We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.
Planar cell polarity (PCP) refers to coordinated polarization of cells within the plane of a cell sheet. A conserved signaling pathway is required for the establishment of PCP in epithelial tissues and for polarized cellular rearrangements known as convergent extension. During PCP signaling, core PCP proteins are sorted asymmetrically along the polarization axis; this sorting is thought to direct coordinated downstream morphogenetic changes across the entire tissue. Here, we show that a gene encoding a ciliary protein (a 'ciliary gene'), Ift88, also known as Polaris, is required for establishing epithelial PCP and for convergent extension of the cochlear duct of Mus musculus. We also show that the proper positioning of ciliary basal bodies and the formation of polarized cellular structures are disrupted in mice with mutant ciliary proteins ('ciliary mutants'), whereas core PCP proteins are partitioned normally along the polarization axis. Thus, our data uncover a distinct requirement for ciliary genes in basal body positioning and morphological polarization during PCP regulation.
The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g −/− mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g fl/fl Myo15-cre +/− mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short-and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.auditory mechanoelectrical transduction | Usher syndrome type 1 | deafness | conditional knockout mice | organ of Corti
Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing, activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging, coupled with gene expression analyses and Notch invalidations, to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary, the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof, activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments.
The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region -the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.
SummarySpatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a “sequential stacking” construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated.
SUMMARYEpithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.
Although the secreted molecule Fgf8 is a key player of the isthmic organiser function, the mechanisms by which it acts remain unclear. Here, we present evidence indicating that Fgf8 is not instructive in establishing zebrafish cerebellar cell identities, although it is required for proliferation and morphogenesis of this territory. We first show that, as in mouse, lack of Otx function in zebrafish leads to transformation of the presumptive mesencephalon into an extended rhombomere 1 (r1). Expanded Fgf8 expression was proposed to be the cause of this fate transformation. However, this report demonstrates that zebrafish embryos lacking both Otx and fgf8 functions retain an extended r1 and display differentiation of at least two cerebellar cell fates. We show that this is not caused by presence of other Fgfs, which implies that in absence of Otx, Fgf function is not necessary for the differentiation of cerebellar cell types. Otx proteins are therefore potent repressors of cerebellar fates, kept out of r1 progeny by Fgf8. Because Otx transcripts are not present in presumptive r1 territory prior to fgf8 expression, Fgf8 is required to maintain, rather than induce, the posterior boundary of Otx expression. This maintenance is enough to allow cerebellar differentiation.
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