Early and transient requirements for FGFR2b/1b ligands in cochlear sensory and neural cell subtype differentiation.

We probed the roles of FGFR2b/1b signaling in mid-gestation cochlear development by inducing dnFGFR2b, a ligand trap that sequesters FGF3 and FGF10. Analyses following E11.5-E18.

: a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia.

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.

Spatial and temporal inhibition of FGFR2b ligands reveals continuous requirements and novel targets in mouse inner ear morphogenesis.

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.

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.

Background: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models.

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.

Genetic rescue of Muenke syndrome model hearing loss reveals prolonged FGF-dependent plasticity in cochlear supporting cell fates.

The stereotyped arrangement of cochlear sensory and supporting cells is critical for auditory function. Our previous studies showed that Muenke syndrome model mice (Fgfr3P244R/+) have hearing loss associated with a supporting cell fate transformation of two Deiters' cells to two pillar cells. We investigated the developmental origins of this transformation and found that two prospective Deiters' cells switch to an outer pillar cell-like fate sequentially between embryonic day 17.

Redundant and dosage sensitive requirements for Fgf3 and Fgf10 in cardiovascular development.

Heart development requires contributions from, and coordinated signaling interactions between, several cell populations, including splanchnic and pharyngeal mesoderm, postotic neural crest and the proepicardium. Here we report that Fgf3 and Fgf10, which are expressed dynamically in and near these cardiovascular progenitors, have redundant and dosage sensitive requirements in multiple aspects of early murine cardiovascular development. Embryos with Fgf3(-/+);Fgf10(-/-), Fgf3(-/-);Fgf10(-/+) and Fgf3(-/-);Fgf10(-/-) genotypes formed an allelic series of increasing severity with respect to embryonic survival, with double mutants dead by E11.

Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish.

The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes.

FGF signaling regulates otic placode induction and refinement by controlling both ectodermal target genes and hindbrain Wnt8a.

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.

Fgf16(IRESCre) mice: a tool to inactivate genes expressed in inner ear cristae and spiral prominence epithelium.

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.

Expression of ERK signaling inhibitors Dusp6, Dusp7, and Dusp9 during mouse ear development.

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.

Netrins promote developmental and therapeutic angiogenesis.

Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrin1a messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development.

Identification of new netrin family members in zebrafish: developmental expression of netrin 2 and netrin 4.

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.

Elastin induces myofibrillogenesis via a specific domain, VGVAPG.

A hallmark of vascular smooth muscle cells (VSMCs) is their dynamic ability to assemble and disassemble contractile proteins into sarcomeric units depending upon their phenotypic state. This phenotypic plasticity plays an important role during vascular development and in obstructive vascular disease. Previously, we showed that the Elastin gene product, tropoelastin, activates myofibrillar organization of VSMCs.

Robo4 is a vascular-specific receptor that inhibits endothelial migration.

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.

Loss of distinct arterial and venous boundaries in mice lacking endoglin, a vascular-specific TGFbeta coreceptor.

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.