Takahiro Ohyama

Wnt signals mediate a fate decision between otic placode and epidermis.

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.

Hey2 Regulation by FGF Provides a Notch-Independent Mechanism for Maintaining Pillar Cell Fate in the Organ of Corti

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.

Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode

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.

BMP Signaling Is Necessary for Patterning the Sensory and Nonsensory Regions of the Developing Mammalian Cochlea

The mammalian inner ear detects sound with the organ of Corti, an intricately patterned region of the cochlea in which one row of inner hair cells and three rows of outer hair cells are surrounded by specialized supporting cells. The organ of Corti derives from a prosensory domain that runs the length of the cochlear duct and is bounded by two nonsensory domains, Köllikers organ on the neural side and the outer sulcus on the abneural side. Although much progress has been made in identifying the signals regulating organ of Corti induction and differentiation, less is known about the mechanisms that establish sensory and nonsensory territories in the cochlear duct. Here, we show that a gradient of bone morphogenetic protein (BMP) signaling is established in the abneural–neural axis of the cochlea. Analysis of compound mutants of Alk3/6 type I BMP receptors shows that BMP signaling is necessary for specification of the prosensory domain destined to form the organ of Corti. Reduction of BMP signaling in Alk3/6 compound mutants eliminates both the future outer sulcus and the prosensory domain, with all cells expressing markers of Köllikers organ. BMP4 upregulates markers of the future outer sulcus and downregulates marker genes of Köllikers organ in cochlear organ cultures in a dose-dependent manner. Our results suggest BMP signaling is required for patterning sensory and nonsensory tissue in the mammalian cochlea.

Expression of mouse Foxi class genes in early craniofacial development.

Recent models of craniofacial development suggest the existence of a common pan‐placodal domain lying next to the neural plate, from which all sensory placodes will arise. In support of this idea, several genes are expressed in the surface ectoderm of the head adjacent to the neural plate, before the appearance of genes in specific cranial placodes. In this study, we examine the expression patterns of the mouse Foxi class genes from embryonic day 6.5 to 10.5. Foxi2 is expressed throughout the cranial ectoderm adjacent to the neural plate from the 4‐somite stage, later becoming excluded from the otic placode. Foxi3 is expressed in a broad region of the pan‐placodal ectoderm adjacent to the neural plate from embryonic day (E) 6.75 to the first somite stage. Its expression becomes restricted to the ectoderm and the endoderm of the branchial pouches at E10.5. Foxi1 expression is first detected in the endolymphatic duct in the otic vesicle at E10.5. These results suggest that the mouse Foxi class genes may play important roles, both during cranial placode specification and in later development of individual cranial sensory structures and other organs derived from the cranial ectoderm. Developmental Dynamics 231:640–646, 2004.

SOX9 Controls Epithelial Branching by Activating RET Effector Genes during Kidney Development

Congenital abnormalities of the kidney and urinary tract are some of the most common defects detected in the unborn child. Kidney growth is controlled by the GDNF/RET signalling pathway, but the molecular events required for the activation of RET downstream targets are still poorly understood. Here we show that SOX9, a gene involved in campomelic dysplasia (CD) in humans, together with its close homologue SOX8, plays an essential role in RET signalling. Expression of SOX9 can be found from the earliest stages of renal development within the ureteric tip, the ureter mesenchyme and in a segment-specific manner during nephrogenesis. Using a tissue-specific knockout approach, we show that, in the ureteric tip, SOX8 and SOX9 are required for ureter branching, and double-knockout mutants exhibit severe kidney defects ranging from hypoplastic kidneys to renal agenesis. Further genetic analysis shows that SOX8/9 are required downstream of GDNF signalling for the activation of RET effector genes such as Sprouty1 and Etv5. At later stages of development, SOX9 is required to maintain ureteric tip identity and SOX9 ablation induces ectopic nephron formation. Taken together, our study shows that SOX9 acts at multiple steps during kidney organogenesis and identifies SOX8 and SOX9 as key factors within the RET signalling pathway. Our results also explain the aetiology of kidney hypoplasia found in a proportion of CD patients.

Canonical Notch Signaling Is Not Necessary for Prosensory Induction in the Mouse Cochlea: Insights from a Conditional Mutant of RBPjκ

The mammalian organ of Corti consists of a highly organized array of hair cells and supporting cells that originate from a common population of prosensory progenitors. Proper differentiation of this complex cellular mosaic requires lateral inhibition mediated by Notch signaling. Several studies have implicated Notch signaling in the earlier induction of the prosensory domain that lies along the length of the cochlear duct, and which forms before the onset of hair cell and supporting cell differentiation. To investigate the role of Notch signaling in prosensory domain formation, we conditionally inactivated the transcriptional mediator of canonical Notch signaling, RBPjκ, throughout the inner ear. Although RBPjκ mutants have severe vestibular defects and a shortened cochlear duct, markers of the prosensory domain appear at the normal time and location in the cochlea of RBPjκ mutants. Despite the lack of RBPjκ, hair cell and supporting cell markers also appear at appropriate times in the cochlea, suggesting that RBPjκ is dispensable for differentiation of the cochlear sensory epithelium. However, we also observed that differentiating hair cells and supporting cells rapidly die in RBPjκ mutants, suggesting a requirement of RBPjκ for cell survival in this tissue. Finally, in contrast to the chick basilar papilla, ectopic activation of Notch signaling did not induce ectopic sensory patches in nonsensory regions of the cochlea. Our results indicate that canonical Notch signaling is not necessary for prosensory specification in the mouse cochlea, suggesting that other signaling pathways may specify this highly derived sensory organ.

Wnt10a Is Involved in AER Formation During Chick Limb Development

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/β‐catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of β‐catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/β‐catenin signaling pathway. Developmental Dynamics 233:282–287, 2005.

Foxi transcription factors promote pharyngeal arch development by regulating formation of FGF signaling centers

The bones of the vertebrate face develop from transient embryonic branchial arches that are populated by cranial neural crest cells. We have characterized a mouse mutant for the Forkhead family transcription factor Foxi3, which is expressed in branchial ectoderm and endoderm. Foxi3 mutant mice are not viable and display severe branchial arch-derived facial skeleton defects, including absence of all but the most distal tip of the mandible and complete absence of the inner, middle and external ear structures. Although cranial neural crest cells of Foxi3 mutants are able to migrate, populate the branchial arches, and display some elements of correct proximo-distal patterning, they succumb to apoptosis from embryonic day 9.75 onwards. We show this cell death correlates with a delay in expression of Fgf8 in branchial arch ectoderm and a failure of neural crest cells in the arches to express FGF-responsive genes. Zebrafish foxi1 is also expressed in branchial arch ectoderm and endoderm, and morpholino knock-down of foxi1 also causes apoptosis of neural crest in the branchial arches. We show that heat shock induction of fgf3 in zebrafish arch tissue can rescue cell death in foxi1 morphants. Our results suggest that Foxi3 may play a role in the establishment of signaling centers in the branchial arches that are required for neural crest survival, patterning and the subsequent development of branchial arch derivatives.

Fgf10 is required for specification of non-sensory regions of the cochlear epithelium.

The vertebrate inner ear is a morphologically complex sensory organ comprised of two compartments, the dorsal vestibular apparatus and the ventral cochlear duct, required for motion and sound detection, respectively. Fgf10, in addition to Fgf3, is necessary for the earliest stage of otic placode induction, but continued expression of Fgf10 in the developing otic epithelium, including the prosensory domain and later in Kolliker׳s organ, suggests additional roles for this gene during morphogenesis of the labyrinth. While loss of Fgf10 was implicated previously in semicircular canal agenesis, we show that Fgf10(-/+) embryos also exhibit a reduction or absence of the posterior semicircular canal, revealing a dosage-sensitive requirement for FGF10 in vestibular development. In addition, we show that Fgf10(-/-) embryos have previously unappreciated defects of cochlear morphogenesis, including a somewhat shortened duct, and, surprisingly, a substantially narrower duct. The mutant cochlear epithelium lacks Reissner׳s membrane and a large portion of the outer sulcus-two non-contiguous, non-sensory domains. Marker gene analyses revealed effects on Reissner׳s membrane as early as E12.5-E13.5 and on the outer sulcus by E15.5, stages when Fgf10 is expressed in close proximity to Fgfr2b, but these effects were not accompanied by changes in epithelial cell proliferation or death. These data indicate a dual role for Fgf10 in cochlear development: to regulate outgrowth of the duct and subsequently as a bidirectional signal that sequentially specifies Reissner׳s membrane and outer sulcus non-sensory domains. These findings may help to explain the hearing loss sometimes observed in LADD syndrome subjects with FGF10 mutations.