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Andrew K. Groves - One of the best experts on this subject based on the ideXlab platform.
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Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction
2016Co-Authors: Lu Yang, Kareen Martin, Juan C Maass, Vassil Vassilev, Raj Ladher, Andrew K. GrovesAbstract:The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the Otic Placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the Otic Placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent Otic Placode formation. To better understand the genetic pathways activated by FGF signaling during Otic Placode induction, we performed microarray experiments to estimate the proportion of chicken Otic Placode genes that can be up-regulated by the FGF pathway in a simple culture model of Otic Placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15 % of chick Otic Placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of Otic Placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in Otic Placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for Otic Placode induction, it demonstrates that other unidentified signalin
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Conserved expression of tfap2a during Otic neurogenesis.
2015Co-Authors: Husniye Kantarci, Renee K Edlund, Andrew K. Groves, Bruce B RileyAbstract:All images show cross-sections of the Otic Placode or vesicle in wild type zebrafish embryos (A-J) or chick embryos (L-Q) with a dorsal up and medial to the left. (A, B) At 14 hpf (10 somites) pax2a (red) marks the precursor cells in the emerging Otic Placode that are co-labeled with tfap2a (blue). (C-H) Cross-sections through the widest part of the neurogenic domain of the Otic vesicle, just posterior to the utricular macula. The outer and inner edges of the Otic vesicle are outlined. Patterns of ngn1 or tfap2a are shown at the indicated times. tfap2a is expressed in the ventrolateral part of the Otic vesicle, which partially overlaps the domain of ngn1 expression. (I-J) Cross-sections passing through the utricular macula of specimens co-stained for Isl1 (red) and tfap2a (blue) at 48 hpf. Expression of tfap2a is not detected in the floor of the Otic vesicle or in the mature SAG neurons at this time. (K) Schematic summary of SAG development in zebrafish, including regional markers. Neuroblasts are specified and delaminate from the Otic vesicle (light purple) adjacent to nascent sensory epithelia (green). Recently delaminated neuroblasts migrate towards hindbrain and continue to proliferate, forming the transit-amplifying pool (blue). Neuroblasts then stop dividing and differentiate into mature neurons (red). Relevant genes expressed in each domain are indicated. Expression of tfap2a (dark purple) overlaps the neurogenic domain, as well as the domain of bmp7a expression. Note that all of the tissues indicated express Fgf-target genes (etv5b and spry4) and transducers of Bmp (smad1 and smad5), but transit amplifying SAG precursors show specific upregulation of smad1 and smad5 [37]. (L-Q) Cross-sections through the Otic vesicle of chick embryos at days 3 and 4 (E3 and E4). The sensory region is labeled with Jagged-1 (green). Tfap2a (red) is expressed in the ventrolateral Otic domain in chick embryos similar to the pattern observed in zebrafish.
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foxi3 is necessary for the induction of the chick Otic Placode in response to fgf signaling
Developmental Biology, 2014Co-Authors: Safia B Khatri, Renee K Edlund, Andrew K. GrovesAbstract:Vertebrate cranial sensory organs are derived from region at the border of the anterior neural plate called the pre-placodal region (PPR). The Otic Placode, the anlagen of the inner ear, is induced from PPR ectoderm by FGF signaling. We have previously shown that competence of embryonic ectoderm to respond to FGF signaling during Otic Placode induction correlates with the expression of PPR genes, but the molecular basis of this competence is poorly understood. Here, we characterize the function of a transcription factor, Foxi3 that is expressed at very early stages in the non-neural ectoderm and later in the PPR of chick embryos. Ablation experiments showed that the underlying hypoblast is necessary for the initiation of Foxi3 expression. Mis-expression of Foxi3 was sufficient to induce markers of non-neural ectoderm such as Dlx5, and the PPR such as Six1 and Eya2. Electroporation of Dlx5, or Six1 together with Eya1 also induced Foxi3, suggesting direct or indirect positive regulation between non-neural ectoderm genes and PPR genes. Knockdown of Foxi3 in chick embryos prevented the induction of Otic Placode markers, and was able to prevent competent cranial ectoderm from expressing Otic markers in response to FGF2. In contrast, Foxi3 expression alone was not sufficient to confer competence to respond to FGF on embryonic ectoderm. Our analysis of PPR and FGF-responsive genes after Foxi3 knockdown at gastrula stages suggests it is not necessary for the expression of PPR genes at these stages, nor for the transduction of FGF signals. The early expression but late requirement for Foxi3 in ear induction suggests it may have some of the properties associated with pioneer transcription factors.
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analysis of fgf dependent and fgf independent pathways in Otic Placode induction
PLOS ONE, 2013Co-Authors: Lu Yang, Raj K. Ladher, Paul M Oneill, Kareen Martin, Juan C Maass, Vassil Vassilev, Andrew K. GrovesAbstract:The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the Otic Placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the Otic Placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent Otic Placode formation. To better understand the genetic pathways activated by FGF signaling during Otic Placode induction, we performed microarray experiments to estimate the proportion of chicken Otic Placode genes that can be up-regulated by the FGF pathway in a simple culture model of Otic Placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick Otic Placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of Otic Placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in Otic Placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for Otic Placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full Otic Placode program.
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notch signaling augments the canonical wnt pathway to specify the size of the Otic Placode
Development, 2008Co-Authors: Chathurani S Jayasena, Takahiro Ohyama, Neil Segil, Andrew K. GrovesAbstract:The inner ear derives from a patch of ectoderm defined by expression of the transcription factor Pax2. We recently showed that this Pax2^+ ectoderm gives rise not only to the Otic Placode but also to the surrounding cranial epidermis, and that Wnt signaling mediates this Placode-epidermis fate decision. We now present evidence for reciprocal interactions between the Wnt and Notch signaling pathways during inner ear induction. Activation of Notch1 in Pax2+ ectoderm expands the placodal epithelium at the expense of cranial epidermis, whereas loss of Notch1 leads to a reduction in the size of the Otic Placode. We show that Wnt signaling positively regulates Notch pathway genes such as Jag1, Notch1 and Hes1, and we have used transgenic Wnt reporter mice to show that Notch signaling can modulate the canonical Wnt pathway. Gain- and loss-of-function mutations in the Notch and Wnt pathways reveal that some aspects of Otic Placode development - such as Pax8 expression and the morphological thickening of the Placode - can be regulated independently by either Notch or Wnt signals. Our results suggest that Wnt signaling specifies the size of the Otic Placode in two ways, by directly upregulating a subset of Otic genes, and by positively regulating components of the Notch signaling pathway, which then act to augment Wnt signaling.
Suzanne L Mansour - One of the best experts on this subject based on the ideXlab platform.
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slc26a9p2acre a new cre driver to regulate gene expression in the Otic Placode lineage and other fgfr2b dependent epithelia
Development, 2020Co-Authors: Lisa D. Urness, Xiaofen Wang, Rolen M Quadros, Donald W Harms, Channabasavaiah B Gurumurthy, Suzanne L MansourAbstract:Pan-Otic CRE drivers enable gene regulation throughout the Otic Placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-Otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-Otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an Otic Placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is Otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the Otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other pan-Otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-Otic CRE drivers, particularly for postnatal analyses.
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slc26a9p2acre a new cre driver to regulate gene expression in the Otic Placode lineage and other fgfr2b dependent epithelia
bioRxiv, 2020Co-Authors: Lisa D. Urness, Xiaofen Wang, Rolen M Quadros, Donald W Harms, Channabasavaiah B Gurumurthy, Suzanne L MansourAbstract:Pan-Otic CRE drivers enable gene regulation throughout the Otic Placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-Otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-Otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We previously identified Slc26a9 as an Otic Placode-specific target of FGFR2b ligands, FGF3 and FGF10. We show here that Slc26a9 is Otic-specific through E10.5, but not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the Otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5-P3. In contrast to analogous mice generated with other pan-Otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-Otic CRE drivers, particularly for postnatal analyses.
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FGF signaling regulates Otic Placode induction and refinement by controlling both ectodermal target genes and hindbrain Wnt8a
Developmental biology, 2010Co-Authors: Lisa D. Urness, Gary C. Schoenwolf, Christian N. Paxton, Xiaofen Wang, Suzanne L MansourAbstract:Abstract The inner ear epithelium, with its complex array of sensory, non-sensory, and neuronal cell types necessary for hearing and balance, is derived from a thickened patch of head ectoderm called the Otic Placode. Mouse embryos lacking both Fgf3 and Fgf10 fail to initiate inner ear development because appropriate patterns of gene expression fail to be specified within the pre-Otic field. To understand the transcriptional “blueprint” initiating inner ear development, we used microarray analysis to identify prospective Placode genes that were differentially expressed in control and Fgf3−/−;Fgf10−/− embryos. Several genes in the down-regulated class, including Hmx3, Hmx2, Foxg1, Sox9, Has2, and Slc26a9 were validated by in situ hybridization. We also assayed candidate target genes suggested by other studies of Otic induction. Two Placode markers, Fgf4 and Foxi3, were down-regulated in Fgf3−/−;Fgf10−/− embryos, whereas Foxi2, a cranial epidermis marker, was expanded in double mutants, similar to its behavior when WNT responses are blocked in the Otic Placode. Assays of hindbrain Wnt genes revealed that only Wnt8a was reduced or absent in FGF-deficient embryos, and that even some Fgf3−/−;Fgf10−/+ and Fgf3−/− embryos failed to express Wnt8a, suggesting a key role for Fgf3, and a secondary role for Fgf10, in Wnt8a expression. Chick explant assays showed that FGF3 or FGF4, but not FGF10, were sufficient to induce Wnt8a. Collectively, our results suggest that Wnt8a provides the link between FGF-induced formation of the pre-Otic field and restriction of the Otic Placode to ectoderm adjacent to the hindbrain.
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mouse fgf15 is the ortholog of human and chick fgf19 but is not uniquely required for Otic induction
Developmental Biology, 2004Co-Authors: Tracy J Wright, Raj K. Ladher, John Mcwhirter, Cornelis Murre, Gary C. Schoenwolf, Suzanne L MansourAbstract:Abstract The inner ear develops from an ectodermal Placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor (Fgf)-19 is expressed in mesoderm underlying the presumptive Otic Placode, and human FGF19 induces expression of Otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of Otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have Otic abnormalities at E9.5–E10.5, suggesting that Fgf15 is not uniquely required for Otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive Otic Placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during Otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse Otic development.
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Fgf3 and Fgf10 are required for mouse Otic Placode induction.
Development (Cambridge England), 2003Co-Authors: Tracy J Wright, Suzanne L MansourAbstract:The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal Placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with Placode induction and Otic vesicle formation, but its absence affects only the later stages of Otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective Otic Placode. Embryos lacking both Fgf3 and Fgf10 fail to form Otic vesicles and have aberrant patterns of Otic marker gene expression, suggesting that FGF signals are required for Otic Placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in Otic induction is to establish normal patterns of gene expression in the prospective Placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in Otic vesicle formation.
Takahiro Ohyama - One of the best experts on this subject based on the ideXlab platform.
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notch signaling augments the canonical wnt pathway to specify the size of the Otic Placode
Development, 2008Co-Authors: Chathurani S Jayasena, Takahiro Ohyama, Neil Segil, Andrew K. GrovesAbstract:The inner ear derives from a patch of ectoderm defined by expression of the transcription factor Pax2. We recently showed that this Pax2^+ ectoderm gives rise not only to the Otic Placode but also to the surrounding cranial epidermis, and that Wnt signaling mediates this Placode-epidermis fate decision. We now present evidence for reciprocal interactions between the Wnt and Notch signaling pathways during inner ear induction. Activation of Notch1 in Pax2+ ectoderm expands the placodal epithelium at the expense of cranial epidermis, whereas loss of Notch1 leads to a reduction in the size of the Otic Placode. We show that Wnt signaling positively regulates Notch pathway genes such as Jag1, Notch1 and Hes1, and we have used transgenic Wnt reporter mice to show that Notch signaling can modulate the canonical Wnt pathway. Gain- and loss-of-function mutations in the Notch and Wnt pathways reveal that some aspects of Otic Placode development - such as Pax8 expression and the morphological thickening of the Placode - can be regulated independently by either Notch or Wnt signals. Our results suggest that Wnt signaling specifies the size of the Otic Placode in two ways, by directly upregulating a subset of Otic genes, and by positively regulating components of the Notch signaling pathway, which then act to augment Wnt signaling.
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the first steps towards hearing mechanisms of Otic Placode induction
The International Journal of Developmental Biology, 2007Co-Authors: Takahiro Ohyama, Andrew K. Groves, Kareen MartinAbstract:The entire inner ear, together with the neurons that innervate it, derive from a simple piece of ectoderm on the side of the embryonic head the Otic Placode. In this review, we describe the current state of the field of Otic Placode induction. Several lines of evidence suggest that all craniofacial sensory organs, including the inner ear, derive from a common "pre-placodal region" early in development. We review data showing that assumption of a pre-placodal cell state correlates with the competence of embryonic ectoderm to respond to Otic Placode inducing signals, such as members of the fibroblast growth factor (FGF) family. We also review evidence for FGF-independent signals that contribute to the induction of the Otic Placode. Finally, we review recent evidence suggesting that Wnt signals may act after FGF signaling to mediate a cell fate decision between Otic Placode and epidermis.
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wnt signals mediate a fate decision between Otic Placode and epidermis
Development, 2006Co-Authors: Takahiro Ohyama, Othman A Mohamed, Makoto Mark Taketo, Daniel Dufort, Andrew K. GrovesAbstract:The Otic Placode, the anlagen of the inner ear, develops from an ectodermal field characterized by expression of the transcription factor Pax2 . Previous fate mapping studies suggest that these Pax2+ cells will give rise to both Otic Placode tissue and epidermis, but the signals that divide the Pax2+ field into placodal and epidermal territories are unknown. We report that Wnt signaling is normally activated in a subset of Pax2+ cells, and that conditional inactivation of β-catenin in these cells causes an expansion of epidermal markers at the expense of the Otic Placode. Conversely, conditional activation of β-catenin in Pax2+ cells causes an expansion of the Otic Placode at the expense of epidermis, and the resulting Otic tissue expresses exclusively dorsal otocyst markers. Together, these results suggest that Wnt signaling acts instructively to direct Pax2+ cells to an Otic placodal, rather than an epidermal, fate and promotes dorsal cell identities in the otocyst.
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wnt signals mediate a fate decision between Otic Placode and epidermis
Development, 2006Co-Authors: Takahiro Ohyama, Othman A Mohamed, Makoto Mark Taketo, Daniel Dufort, Andrew K. GrovesAbstract:The Otic Placode, the anlagen of the inner ear, develops from an ectodermal field characterized by expression of the transcription factor Pax2. Previous fate mapping studies suggest that these Pax2(+) cells will give rise to both Otic Placode tissue and epidermis, but the signals that divide the Pax2(+) field into placodal and epidermal territories are unknown. We report that Wnt signaling is normally activated in a subset of Pax2(+) cells, and that conditional inactivation of beta-catenin in these cells causes an expansion of epidermal markers at the expense of the Otic Placode. Conversely, conditional activation of beta-catenin in Pax2(+) cells causes an expansion of the Otic Placode at the expense of epidermis, and the resulting Otic tissue expresses exclusively dorsal otocyst markers. Together, these results suggest that Wnt signaling acts instructively to direct Pax2(+) cells to an Otic placodal, rather than an epidermal, fate and promotes dorsal cell identities in the otocyst.
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generation of pax2 cre mice by modification of a pax2 bacterial artificial chromosome
Genesis, 2004Co-Authors: Takahiro Ohyama, Andrew K. GrovesAbstract:Summary: The Pax2 gene is expressed in the developing otocyst, kidney, and midbrain–hindbrain boundary. We generated Pax2-Cre transgenic lines by modification of a Pax2 bacterial artificial chromosome (BAC). In one Pax2-Cre line, Cre mRNA starts to be expressed in the Otic Placode at the late presomite stage. R26R reporter mouse analysis revealed that the Cre expression is sufficient to delete the loxP-flanked sequences in most of the cells in the inner ear. Reporter-positive cells are also detected in other Pax2-expressing tissues such as midbrain, cerebellum, olfactory bulb, and kidney, suggesting that these cells are the descendants of Pax2-expressing cells in these tissues and that Pax2-Cre transgenic mice can delete genes efficiently in these tissues. genesis 38:195–199, 2004. © 2004 Wiley-Liss, Inc.
Bruce B Riley - One of the best experts on this subject based on the ideXlab platform.
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Conserved expression of tfap2a during Otic neurogenesis.
2015Co-Authors: Husniye Kantarci, Renee K Edlund, Andrew K. Groves, Bruce B RileyAbstract:All images show cross-sections of the Otic Placode or vesicle in wild type zebrafish embryos (A-J) or chick embryos (L-Q) with a dorsal up and medial to the left. (A, B) At 14 hpf (10 somites) pax2a (red) marks the precursor cells in the emerging Otic Placode that are co-labeled with tfap2a (blue). (C-H) Cross-sections through the widest part of the neurogenic domain of the Otic vesicle, just posterior to the utricular macula. The outer and inner edges of the Otic vesicle are outlined. Patterns of ngn1 or tfap2a are shown at the indicated times. tfap2a is expressed in the ventrolateral part of the Otic vesicle, which partially overlaps the domain of ngn1 expression. (I-J) Cross-sections passing through the utricular macula of specimens co-stained for Isl1 (red) and tfap2a (blue) at 48 hpf. Expression of tfap2a is not detected in the floor of the Otic vesicle or in the mature SAG neurons at this time. (K) Schematic summary of SAG development in zebrafish, including regional markers. Neuroblasts are specified and delaminate from the Otic vesicle (light purple) adjacent to nascent sensory epithelia (green). Recently delaminated neuroblasts migrate towards hindbrain and continue to proliferate, forming the transit-amplifying pool (blue). Neuroblasts then stop dividing and differentiate into mature neurons (red). Relevant genes expressed in each domain are indicated. Expression of tfap2a (dark purple) overlaps the neurogenic domain, as well as the domain of bmp7a expression. Note that all of the tissues indicated express Fgf-target genes (etv5b and spry4) and transducers of Bmp (smad1 and smad5), but transit amplifying SAG precursors show specific upregulation of smad1 and smad5 [37]. (L-Q) Cross-sections through the Otic vesicle of chick embryos at days 3 and 4 (E3 and E4). The sensory region is labeled with Jagged-1 (green). Tfap2a (red) is expressed in the ventrolateral Otic domain in chick embryos similar to the pattern observed in zebrafish.
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Conditions that influence the response to Fgf during Otic Placode induction.
Developmental biology, 2012Co-Authors: Mahesh S Padanad, Neha Bhat, Biwei Guo, Bruce B RileyAbstract:Despite the vital importance of Fgf for Otic induction, previous attempts to study Otic induction through Fgf misexpression have yielded widely varying and contradictory results. There are also discrepancies regarding the ability of Fgf to induce Otic tissue in ectopic locations, raising questions about the sufficiency of Fgf and the degree to which other local factors enhance or restrict Otic potential. Using heat shock-inducible transgenes to misexpress Fgf3 or Fgf8 in zebrafish, we found that the stage, distribution and level of misexpression strongly influence the response to Fgf. Fgf misexpression during gastrulation can inhibit or promote Otic development, depending on context, whereas misexpression after gastrulation leads to expansion of Otic markers throughout preplacodal ectoderm surrounding the head. Elevated Fgf also expands expression of the putative competence factor Foxi1, which is required for Fgf to expand other Otic markers. Misexpression of downstream factors Pax2a or Pax8 also expands Otic markers but cannot bypass the requirement for Fgf or Foxi1. Co-misexpression of Pax2/8 with Fgf8 potentiates formation of ectopic Otic vesicles expressing a full range of Otic markers. These findings document the variables critically affecting the response to Fgf and clarify the roles of foxi1 and pax2/8 in the Otic response.
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pax2 8 proteins coordinate sequential induction of Otic and epibranchial Placodes through differential regulation of foxi1 sox3 and fgf24
Developmental Biology, 2011Co-Authors: Mahesh S Padanad, Bruce B RileyAbstract:Vertebrate cranial Placodes contribute vitally to development of sensory structures of the head. Amongst posterior Placodes, the Otic Placode forms the inner ear whereas nearby epibranchial Placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these Placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common Otic/epibranchial field, which later subdivides in response to other signals. However, we find that Otic and epibranchial Placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces Otic expression of pax8 and sox3, which cooperate synergistically to establish Otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate Otic expression of foxi1, a necessary step for further Otic development. Additionally, pax2/8 activate Otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on Otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the Otic Placode forms first and induces epibranchial Placodes through an Fgf-relay.
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zebrafish pax8 is required for Otic Placode induction and plays a redundant role with pax2 genes in the maintenance of the Otic Placode
Development, 2005Co-Authors: Melinda D Mackereth, Sujin Kwak, Andreas Fritz, Bruce B RileyAbstract:Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of Otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8 , we completed sequencing of the entire gene, including the 5′ and 3′ UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and Otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8 , or another early regulator, dlx3b , which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1 , which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8 . Disrupting pax8, pax2a and pax2b did not further impair Otic induction relative to loss of pax8 alone. However, the amount of Otic tissue gradually decreased in pax8-pax2a-pax2b -deficient embryos such that no Otic tissue was detectable by 24 hours post-fertilization. Loss of Otic tissue did not correlate with increased cell death, suggesting that Otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal Otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain Otic fate.
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A direct role for Fgf but not Wnt in Otic Placode induction.
Development (Cambridge England), 2004Co-Authors: Bryan T. Phillips, Elly M. Storch, Arne C. Lekven, Bruce B RileyAbstract:Induction of the Otic Placode, which gives rise to all tissues comprising the inner ear, is a fundamental aspect of vertebrate development. A number of studies indicate that fibroblast growth factor (Fgf), especially Fgf3, is necessary and sufficient for Otic induction. However, an alternative model proposes that Fgf must cooperate with Wnt8 to induce Otic differentiation. Using a genetic approach in zebrafish, we tested the roles of Fgf3, Fgf8 and Wnt8. We demonstrate that localized misexpression of either Fgf3 or Fgf8 is sufficient to induce ectopic Otic Placodes and vesicles, even in embryos lacking Wnt8. Wnt8 is expressed in the hindbrain around the time of Otic induction, but loss of Wnt8 merely delays expression of preOtic markers and Otic vesicles form eventually. The delay in Otic induction correlates closely with delayed expression of fgf3 and fgf8 in the hindbrain. Localized misexpression of Wnt8 is insufficient to induce ectopic Otic tissue. By contrast, global misexpression of Wnt8 causes development of supernumerary Placodes/vesicles, but this reflects posteriorization of the neural plate and consequent expansion of the hindbrain expression domains of Fgf3 and Fgf8. Embryos that misexpress Wnt8 globally but are depleted for Fgf3 and Fgf8 produce no Otic tissue. Finally, cells in the preOtic ectoderm express Fgf (but not Wnt) reporter genes. Thus, preOtic cells respond directly to Fgf but not Wnt8. We propose that Wnt8 serves to regulate timely expression of Fgf3 and Fgf8 in the hindbrain, and that Fgf from the hindbrain then acts directly on preplacodal cells to induce Otic differentiation.
Tanya T Whitfield - One of the best experts on this subject based on the ideXlab platform.
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Sculpting the labyrinth: Morphogenesis of the developing inner ear
Seminars in Cell and Developmental Biology, 2017Co-Authors: Berta Alsina, Tanya T WhitfieldAbstract:The vertebrate inner ear is a precision sensory organ, acting as both a microphone to receive sound and an accelerometer to detect gravity and motion. It consists of a series of interlinked, fluid-filled chambers containing patches of sensory epithelia, each with a specialised function. The ear contains many different differentiated cell types with distinct morphologies, from the flask-shaped hair cells found in thickened sensory epithelium, to the thin squamous cells that contribute to non-sensory structures, such as the semicircular canal ducts. Nearly all cell types of the inner ear, including the afferent neurons that innervate it, are derived from the Otic Placode, a region of cranial ectoderm that develops adjacent to the embryonic hindbrain. As the ear develops, the Otic epithelia grow, fold, fuse and rearrange to form the complex three-dimensional shape of the membranous labyrinth. Much of our current understanding of the processes of inner ear morphogenesis comes from genetic and pharmacological manipulations of the developing ear in mouse, chicken and zebrafish embryos. These traditional approaches are now being supplemented with exciting new techniques—including force measurements and light-sheet microscopy—that are helping to elucidate the mechanisms that generate this intricate organ system.
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Development of the inner ear
Current opinion in genetics & development, 2015Co-Authors: Tanya T WhitfieldAbstract:The vertebrate inner ear is a sensory organ of exquisite design and sensitivity. It responds to sound, gravity and movement, serving both auditory (hearing) and vestibular (balance) functions. Almost all cell types of the inner ear, including sensory hair cells, sensory neurons, secretory cells and supporting cells, derive from the Otic Placode, one of the several ectodermal thickenings that arise around the edge of the anterior neural plate in the early embryo. The developmental patterning mechanisms that underlie formation of the inner ear from the Otic Placode are varied and complex, involving the reiterative use of familiar signalling pathways, together with roles for transcription factors, transmembrane proteins, and extracellular matrix components. In this review, I have selected highlights that illustrate just a few of the many recent discoveries relating to the development of this fascinating organ system.
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fgf and hh signalling act on a symmetrical pre pattern to specify anterior and posterior identity in the zebrafish Otic Placode and vesicle
Development, 2011Co-Authors: Katherine L Hammond, Tanya T WhitfieldAbstract:Specification of the Otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior Otic identity between the 10 somite (Otic Placode) and 20 somite (early Otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior Otic specification during a similar period, a function that is completely separable from its earlier role in Otic Placode induction. In lia–/– (fgf3–/–) mutants, anterior Otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior Otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior Otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical Otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical Otic pre-pattern to specify anterior and posterior Otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.
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axial patterning in the developing vertebrate inner ear
The International Journal of Developmental Biology, 2007Co-Authors: Tanya T Whitfield, Katherine L HammondAbstract:Axial patterning in the vertebrate inner ear has been studied for over eighty years, and recent work has made great progress towards an understanding of the molecular mechanisms responsible for establishing asymmetries about the Otic axes. Tissues extrinsic to the ear provide sources of signalling molecules that are active early in development, at or before Otic Placode stages, while intrinsic factors interpret these signals to establish and maintain axial pattern. Key features of dorsoventral Otic patterning in amniote embryos involve Wnt and Fgf signalling from the hindbrain and Hh signalling from midline tissues (notochord and floorplate). Mutual antagonism between these pathways and their downstream targets within the Otic epithelium help to refine and maintain dorsoventral axial patterning in the ear. In the zebrafish ear, the same tissues and signals are implicated, but appear to play a role in anteroposterior, rather than dorsoventral, Otic patterning. Despite this paradox, conservation of mechanisms may be higher than is at first apparent.
