hypothesis in which initial inductive events establish the pre-placodal domain, followed later by more local signaling events which induce specific cranial placodes (Bailey and Streit, 2006; Baker and Bronner-Fraser, 2001; Brugmann and Moody, 2005). In the case of otic placode induction, Jacobson also found that underlying endoderm and mesoderm, and hindbrain adjacent to the presumptive otic ectoderm have otic inducing activities in different time periods (Jacobson, 1963, 1966). In recent decades, the application of molecular and cell biological techniques allowed the discovery of a number of inducers for the otic placode. For example, fibroblast growth factors (FGFs) that are expressed in the hindbrain and mesoderm at appropriate times have been shown to be necessary and sufficient to induce the otic placode in different model organisms (reviewed in Groves, 2005; Ohyama et al., 2007; Riley and Phillips, 2003). In the mouse, Fgf8 expressed in the underlying endoderm, Fgf10 expressed in the underlying mesoderm and Fgf3 expressed in the hindbrain are considered as potential inducers since mutants of both Fgf3 and Fgf10 or mutants of both Fgf3 and Fgf8 fail to form the otic vesicles or form microvesicles (Ladher et al., 2005; Wright and Mansour, 2003). Moreover, several genes have been shown to be expressed in the presumptive otic ectoderm prior to placodal
3. Lineage tracing of Pax2+ ectoderm by Pax2-Cre mice
4. 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 hindbrainplacode boundary such as Wnt1, Wnt3a and Wnt6 (Jayasena et al., 2008; Parr et al., 1993; Wilkinson et al., 1987) around the onset of otic placode induction. We used Wnt reporter transgenic mice (Mohamed et al., 2004) to identify a gradient of Wnt activity from the medial side of the pre-otic field next
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to the hindbrain, declining towards the lateral edge of the otic placode (Ohyama et al., 2006). To determine whether Wnt signals play roles in otic placode induction, we used the Pax2Cre mice as a tool to manipulate the Wnt pathway in the entire pre-otic field. We conditionally inactivated or activated βcatenin, a downstream effector of the canonical Wnt signaling pathway in Pax2+ pre-otic field cells. In β-catenin conditional knockout (CKO) mice, the size of the otic placode is significantly reduced. Conversely, conditional stabilization of βcatenin (cAct) results in an expansion of a placode-like thickening at the expense of non-placodal ectoderm. Therefore, we proposed that canonical Wnt signals mediate a placode-epidermis fate decision within the pre-otic field (Ohyama et al., 2006). We next examined whether Notch signals are involved in the placode-epidermis fate decision in the pre-otic field downstream of canonical Wnt signals. This was motivated by the expression of several Notch signal component genes in the pre-otic field after Wnt signals are first detected
(Jayasena et al., 2008). In the β-catenin cAct mutants, several Notch component genes such as Notch1, Jagged1 (Jag1) and a Notch target gene Hes1 (Kageyama et al., 2007) are ectopically expanded in the expanded placode. We also activated Notch1 in the pre-otic field by crossing Pax2-Cre mice with transgenic mice in which expression of a constitutively active Notch1 intracellular domain (N1ICD) can be controlled by the CreloxP system (Murtaugh et al., 2003). In conditionally activated N1ICD mutants (cN1ICD), a placode-like thickening is expanded at the expense of epidermis similar to the phenotype of β-catenin cAct mutants. However, not all genes that are expanded in the β-catenin cAct mutants are up-regulated in cN1ICD mice. For instance, Pax8 is upregulated widely in the expanded placode, while several otic marker genes such as Pax2, Gbx2 and Sox9 are not expanded. These results suggest that some of the otic markers such as Pax2, Gbx2 and Sox9 are regulated by Wnt signaling but not Notch signaling, while some genes such as Pax8 and the morphological change of the placodal
Fig. 1 – Wnt and Notch signals mediate size of the otic placode. (A) Graded signals of Wnt genes mediate a placode-epidermis fate decision within the pre-otic field by positively regulating the expression of Dlx5, Sox9, Gbx2, Pax2, Pax8 and components of the canonical Notch signals such as Notch1, Jagged1 (Jag1) and Hes1. Notch signals are activated by the expression of Jag1, then positively feed back to augment Wnt signals. Notch signals can also activate some genes such as Pax8 and Hes1 independently of Wnt signals. (B) Schematic drawing of the relationship between Wnt signals and placode size in different mutant genotypes. A gradient of Wnt activity from the hindbrain is established across the mediolateral axis of the pre-otic field. In wildtype (WT, blue line), cells exposed a certain threshold of Wnt signals express Jag1, which initiates activation of the Notch pathway. Notch signaling augments Wnt signals in the Jag1+ domain which is eventually committed to become the otic placode (blue rectangle). The rest of the pre-otic field, which receives lower Wnt signals below the placode threshold becomes epidermis (gray rectangle). In the absence of Notch1 (Notch1 KO, gray line), Wnt signaling is not augmented, resulting a smaller placode size and expansion of epidermis. With the activation of Notch signals in the entire pre-otic field (cN1ICD, orange line), Wnt signaling is augmented throughout the pre-otic field, resulting a slight expansion of the placode. The rest of the pre-otic field becomes a placode-like structure that expresses some genes (like Pax8) and thickens morphologically, but does not express a full complement of otic genes (like Pax2, Dlx5, Gbx2; Jayasena et al., 2008). In the absence of Wnt signals (β-cat CKO, light gray line), the entire pre-otic field becomes epidermis at the expense of the otic placode. With the constitutive activation of Wnt signals in the entire pre-otic field (β-cat cAct, purple line), Wnt is activated throughout the pre-otic field, resulting an expansion of the otic placode at the expense of epidermis.
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activated Notch signals can augment Wnt signals by positive-feedback (Fig. 1, Jayasena et al., 2008).
5.
Fig. 2 – The three-step model. Otic induction is a series of inducing events divided by three major steps. Step 1: Ectoderm adjacent to the neural plate (NP), or the pre-placodal domain, acquires the competence to give rise to most of cranial sensory placodes. Step 2: Subsequent FGF signaling induces early otic genes such as Pax2 and Pax8 (green) to establish the pre-otic field. Step 3: The pre-otic field is partitioned into epidermis and committed otic placode (magenta) through the actions of Wnt and Notch signals.
thickening are regulated by both Wnt and Notch signals. Consistent with previous studies (Estrach et al., 2006; Katoh and Katoh, 2006), our results suggest that Jag1 is a direct target of Wnt signaling. Therefore, Wnt can activate Notch signaling through up-regulation of Jag1 expression. We also analyzed the genetic interaction between Wnt and Notch signals during the placode-epidermis fate decision by generating β-catenin CKO; cN1ICD compound mutants. In the compound mutants, we still observed the placodal thickening and expansion of Pax8 and Jag1 genes. These results suggest that Notch signals can regulate these aspects of otic development independently from Wnt signals. We next tested whether Notch signaling is necessary for placodal thickening and/or expression of otic genes by analyzing several Notch related mutants such as Notch1-null (Conlon et al., 1995), Pofut1-null (Shi and Stanley, 2003) and Rbpj1-CKO (Tanigaki et al., 2002). In these mutants, the otic placode is formed, but both the area of Pax2/8 expression and the size of the otic placode are significantly reduced due to a fate change between placode and epidermis. Notably, a Notch signaling target, Hes1 is initially expressed during early placode stages in the mutant, but is not maintained at the otic cup stage while Jag1 expression remains. These results suggest that canonical Notch signaling is not required for the initial induction of the otic placode, but is involved in size control of the placode and maintenance of some of the otic genes. We further analyzed the difference between Wnt-driven and Notch-driven expansion of the otic placode. We measured Wnt reporter activity (Mohamed et al., 2004) under cN1ICD or Notch1-null background. Although expansion of the thickening ectoderm with Pax8 expression to the level of pharynx is observed in the cN1ICD mice, the size of the otic cup in cN1ICD is only slightly larger with a modest expansion of the Wnt reporter signal. Conversely, Wnt reporter signal is significantly reduced in Notch1-null background. Taken together, our results indicate that expression of the Notch component genes is activated by the Wnt signals, then the
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 such as Pax2 and Pax8 in response to the inductive signals is often considered as a specification of the otic fate and thus, as the moment of induction. As described above, however, the lineage analysis of genetically labeled Pax2+ ectoderm by our Pax2-Cre mice clearly showed that this region gives rise to both otic and epidermal populations. Therefore, we propose to define this region as a pre-otic field (Step 2 in Fig. 2) which is patterned by a gradient of Wnt signaling such that high levels of Wnt signals induce otic tissue, whereas lower (or no) levels of Wnt signals cause the formation of epidermis. The graded Wnt signal is augmented by Notch signaling which helps to fine-tune a binary fate decision between placode and epidermis (Fig. 1). Finally, some cells within the pre-otic field are committed to become the otic placode (Step 3 in Fig. 2). As Jacobson suggested more than forty years ago, otic induction is a series of sequential events directed by multiple spatiotemporal signals and there is no need to invoke a single unitary moment of otic induction. Manipulation of genes in the inner ear with Pax2-Cre mice has provided information that has changed our understanding of the mechanisms of otic induction. Although new mouse lines are emerging that enable more precise gene control in different cell populations at different point during ear development, the Pax2-Cre line remains a useful tool to test gene function in the mouse inner ear.
6.
Experimental procedures
6.1.
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 Resource Centers (www.mmrrc.org).
Acknowledgments I thank Dr. Andrew Groves (Baylor College of Medicine, Houston, TX) for discussion and comments.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Review
Unraveling inner ear induction by gene manipulation using Pax2-Cre BAC transgenic mice Takahiro Ohyama⁎ Division of Cell Biology and Genetics, House Ear Institute, 2100 West Third Street, Los Angeles, CA 90057, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
One of the biggest drawbacks of conventional mouse knockout techniques in the study of the
Accepted 10 February 2009
inner ear is that loss of a gene of interest may cause embryonic lethality before the inner ear
Available online 2 March 2009
develops. Thus, there is a need for an inner ear-specific gene manipulation system for lossand gain-of-function analysis in the mouse inner ear. We generated a Pax2-Cre BAC transgenic
Keywords:
line in which Cre recombinase expression recapitulates Pax2 expression in the presumptive
Otic placode
otic ectoderm. Here, we present a brief summary of a recent model of inner ear induction
Inner ear
suggested by the results of inner ear-specific gene modification using Pax2-Cre mice.
Induction
© 2009 Elsevier B.V. All rights reserved.
FGF Wnt Notch Conditional knockout mouse
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of the otic placode . . . . . . . . . . . . . . . . . . . . . Lineage tracing of Pax2+ ectoderm by Pax2-Cre mice . . . . . . . . Inner ear-specific gene manipulation of Wnt and Notch signaling The three-step model . . . . . . . . . . . . . . . . . . . . . . . . . Experimental procedures . . . . . . . . . . . . . . . . . . . . . . . 6.1 Pax2-Cre BAC transgenic strain . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
Isolation of embryonic stem (ES) cells and gene-targeting techniques have dramatically improved mouse genetic study
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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
⁎ Fax: +1 213 273 8088. E-mail address: [email protected]. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.036
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organism. Improvements upon the technology using the CreLox 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 tissuespecific 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).
thickening such as Pax2/8 and zebrafish foxi1 (Hans et al., 2004, 2007; Mackereth et al., 2005; Riley et al., 1999; Solomon et al., 2003, 2004; Torres et al., 1996). These genes are necessary for otic placode formation and thus are considered as early otic markers of placode induction. With these markers in hand, the classic grafting experiments were reexamined in the chick to reveal the timing of otic specification and commitment (Groves and Bronner-Fraser, 2000; Martin and Groves, 2006; Ohyama et al., 2007). Four decades ago, Jacobson wrote in his 1966 review: But the concept of induction, once separated out and named, has suffered reification. A number of papers imply or refer to “the moment of induction,” and attention has prematurely shifted from study of the process of embryonic induction to a search for “the inductor substance” (Jacobson, 1966). Indeed, we often refer to the expression of early otic marker genes as “the moment of otic induction.” In order to test this assertion, we determined whether the expression of one of the early otic genes, Pax2, represents the moment of otic induction.
2.
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. Surprisingly, we found that reporter-positive cells give rise to both otic placode and epidermis (Ohyama and Groves, 2004; Ohyama et al., 2007). In chick, similar results have been observed by lineage analysis of dye-labeled cells in the presumptive otic ectoderm (Streit, 2002). These results strongly suggest that the induction of the early otic marker genes induced by FGFs is not equivalent to the induction of the inner ear and thus, we described the domain marked by the early otic marker genes such as Pax2 and Pax8 as a ‘pre-otic field’, distinct from the ‘otic placode’ (Ohyama et al., 2007).
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, 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 all cranial sensory placodes. A number of later experiments support this hypothesis in which initial inductive events establish the pre-placodal domain, followed later by more local signaling events which induce specific cranial placodes (Bailey and Streit, 2006; Baker and Bronner-Fraser, 2001; Brugmann and Moody, 2005). In the case of otic placode induction, Jacobson also found that underlying endoderm and mesoderm, and hindbrain adjacent to the presumptive otic ectoderm have otic inducing activities in different time periods (Jacobson, 1963, 1966). In recent decades, the application of molecular and cell biological techniques allowed the discovery of a number of inducers for the otic placode. For example, fibroblast growth factors (FGFs) that are expressed in the hindbrain and mesoderm at appropriate times have been shown to be necessary and sufficient to induce the otic placode in different model organisms (reviewed in Groves, 2005; Ohyama et al., 2007; Riley and Phillips, 2003). In the mouse, Fgf8 expressed in the underlying endoderm, Fgf10 expressed in the underlying mesoderm and Fgf3 expressed in the hindbrain are considered as potential inducers since mutants of both Fgf3 and Fgf10 or mutants of both Fgf3 and Fgf8 fail to form the otic vesicles or form microvesicles (Ladher et al., 2005; Wright and Mansour, 2003). Moreover, several genes have been shown to be expressed in the presumptive otic ectoderm prior to placodal
3. Lineage tracing of Pax2+ ectoderm by Pax2-Cre mice
4. 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 hindbrainplacode boundary such as Wnt1, Wnt3a and Wnt6 (Jayasena et al., 2008; Parr et al., 1993; Wilkinson et al., 1987) around the onset of otic placode induction. We used Wnt reporter transgenic mice (Mohamed et al., 2004) to identify a gradient of Wnt activity from the medial side of the pre-otic field next
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to the hindbrain, declining towards the lateral edge of the otic placode (Ohyama et al., 2006). To determine whether Wnt signals play roles in otic placode induction, we used the Pax2Cre mice as a tool to manipulate the Wnt pathway in the entire pre-otic field. We conditionally inactivated or activated βcatenin, a downstream effector of the canonical Wnt signaling pathway in Pax2+ pre-otic field cells. In β-catenin conditional knockout (CKO) mice, the size of the otic placode is significantly reduced. Conversely, conditional stabilization of βcatenin (cAct) results in an expansion of a placode-like thickening at the expense of non-placodal ectoderm. Therefore, we proposed that canonical Wnt signals mediate a placode-epidermis fate decision within the pre-otic field (Ohyama et al., 2006). We next examined whether Notch signals are involved in the placode-epidermis fate decision in the pre-otic field downstream of canonical Wnt signals. This was motivated by the expression of several Notch signal component genes in the pre-otic field after Wnt signals are first detected
(Jayasena et al., 2008). In the β-catenin cAct mutants, several Notch component genes such as Notch1, Jagged1 (Jag1) and a Notch target gene Hes1 (Kageyama et al., 2007) are ectopically expanded in the expanded placode. We also activated Notch1 in the pre-otic field by crossing Pax2-Cre mice with transgenic mice in which expression of a constitutively active Notch1 intracellular domain (N1ICD) can be controlled by the CreloxP system (Murtaugh et al., 2003). In conditionally activated N1ICD mutants (cN1ICD), a placode-like thickening is expanded at the expense of epidermis similar to the phenotype of β-catenin cAct mutants. However, not all genes that are expanded in the β-catenin cAct mutants are up-regulated in cN1ICD mice. For instance, Pax8 is upregulated widely in the expanded placode, while several otic marker genes such as Pax2, Gbx2 and Sox9 are not expanded. These results suggest that some of the otic markers such as Pax2, Gbx2 and Sox9 are regulated by Wnt signaling but not Notch signaling, while some genes such as Pax8 and the morphological change of the placodal
Fig. 1 – Wnt and Notch signals mediate size of the otic placode. (A) Graded signals of Wnt genes mediate a placode-epidermis fate decision within the pre-otic field by positively regulating the expression of Dlx5, Sox9, Gbx2, Pax2, Pax8 and components of the canonical Notch signals such as Notch1, Jagged1 (Jag1) and Hes1. Notch signals are activated by the expression of Jag1, then positively feed back to augment Wnt signals. Notch signals can also activate some genes such as Pax8 and Hes1 independently of Wnt signals. (B) Schematic drawing of the relationship between Wnt signals and placode size in different mutant genotypes. A gradient of Wnt activity from the hindbrain is established across the mediolateral axis of the pre-otic field. In wildtype (WT, blue line), cells exposed a certain threshold of Wnt signals express Jag1, which initiates activation of the Notch pathway. Notch signaling augments Wnt signals in the Jag1+ domain which is eventually committed to become the otic placode (blue rectangle). The rest of the pre-otic field, which receives lower Wnt signals below the placode threshold becomes epidermis (gray rectangle). In the absence of Notch1 (Notch1 KO, gray line), Wnt signaling is not augmented, resulting a smaller placode size and expansion of epidermis. With the activation of Notch signals in the entire pre-otic field (cN1ICD, orange line), Wnt signaling is augmented throughout the pre-otic field, resulting a slight expansion of the placode. The rest of the pre-otic field becomes a placode-like structure that expresses some genes (like Pax8) and thickens morphologically, but does not express a full complement of otic genes (like Pax2, Dlx5, Gbx2; Jayasena et al., 2008). In the absence of Wnt signals (β-cat CKO, light gray line), the entire pre-otic field becomes epidermis at the expense of the otic placode. With the constitutive activation of Wnt signals in the entire pre-otic field (β-cat cAct, purple line), Wnt is activated throughout the pre-otic field, resulting an expansion of the otic placode at the expense of epidermis.
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activated Notch signals can augment Wnt signals by positive-feedback (Fig. 1, Jayasena et al., 2008).
5.
Fig. 2 – The three-step model. Otic induction is a series of inducing events divided by three major steps. Step 1: Ectoderm adjacent to the neural plate (NP), or the pre-placodal domain, acquires the competence to give rise to most of cranial sensory placodes. Step 2: Subsequent FGF signaling induces early otic genes such as Pax2 and Pax8 (green) to establish the pre-otic field. Step 3: The pre-otic field is partitioned into epidermis and committed otic placode (magenta) through the actions of Wnt and Notch signals.
thickening are regulated by both Wnt and Notch signals. Consistent with previous studies (Estrach et al., 2006; Katoh and Katoh, 2006), our results suggest that Jag1 is a direct target of Wnt signaling. Therefore, Wnt can activate Notch signaling through up-regulation of Jag1 expression. We also analyzed the genetic interaction between Wnt and Notch signals during the placode-epidermis fate decision by generating β-catenin CKO; cN1ICD compound mutants. In the compound mutants, we still observed the placodal thickening and expansion of Pax8 and Jag1 genes. These results suggest that Notch signals can regulate these aspects of otic development independently from Wnt signals. We next tested whether Notch signaling is necessary for placodal thickening and/or expression of otic genes by analyzing several Notch related mutants such as Notch1-null (Conlon et al., 1995), Pofut1-null (Shi and Stanley, 2003) and Rbpj1-CKO (Tanigaki et al., 2002). In these mutants, the otic placode is formed, but both the area of Pax2/8 expression and the size of the otic placode are significantly reduced due to a fate change between placode and epidermis. Notably, a Notch signaling target, Hes1 is initially expressed during early placode stages in the mutant, but is not maintained at the otic cup stage while Jag1 expression remains. These results suggest that canonical Notch signaling is not required for the initial induction of the otic placode, but is involved in size control of the placode and maintenance of some of the otic genes. We further analyzed the difference between Wnt-driven and Notch-driven expansion of the otic placode. We measured Wnt reporter activity (Mohamed et al., 2004) under cN1ICD or Notch1-null background. Although expansion of the thickening ectoderm with Pax8 expression to the level of pharynx is observed in the cN1ICD mice, the size of the otic cup in cN1ICD is only slightly larger with a modest expansion of the Wnt reporter signal. Conversely, Wnt reporter signal is significantly reduced in Notch1-null background. Taken together, our results indicate that expression of the Notch component genes is activated by the Wnt signals, then the
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 such as Pax2 and Pax8 in response to the inductive signals is often considered as a specification of the otic fate and thus, as the moment of induction. As described above, however, the lineage analysis of genetically labeled Pax2+ ectoderm by our Pax2-Cre mice clearly showed that this region gives rise to both otic and epidermal populations. Therefore, we propose to define this region as a pre-otic field (Step 2 in Fig. 2) which is patterned by a gradient of Wnt signaling such that high levels of Wnt signals induce otic tissue, whereas lower (or no) levels of Wnt signals cause the formation of epidermis. The graded Wnt signal is augmented by Notch signaling which helps to fine-tune a binary fate decision between placode and epidermis (Fig. 1). Finally, some cells within the pre-otic field are committed to become the otic placode (Step 3 in Fig. 2). As Jacobson suggested more than forty years ago, otic induction is a series of sequential events directed by multiple spatiotemporal signals and there is no need to invoke a single unitary moment of otic induction. Manipulation of genes in the inner ear with Pax2-Cre mice has provided information that has changed our understanding of the mechanisms of otic induction. Although new mouse lines are emerging that enable more precise gene control in different cell populations at different point during ear development, the Pax2-Cre line remains a useful tool to test gene function in the mouse inner ear.
6.
Experimental procedures
6.1.
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 Resource Centers (www.mmrrc.org).
Acknowledgments I thank Dr. Andrew Groves (Baylor College of Medicine, Houston, TX) for discussion and comments.
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