Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development.     

Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.     

Expression of Frizzled10 in mouse central nervous system

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.     

Spatiotemporal expression of Notch receptors and ligands in developing mouse placenta

Notch signaling is involved in cell lineage specification in many developing organs. In mice there are four known Notch receptor genes (Notch1–4) and five ligands genes (Dll1, 3, 4 and Jagged1 and 2). Notch2 is essential for development of placenta, an organ that mediates feto-maternal nutrient and gas exchange as well as maternal adaptations to pregnancy. However the role of other Notch receptors and ligands in placentation is not known. In order to gain better insight into the role of Notch signaling in mouse placenta we thoroughly analyzed mRNA expression of all Notch receptors and ligands in all trophoblast cell types from the embryonic day (E) 7.5 to E12.5, the period during which all of the substructures of the placenta develop. Here we show that Notch receptors and ligands are specifically and dynamically expressed in multiple cell layers of developing placenta. We found that the Notch2 receptor and Jagged1 and Jagged2 ligand genes are complementarily expressed in trophoblast cells of the chorion and its later derivatives in the labyrinth. Dll4 and Notch2 expression complement each other in the ectoplacental cone, while Dll1 and Notch2 are expressed in an ectoplacental cone derivative, the junctional zone. Moreover Dll4 and Notch2 are expressed at the ectoplacental cone–decidua interface at early stages of placentation. Additionally we show that Notch2 is dynamically expressed in all trophoblast giant cell subtypes, which is consistent with previous reports. Overall these expression pattern results suggest that Notch signaling may play several diverse roles during placenta development.     

Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear

The vertebrate inner ear, a complex sensory organ with vestibular and auditory functions, is derived from a single ectoderm structure called the otic placode. Currently, the molecular mechanisms governing the differentiation and specification of the otic epithelium are poorly understood. We present here a detailed expression study of LMO1-4 in the developing mouse inner ear using a combination of in situ hybridization and immunohistochemistry. LMO1 is specifically expressed in the vestibular and cochlear hair cells as well as the vestibular ganglia of the developing inner ear. LMO2 expression is detected in the periotic mesenchyme of the developing mouse cochlea from E12.5 to E14.5. The expression of LMO3 expression is first observed in the cochlea at E13.5 and becomes confined to the lesser epithelial ridge (LER) from E14.5 to E17.5. LMO3 is also expressed in some of the vestibular ganglion cells. LMO4 is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista, and the spiral ganglia throughout embryogenesis. Thus, the regionalized expression patterns of LMO1-4 are closely associated with the morphogenesis of the inner ear.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Intercellular signals and cell-fate choices in the developing inner ear: origins of global and of fine-grained pattern

The major regions of the inner ear begin to be distinguishable by their patterns of gene expression very early, before the otocyst has closed. Later, individual cells within a neurogenic or sensory patch become committed to specific pathways of differentiation. Insights gained from homologies with invertebrates and from studies of tissues other than the ear, combined with discoveries from screens for mutations affecting development in the zebrafish, are beginning to reveal the genes and signalling mechanisms that control these cell-fate choices in the developing inner ear.