Enological Suitability of Indigenous Yeast Strains for 'Verdejo' Wine Production.

The use of indigenous yeasts for the production of wines is a tool to defend the typicity of a particular region. The selection of appropriate indigenous yeasts ensures the maintenance of oenological characteristics by simulating spontaneous alcoholic fermentation (AF) while avoiding the risks of stuck or sluggish fermentations. In this study, autochthonous yeasts from Verdejo grape juice (Appellation of Origin Rueda) were selected, identified, and characterized to exploit the characteristics of the 'terroir'. The fermentation capacity of seven strains was studied individually at the laboratory scale. The most suitable strains (Saccharomyces cerevisiae: Sacch 1, Sacch 2, Sacch 4, and Sacch 6) and Sacch 6 co-inoculated with Metschnikowia pulcherrima were characterized at the pilot scale. The fermentation kinetics, bioproduct release, volatile composition, and sensory profile of the wines were evaluated. Significant differences were found, especially in the aroma profile. In particular, Sacch 6 and Sacch 6 co-inoculated with M. pulcherrima produced higher amounts of ethyl esters and acetates and lower amounts of higher alcohols than the spontaneous AF. Wines inoculated with indigenous yeasts had higher sensory scores for fruit aromas and overall rating. The selection of indigenous yeasts improved the aroma of Verdejo wines and could contribute to determining the wine typicity of the wine region.

Inactivation of Fgf3 and Fgf4 within the Fgf3/Fgf4/Fgf15 gene cluster reveals their redundant requirement for mouse inner ear induction and embryonic survival.

BACKGROUND: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. RESULTS: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. CONCLUSIONS: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival.

Meis2 Is Required for Inner Ear Formation and Proper Morphogenesis of the Cochlea.

Meis genes have been shown to control essential processes during development of the central and peripheral nervous system. Here we have explored the roles of the Meis2 gene during vertebrate inner ear induction and the formation of the cochlea. Meis2 is expressed in several tissues required for inner ear induction and in non-sensory tissue of the cochlear duct. Global inactivation of Meis2 in the mouse leads to a severely reduced size of the otic vesicle. Tissue-specific knock outs of Meis2 reveal that its expression in the hindbrain is essential for otic vesicle formation. Inactivation of Meis2 in the inner ear itself leads to an aberrant coiling of the cochlear duct. By analyzing transcriptomes obtained from Meis2 mutants and ChIPseq analysis of an otic cell line, we define candidate target genes for Meis2 which may be directly or indirectly involved in cochlear morphogenesis. Taken together, these data show that Meis2 is essential for inner ear formation and provide an entry point to unveil the network underlying proper coiling of the cochlear duct.

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea.

Roles of Wnt8a during formation and patterning of the mouse inner ear.

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle.

N-myc controls proliferation, morphogenesis, and patterning of the inner ear.

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27(Kip1) and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

mab21l2 transgenics reveal novel expression patterns of mab21l1 and mab21l2, and conserved promoter regulation without sequence conservation.

mab21l1 and mab21l2 paralogs have widespread and dynamic expression patterns during vertebrate development. Both genes are expressed in the developing eye, midbrain, neural tube, and branchial arches. Our goal was to identify promoter regions with activity in mab21l2 expression domains. Assays of mab21l2 promoter-EGFP constructs in zebrafish embryos confirm that constructs containing 7.2 or 4.9 kb of mab21l2 promoter region are sufficient to drive expression in known (e.g., tectum, branchial arches) and unexpected domains (e.g., lens and retinal amacrine cells). A comparative analysis identifies complementary and novel expression domains of endogenous mab21l2 (e.g., lens and ventral iridocorneal canal) and mab21l1 (e.g., retinal amacrine and ganglion cells). Interestingly, therefore, despite the absence of conserved non-coding elements, a 4.9-kb mab21l2 promoter is sufficient to recapitulate expression in tissues unique to mab21l1 or mab21l2.

Zebrafish Tg(7.2mab21l2:EGFP)ucd2 transgenics reveal a unique population of retinal amacrine cells.

PURPOSE: Amacrine cells constitute a diverse, yet poorly characterized, cell population in the inner retina. Here, the authors sought to characterize the morphology, molecular physiology, and electrophysiology of a subpopulation of EGFP-expressing retinal amacrine cells identified in a novel zebrafish transgenic line. METHODS: After 7.2 kb of the zebrafish mab21l2 promoter was cloned upstream of EGFP, it was used to create the Tg(7.2mab21l2:EGFP)ucd2 transgenic line. Transgenic EGFP expression was analyzed by fluorescence microscopy in whole mount embryos, followed by detailed analysis of EGFP-expressing amacrine cells using fluorescence microscopy, immunohistochemistry, and electrophysiology. RESULTS: A 7.2-kb fragment of the mab21l2 promoter region is sufficient to drive transgene expression in the developing lens and tectum. Intriguingly, EGFP was also observed in differentiated amacrine cells. EGFP-labeled amacrine cells in Tg(7.2mab21l2:EGFP)ucd2 constitute a novel GABA- and glycine-negative amacrine subpopulation. Morphologically, EGFP-expressing cells stratify in sublamina 1 to 2 (type 1 OFF) or sublamina 3 to 4 (type 1 ON) or branch diffusely (type 2). Electrophysiologically, these cells segregate into amacrine cells with somas in the vitreal part of the INL and linear responses to current injection or, alternatively, amacrine cells with somas proximal to the IPL and active oscillatory voltage signals. CONCLUSIONS; The novel transgenic line Tg(7.2mab21l2:EGFP)ucd2 uncovers a unique subpopulation of retinal amacrine cells.

Tissue-specific requirements for FGF8 during early inner ear development.

Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates.

Gene expression analysis of canonical Wnt pathway transcriptional regulators during early morphogenesis of the facial region in the mouse embryo.

Structures and features of the face, throat and neck are formed from a series of branchial arches that grow out along the ventrolateral aspect of the embryonic head. Multiple signalling pathways have been implicated in patterning interactions that lead to species-specific growth and differentiation within the branchial region that sculpt these features. A direct role for Wnt signalling in particular has been shown. The spatial and temporal distribution of Wnt pathway components contributes to the operation of the signalling system. We present the precise distribution of gene expression of canonical Wnt pathway transcriptional regulators, Tcf1, Lef1, Tcf3, Tcf4 and beta-catenin between embryonic day (E) 9.5 and 11.5. In situ hybridization combined with Optical Projection Tomography was used to record and compare distribution of transcripts in 3D within the developing branchial arches. This shows widespread yet very specific expression of the gene set indicating that all genes contribute to proper patterning of the region. Tcf1 and Lef1 are more prominent in rostral arches, particularly at later ages, and Tcf3 and Tcf4 are in general expressed more deeply (medial/endodermal aspect) in the arches than Tcf1 and Lef1. Comparison with Wnt canonical pathway readout patterns shows that the relationship between the expression of individual transcription factors and activation of the pathway is not simple, indicating complexity and flexibility in the signalling system.

Duplication and divergence of zebrafish CRALBP genes uncovers novel role for RPE- and Muller-CRALBP in cone vision.

PURPOSE: During vertebrate phototransduction 11-cis-retinal is isomerized to all-trans-retinal. Light sensitivity is restored by recombination of apo-opsin with 11-cis-retinal to regenerate visual pigments. The conversion of all-trans retinal back to 11-cis-retinal is known as the visual cycle. Within the retina, cellular retinal-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Müller glia. CRALBP expressed in the RPE is known to facilitate the rate of the rod visual cycle. Recent evidence suggests a role for Müller glia in an alternate cone visual cycle. In this study, the role of RPE- and Müller-CRALBP in cone vision was characterized. METHODS: The CRALBP orthologues rlbp1a and rlbp1b were identified in zebrafish by bioinformatic methods. The spatial and developmental expression of rlbp1a and rlbp1b was determined by in situ hybridization and immunohistochemistry. Depletion of the expression of the corresponding Cralbp a and Cralbp b proteins was achieved by microinjection of antisense morpholinos. Visual function was analyzed in 5-day post fertilization (dpf) larvae using the optokinetic response assay. RESULTS: The zebrafish genome contains two CRALBP ohnologues, rlbp1a and rlbp1b. These genes have functionally diverged, exhibiting differential expression at 5 dpf in RPE and Müller glia, respectively. Depletion of CRALBP in the RPE or Müller glia results in abnormal cone visual behavior. CONCLUSIONS: The results suggest that cone photoreceptors incorporate 11-cis-retinoids derived from the rod and cone visual cycles into their visual pigments and that Müller-CRALBP participates in the cone visual cycle.

Differential requirements for FGF3, FGF8 and FGF10 during inner ear development.

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.

FGF signalling controls expression of vomeronasal receptors during embryogenesis.

Fibroblast growth factors (FGFs) have been shown to control formation and differentiation of multiple organ systems in the developing vertebrate embryo. The analysis of differential gene expression during embryogenesis is, therefore, a potent tool to identify novel target genes regulated by FGF signalling. Here, we have applied microarray analysis to identify differentially regulated genes in FGF mutant mouse embryos. Surprisingly, transcripts corresponding to vomeronasal receptors (VRs), which so far have been only detected in the vomeronasal organ (VNO), were found to be downregulated in FGF mutant embryos. VR expression was detected in the developing olfactory pit and the anlage of the VNO. Interestingly, several FGFs can be detected in the developing olfactory pit during mouse embryogenesis [Bachler, M., Neubuser, A. 2001. Expression of members of the Fgf family and their receptors during midfacial development. Mech. Dev. 100, 313-316]. FGF signalling may thus control expression of VRs in the olfactory pit and formation of the VNO. Moreover, VR expression was detected in unexpected locations within the developing embryo including retina, dorsal root ganglia and neural tube. The relevance of VR expression in these structures and for formation of the VNO is discussed.

Requirements for FGF3 and FGF10 during inner ear formation.

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.