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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.02.016 Byline: Lisa D. Urness (a), Christian N. Paxton (b), Xiaofen Wang (a), Gary C. Schoenwolf (b), Suzanne L. Mansour (a)(b) Keywords: FGF signaling; Otic placode induction; WNT signaling; Microarray analysis 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.sup.- .sup./ .sup.- ;Fgf10.sup.- .sup./ .sup.- 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.sup.- .sup./ .sup.- ;Fgf10.sup.- .sup./ .sup.- 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.sup.- .sup./ .sup.- ;Fgf10.sup.- .sup./+ and Fgf3.sup.- .sup./ .sup.- 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. Author Affiliation: (a) Department of Human Genetics, University of Utah, 15 N 2030 E, RM 2100, Salt Lake City, UT 84112-5330, USA (b) Department of Neurobiology and Anatomy, University of Utah, 30 N 1900 E, RM 2R066 SOM, Salt Lake City, UT 84132-3401, USA Article History: Received 13 January 2010; Revised 10 February 2010; Accepted 10 February 2010

Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting. In contrast to known neuronal Robo family members, the arrangement of the extracellular domains of Robo4 diverges significantly from that of all other Robo family members. Moreover, Robo4 is specifically expressed in the vascular endothelium during murine embryonic development. We show that Robo4 binds Slit and inhibits cellular migration in a heterologous expression system, analogous to the role of known Robo receptors in the nervous system. Immunoprecipitation studies indicate that Robo4 binds to Mena, a known effector of Robo-Slit signaling. Finally, we show that Robo4 is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. These data demonstrate that Robo4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development. Keywords: Vascular development; Angiogenesis; Endothelial migration; Vascular sprouting; Activin receptor-like kinase 1 (Alk1); HHT; Neuronal/vascular guidance; Roundabout (Robo); Slit; Vascular patterning

Several characteristic morphological and functional differences distinguish arteries from veins. It was thought that hemodynamic forces shaped these differences; however, increasing evidence suggests that morphogenetic programs play a central role in blood vessel differentiation. Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by the inappropriate fusion of arterioles with venules. The genes implicated in this disease, ALK1 and endoglin, may be involved in defining the fundamental boundaries between arteries and veins. We previously showed that mice lacking Alk1 lost structural, molecular, and functional distinctions between arteries and veins [Nat. Genet. 26 (2000), 328]. Here, we report that mice lacking endoglin develop arterial-venous malformations and fail to confine intraembryonic hematopoiesis to arteries. In contrast to Alk1 mutants, endoglin mutants do not show profound vessel dilation or downregulation of arterial ephrinB2. Finally, our data indicate that a failure in cardiac cushion formation observed in both strains may be secondary to the peripheral vasculature defect. The phenotypic similarities, yet reduced severity, implicates endoglin as an accessory coreceptor that specifically modulates Alk1 signaling. We propose that endoglin and Alk1 are necessary for the maintenance of distinct arterial-venous vascular beds and that attenuation of the Alk1 signaling pathway is the precipitating event in the etiology of HHT. Keywords: Alk1; Acvrl1: Endoglin: Hereditary hemorrhagic telangiectasia (HHT); Arteriovenous malformations: Arteriovenous identity

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 panotic 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. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors play important roles in inner ear development. Previous studies showed that mouse Fgf16 is expressed asymmetrically during the otic cup and vesicle stages of development, suggesting roles in regulating or responding to anteroposterior axial cues. Here, we studied otic Fgf16 expression throughout embryonic development and found transcripts in the developing cristae and in a few cells in the lateral wall of the cochlear duct. To determine the otic function of Fgf16 and to follow the fate of Fgf16-expressing cells, we generated an Fgf16 allele. We show that Fgf16 does not have a unique role in inner ear development and that the Fgf16 lineage is found throughout the three cristae, in portions of the semicircular canal ducts, and in the cochlear spiral prominence epithelial cells. This strain will be useful for gene ablations in these tissues. Developmental Dynamics 238:358-366, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The levels of fibroblast growth factor (FGF) signaling play important roles in coordinating development of the mouse inner, middle, and outer ears. Extracellular signal-regulated kinases (ERKs) are among the effectors that transduce the FGF signal to the nucleus and other cellular compartments. Attenuation of ERK activity by dephosphorylation is necessary to modulate the magnitude and duration of the FGF signal. Recently, we showed that inactivation of the ERK phosphatase, dual specificity phosphatase 6 (DUSP6), causes partially penetrant postnatal lethality, hearing loss and skeletal malformations. To determine whether other Dusps may function redundantly with Dusp6 during otic development, we surveyed the expression domains of the three ERK-specific DUSP transcripts, Dusp6, Dusp7, and Dusp9, in the embryonic mouse ear. We show that each is expressed in partially overlapping patterns that correspond to regions of active FGF signaling, suggesting combinatorial roles in negative regulation of this pathway during ear development. Developmental Dynamics 237:163-169, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways, and disruptions cause abnormalities of hearing and/or balance. The FGFR2b ligands FGF3 and FGF10 are expressed throughout otic development and are required individually for normal morphogenesis, but their prior and redundant roles in otic placode induction complicates investigation of subsequent combinatorial functions in morphogenesis. To interrogate these roles and identify new effectors of FGF3 and FGF10 signaling at the earliest stages of otic morphogenesis, we used conditional gene ablation after otic placode induction, and temporal inhibition of signaling with a secreted, dominant-negative FGFR2b ectodomain. We show that both ligands are required continuously after otocyst formation for maintenance of otic neuroblasts and for patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genome-wide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Fibroblast growth factors play important roles in inner ear development. Previous studies showed that mouse Fgf16 is expressed asymmetrically during the otic cup and vesicle stages of development, suggesting roles in regulating or responding to anteroposterior axial cues. Here, we studied otic Fgf16 expression throughout embryonic development and found transcripts in the developing cristae and in a few cells in the lateral wall of the cochlear duct. To determine the otic function of Fgf16 and to follow the fate of Fgf16-expressing cells, we generated an Fgf16(IRESCre) allele. We show that Fgf16 does not have a unique role in inner ear development and that the Fgf16 lineage is found throughout the three cristae, in portions of the semicircular canal ducts, and in the cochlear spiral prominence epithelial cells. This strain will be useful for gene ablations in these tissues.

Netrin 1 is a diffusible factor that attracts commissural axons to the floor plate of the spinal cord. Recent evidence indicates that Netrin 1 is widely expressed and functions in the development of multiple organ systems. In mammals, there are three genes encoding Netrins, whereas in zebrafish, only the Netrin 1 orthologs netrin 1a and netrin 1b have been identified. Here, we have cloned two new zebrafish Netrins, netrin 2 and netrin 4, and present a comparative sequence and expression analysis. Despite significant sequence similarity with netrin 1a/netrin 1b, netrin 2 displays a unique expression pattern. Netrin 2 transcript is first detected in the notochord and in developing somites at early somitogenesis. By late somitogenesis, netrin 2 is expressed in the fourth rhombomere and is subsequently expressed in the hindbrain and otic vesicles. In contrast, netrin 4 is detected only at very low levels during early development. The nonoverlapping expression patterns of these four Netrins suggest that they may play unique roles in zebrafish development., (Developmental Dynamics 234:726-731, 2005. (c) 2005 Wiley-Liss, Inc.)