Shh signaling and regulatory gene expression in mouse taste buds.
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Gene and alternative splicing annotation with AIR.
Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.
Alternative Splicing↗
Intrinsic quantal variability due to stochastic properties of receptor-transmitter interactions.
Synaptic events at the neuromuscular junction are integer multiples of a quantum, the postsynaptic response to transmitter released from one presynaptic vesicle. At central synapses where quanta are small, it has been suggested they are invariant due to occupation of all postsynaptic receptors, a concept neglecting inherent fluctuations in channel behavior. If this did occur, the quantal release model would not apply there and could not be used to localize sites of synaptic modification. Monte Carlo simulations of quanta include transmitter diffusion and interactions with postsynaptic receptors that are treated probabilistically. These models suggest that when there are few postsynaptic channels available at a synapse, their stochastic behavior produces significant intrinsic variance in response amplitude and kinetics, and saturation does not occur. These results were confirmed by analysis of inhibitory quanta in embryonic and adult Mauthner cells involving a small and large number of channels, respectively. The findings apply to excitatory synapses as well.
Animals↗
Insight into podocyte differentiation from the study of human genetic disease: nail-patella syndrome and transcriptional regulation in podocytes.
In recent years, our understanding of the molecular basis of kidney development has benefited from the study of rare genetic diseases affecting renal function. This has especially been the case with the differentiation of the highly specialized podocyte in the pathogenesis of human disorders and mouse phenotypes affecting the renal filtration barrier. This filtration barrier represents the end product of a complex series of signaling events that produce a tripartite structure consisting of interdigitating podocyte foot processes with intervening slit diaphragms, the glomerular basement membrane, and the fenestrated endothelial cell. Dysregulation of unique cytoskeletal and extracellular matrix proteins in genetic forms of nephrotic syndrome has shown how specific structural proteins contribute to podocyte function and differentiation. However, much less is known about the transcriptional determinants that both specify and maintain this differentiated cell. Our studies of a skeletal malformation syndrome, nail-patella syndrome, have shown how the LIM homeodomain transcription factor, Lmx1b, contributes to transcriptional regulation of glomerular basement membrane collagen expression by podocytes. Moreover, they raise intriguing questions about more global transcriptional regulation of podocyte morphogenesis.
Animals↗
Nkx2.2-repressor activity is sufficient to specify alpha-cells and a small number of beta-cells in the pancreatic islet.
The homeodomain protein Nkx2.2 (Nkx2-2) is a key regulator of pancreatic islet cell specification in mice; Nkx2.2 is essential for the differentiation of all insulin-producing beta-cells and of the majority of glucagon-producing alpha-cells, and, in its absence, these cell types are converted to a ghrelin cell fate. To understand the molecular functions of Nkx2.2 that regulate these early cell-fate decisions during pancreatic islet development, we created Nkx2.2-dominant-derivative transgenic mice. In the absence of endogenous Nkx2.2, the Nkx2.2-Engrailed-repressor derivative is sufficient to fully rescue glucagon-producing alpha-cells and to partially rescue insulin-producing beta-cells. Interestingly, the insulin-positive cells that do form in the rescued mice do not express the mature beta-cell markers MafA or Glut2 (Slc2a2), suggesting that additional activator functions of Nkx2.2 are required for beta-cell maturation. To explore the mechanism by which Nkx2.2 functions as a repressor in the islet, we assessed the pancreatic expression of the Groucho co-repressors, Grg1, Grg2, Grg3 and Grg4 (Tle1-Tle4), which have been shown to interact with and modulate Nkx2.2 function. We determined that Grg3 is highly expressed in the embryonic pancreas in a pattern similar to Nkx2.2. Furthermore, we show that Grg3 physically interacts with Nkx2.2 through its TN domain. These studies suggest that Nkx2.2 functions predominantly as a transcriptional repressor during specification of endocrine cell types in the pancreas.
Animals↗
The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain.
In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.
Animals↗
What do animal models have to tell us regarding Duchenne muscular dystrophy?
Animal models of DMD have played, and will continue to play, a key role in the understanding of the pathogenesis and treatment of Duchenne muscular dystrophy (DMD). The mdx mouse and GRMD dog are spontaneous dystrophin deficient mutants and have been the most widely used models to date. A number of other murine models have been created by exposure to mutagens or genetic manipulation. The animal models have allowed the development of a number of promising experimental therapeutic approaches to DMD that are now entering clinical trial, the majority of which would not have been developed without their use. However, there has been much debate about the merits of the different animal models, which will only be finally clear as we learn from the initial human clinical trials.
Animals↗
Light-induced Ca2+ release in the visible cones of the zebrafish.
We used suction-pipette recording and fluo-4 fluorescence to study light-induced Ca2+ release from the visible double cones of zebrafish. In Ringer, light produces a slow decrease in fluorescence which can be fitted by the sum of two decaying exponentials with time constants of 0.5 and 3.8 s. In 0Ca2+-0Na+ solution, for which fluxes of Ca2+ across the outer segment plasma membrane are greatly reduced, light produces a slow increase in fluorescence. Both the decrease and increase are delayed after incorporation of the Ca2+ chelator BAPTA, indicating that both are produced by a change in Ca2+. If the Ca2+ pool is first released by bright light in 0Ca2+-0Na+ solution and the cone returned to Ringer, the time course of Ca2+ decline is much faster than in Ringer without previous light exposure. This indicates that the time constants of 0.5 and 3.8 s actually reflect a sum of Na+/Ca2+-K+ exchange and light-induced release of Ca2+. The Ca2+ released by light appears to come from at least two sites, the first comprising 66% of the total pool and half-released by bleaching 4.8% of the pigment. Release of the remaining Ca2+ from the second site requires the bleaching of nearly all of the pigment. If, after release, the cone is maintained in darkness, a substantial fraction of the Ca2+ returns to the release pool even in the absence of pigment regeneration. The light-induced release of Ca2+ can produce a modulation of the dark current as large as 0.75 pA independently of the normal transduction cascade, though the rise time of the current is considerably slower than the normal light response. These experiments show that Ca2+ can be released within the cone outer segment by light intensities within the physiological range of photopic vision. The role this Ca2+ release plays remains unresolved.
Aniline Compounds↗
Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis.
Exposure to ethanol during development can lead to a constellation of congenital anomalies, resulting in prenatal and postnatal failure to thrive, central nervous system (CNS) deficits, and a number of patterning defects that lead to defects in the cardiovascular system, facial structures, and limbs. The cellular, biochemical, and molecular mechanisms by which ethanol exerts its developmental toxicity and the genes that influence sensitivity to developmental ethanol exposure have yet to be discovered, despite being one of the more common nongenetic causes of birth defects. The zebrafish undergoes much the same patterning and morphogenesis as other vertebrate embryos do--including humans--that are distinct and cannot be studied in invertebrates. Developmental processes in zebrafish are affected by ethanol exposure in a dose-dependent manner, resulting in learning and memory deficits, cell death in the CNS, skeletal dysmorphogenesis, and alterations in startle reflex responses. Interestingly, significant ethanol effects on learning and behavioral endpoints occurred at concentrations well below those that induced cell death in the CNS. This work provides the foundation for identifying genes and pathways involved in developmental alcohol toxicity in vertebrates, leading to a more complete mechanistic understanding of fetal alcohol disorders in humans.
Alcohol-Induced Disorders, Nervous System↗
Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm.
Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, gene encoding a transcription factor required for mesodermal morphogenetic movements. Further, we show that the floating head homeobox gene is required in axial mesoderm to repress the expression of both spadetail and papc, promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface protein may provide a link between regulatory transcription factors and the actual cell biological behaviors that execute morphogenesis during gastrulation.
Amino Acid Sequence↗
Regulation of axon growth in vivo by activity-based competition.
The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.
Animals↗
Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.
Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.
Adenoviridae↗
Nephrin expression and three-dimensional morphogenesis of the Xenopus pronephric glomus.
Nephrin (NHPS1) encodes a transmembrane protein of approximately 1,200 amino acids that plays a critical role in podocyte slit-diaphragm formation and the development of functional mammalian glomerular filtration barriers. In humans and mice with congenital defects in the nephrin gene, the glomerular filtration barrier is defective and protein leakage into the kidney filtrate causes a life-threatening proteinuria. This protein also plays an essential role in the formation of the stellate cells of the Drosophila Malpighian tubules. In this report, the sequence and expression of a Xenopus ortholog of nephrin is described using both conventional and novel three-dimensional (3D) visualization methodologies. Xenopus nephrin encodes a protein of 1,238 amino acids and is expressed at high levels in the forming pronephric kidney glomus, the equivalent of the mammalian glomerulus. Expression commences at stage 25 and is specific to the pronephric glomus up until at least tadpole feeding stages. Two-color fluorescent whole-mount in situ analysis of nephrin expression allowed the 3D shape of the glomus to be imaged and contrasted to the pronephric tubules throughout its morphogenesis. Confocal data processing pipelines were established to generate both volumetric and surface models of the developing pronephros, and a Web-based visualization system was used to generate dynamic and manipulable models of the forming nephric organs. This system allows simple on-line morphometric analysis of the developing pronephric components. As in fish embryos, the glomera first form laterally then migrate medially as the pronephros matures. Unlike in the zebrafish, in Xenopus, this migration stops short of complete fusion of the two glomera at the midline, but a nephrin-positive glomeral nexus does form anteriorly and links the two structures from stage 38 onward.
Amino Acid Sequence↗
Lefty proteins are long-range inhibitors of squint-mediated nodal signaling.
The regulation of signaling pathways by feedback inhibitors has become an emerging theme in the control of pattern formation during development. Nodal and Lefty proteins belong to divergent subfamilies of the TGF-beta family. Nodal signals promote mesendoderm induction in vertebrates, and Lefty proteins antagonize it. In zebrafish, Squint functions as a long-range Nodal signal during mesoderm induction. We report that the range over which Squint induces mesoderm is reduced by Lefty proteins. In contrast, the activity range of the short-range Nodal signal Cyclops is not regulated by Lefty activity. We present three lines of evidence that Lefty proteins diminish the range of Squint signaling by acting not only as antagonists of Squint autoregulation but also as long-range inhibitors of Squint activity. First, Lefty can block Nodal signaling at a distance. Second, Lefty regulates the range of Squint signaling before regulating squint expression. Third, Lefty restricts the range of Squint activity in squint mutant embryos, in which the endogenous gene is not subject to autoregulation. We also find that Lefty restricts the response to both high and low levels of Nodal signaling. These results indicate that Lefty proteins restrict the activity range of Nodal signals by dampening Nodal signaling in surrounding cells.
Animals↗
Characterization and expression pattern of zebrafish Anti-Müllerian hormone (Amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development.
The role of Anti-Müllerian hormone (Amh) during gonad development has been studied extensively in mammals, but is less well understood in other vertebrates. In male mammalian embryos, Sox9 activates expression of Amh, which initiates the regression of the Mullerian ducts and inhibits the expression of aromatase (Cyp19a1), the enzyme that converts androgens to estrogens. To better understand shared features of vertebrate gonadogenesis, we cloned amh cDNA from zebrafish, characterized its genomic structure, mapped it, analyzed conserved syntenies, studied its expression pattern in embryos, larvae, juveniles, and adults, and compared it to the expression patterns of sox9a, sox9b and cyp19a1a. We found that the onset of amh expression occurred while gonads were still undifferentiated and sox9a and cyp19a1a were already expressed. In differentiated gonads of juveniles, amh showed a sexually dimorphic expression pattern. In 31 days post-fertilization juveniles, testes expressed amh and sox9a, but not cyp19a1a, while ovaries expressed cyp19a1a and sox9b, but not amh. In adult testes, amh and sox9a were expressed in presumptive Sertoli cells. In adult ovaries, amh and cyp19a1a were expressed in granulosa cells surrounding the oocytes, and sox9b was expressed in a complementary fashion in the ooplasm of oocytes. The observed expression patterns of amh, sox9a, sox9b, and cyp19a1a in zebrafish correspond to the patterns expected if their regulatory interactions have been conserved with mammals. The finding that zebrafish sox9b and sox8 were not co-expressed with amh in oocytes excludes the possibility that amh expression in zebrafish granulosa cells is directly regulated by either of these two genes.
Amino Acid Sequence↗
Heat- and cold-inducible regulation of HSP70 expression in zebrafish ZF4 cells.
Elevated temperature induces a rapid heat shock transcription factor (HSFs)-mediated expression of heat shock (hsp) genes. The effect of cold exposure on hsp gene expression has hardly been investigated, although ectothermic animals experience both cold and heat stress. We have previously shown in zebrafish that the expression of hsf1a and a unique isoform hsf1b vary in a tissue-specific manner upon heat stress. In the current study, using a zebrafish (Danio rerio) embryonic cell line (ZF4), we have compared the effects of heat shock (28-->37 degrees C) vs. cold shock (28-->20 degrees C) on the expression of ahsf1a, zhsf1b and hsp70. Concomitantly, the suitability of the ZF4 cells as a model system was verified. The expression pattern of HSP70 proteins following heat or cold exposure is distinct, and the total HSP70 level is upregulated or stable, respectively. Moreover, heat exposure specifically increases the ratio of zhsf1a/b expression (10-fold), whereas cold exposure decreases it to one half. These data suggest that the zhsf1a/zhsf1b ratio is regulated in a temperature-dependent manner, and the ratio may be indicative of the stressor-specific HSP70 expression. Furthermore, the response in ZF4 cells upon heat shock resembles the response observed in zebrafish liver and thus, supports the use of this cell line in stress response studies.
Animals↗
Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process.
The origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain--especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly "patrolling" for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into "early (amoeboid) microglia": they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier.
Animals↗
Regulation of iro3 expression in the zebrafish spinal cord.
Specification of spinal cord neurons is regulated by several different transcription factors. In this study, we analyze expression and regulation of the transcription factor iro3 in zebrafish spinal cord. In addition to its broad expression in the progenitor domain of intermediate spinal cord, iro3 is also expressed in postmitotic ventral neurons, starting at early somitogenesis stages. Initially, this expression is only in two primary motoneurons, CaP and VaP, but by 24 hr postfertilization, iro3 is expressed by all classes of zebrafish spinal motoneurons as well as by a ventral interneuron called VeLD. iro3 expression in the progenitor domain of intermediate spinal cord is regulated independently from its expression in ventral neurons. Hedgehog (Hh) signaling is unnecessary for iro3 expression in intermediate spinal cord, but it is required to repress iro3 expression in the progenitor domain of ventral spinal cord. We also show that the basic helix-loop-helix transcription factor Olig2 is required for repression of iro3 expression in the progenitor domain of ventral spinal cord. We discuss our findings in the context of previous studies, suggesting that iro3 represses formation of motoneurons and promotes formation of interneurons.
Alleles↗