High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence ... More
Fluorescence imaging of the desorption of dye from fused silica versus silica gel.
AuthorsLudes MD, Anthony SR, Wirth MJ
JournalAnal Chem
PubMed ID12964753
The desorption rate constants for a cationic dye from strong adsorption sites are compared for the same chromatographic interface but for two different substrates, fused silica and chromatographic silica gel. The dye is 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI). The interface consists of acetonitrile and a hydrocarbon monolayer (C8) covalently bound to the ... More
Distance measurements within a concatamer of the plasma membrane Cl?/HCO3? exchanger, AE1.
AuthorsBasu A, Mazor S, Casey JR,
JournalBiochemistry
PubMed ID20828148
'AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl?/HCO3? exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed ... More
Characterization of myenteric interneurons with somatostatin immunoreactivity in the guinea-pig small intestine.
AuthorsSong ZM, Brookes SJ, Ramsay GA, Costa M
JournalNeuroscience
PubMed ID9276502
'The projections, connections, morphology and electrophysiological features of the myenteric interneurons with somatostatin immunoreactivity in the guinea-pig small intestine have been established using retrograde tracing, immunohistochemistry, confocal microscopy and intracellular recording. After application of the fluorescent dye, 1,1''-didodecyl-3,3,3'',3''-tetramethyl indocarbocyanine perchlorate (DiI), to the myenteric plexus, up to 900 nerve cell ... More
Regulation of apoptosis in the endocardial cushions of the developing chick heart.
AuthorsKeyes WM, Sanders EJ
JournalAm J Physiol Cell Physiol
PubMed ID11997250
'During the early stages of heart development, there are two main foci of cell death: outflow tract (OT) and atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We ... More
Measurement and simulation of tailing zones of a cationic dye in analytical-scale reversed phase chromatography.
AuthorsWirth MJ, Smith EA, Anthony SR
JournalJ Chromatogr A
PubMed ID15116915
'A quantitative physical description of tailing is reported here for analytical-scale reversed phase chromatography with Type B silica. Simulations of experimental chromatograms for a cationic dye, 1,1''-didodecyl-3,3,3''3''-tetramethylindocarbocyanine perchlorate (DiI) were performed as a function of DiI concentration and flow rate, revealing nonlinear tailing due to a bi-Langmuir adsorption isotherm. The ... More
Cell death-associated translocation of plasma membrane components induced by CTL.
AuthorsKawasaki Y, Saito T, Shirota-Someya Y, Ikegami Y, Komano H, Lee MH, Froelich CJ, Shinohara N, Takayama H
JournalJ Immunol
PubMed ID10779768
'In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridin ium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated ... More
Intrinsic innervation patterns of the smooth muscle in the rumen and reticulum of lambs.
AuthorsPfannkuche H, Schellhorn C, Schemann M, Gäbel G
JournalJ Anat
PubMed ID15061755
'The rumen and reticulum of sheep serve as a fermentation chamber. Both compartments exhibit specific motility patterns. With developmental changes, the size of the reticulorumen dramatically increases when newborn lambs mature to adult sheep. This makes it possible to investigate the intrinsic innervation of the reticuloruminal muscles in lambs by ... More
Mucosal projections of enteric neurons in the porcine small intestine.
AuthorsHens J, Schrödl F, Brehmer A, Adriaensen D, Neuhuber W, Scheuermann DW, Schemann M, Timmermans JP
JournalJ Comp Neurol
PubMed ID10813797
'In the present study, a combination of immunohistochemistry and retrograde 1,1;-didodecyl-3,3,3;,3;-tetramethylindocarbocyanine perchlorate (DiI) tracing was used to unravel the morphology, distribution, and neurochemical coding of submucous and myenteric neurons with axonal projections to the mucosa of the porcine small intestine. The majority of traced neurons was located in the inner ... More
Carbocyanine dyes with long alkyl side-chains: broad spectrum inhibitors of mitochondrial electron transport chain activity.
AuthorsAnderson WM, Trgovcich-Zacok D
JournalBiochem Pharmacol
PubMed ID7763312
'Certain indocarbocyanine, thiacarbocyanine, and oxacarbocyanine dyes possessing short alkyl side-chains (one to five carbons) are potent inhibitors of mammalian mitochondrial NADH-ubiquinone reductase (EC 1.6.99.3) activity (Anderson et al., Biochem Pharmacol 41: 677-684, 1991; Anderson et al., Biochem Pharmacol 45: 691-696, 1993; Anderson et al., Biochem Pharmacol 45: 2115-2122, 1993), and ... More
Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2.
AuthorsKim YJ, Kim KP, Han SK, Munoz NM, Zhu X, Sano H, Leff AR, Cho W
JournalJ Biol Chem
PubMed ID12124392
'We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. ... More
Probing the dynamic guest-host interactions in sol-gel films using single molecule spectroscopy.
AuthorsViteri CR, Gilliland JW, Yip WT
JournalJ Am Chem Soc
PubMed ID12580626
'Organic dyes usually exhibit enhanced photostability when trapped inside sol-gel silicates. The enhanced photostability is attributed to the reduction of intramolecular motions that facilitate photodegradation. We report the simultaneous detection of mobility and photostability of sol-gel encapsulated didodecyl-3,3,3'',3''-tetramethylindocarbocyanine (DiI) using single molecule spectroscopy. Fluorescence from DiI was resolved into parallel ... More
Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine.
AuthorsSong ZM, Costa M, Brookes SJ
JournalJ Comp Neurol
PubMed ID9721907
'The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1''-didodecyl-3,3,3'',3''-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of ... More
Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites.
AuthorsZakharenko S, Popov S
JournalJ Cell Biol
PubMed ID9817763
'Nerve growth depends on the delivery of cell body-synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body- derived vesicles ... More
The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria.
AuthorsFreedman JC, Hoffman JF
JournalJ Gen Physiol
PubMed ID39969
'The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc ... More
Toxicity of "DiI" for embryonic rat motoneurons and sensory neurons in vitro.
AuthorsSt John PA
JournalLife Sci
PubMed ID1749311
'The carbocyanine dye DiIC18(3) ("DiI") is commonly used for both anterograde and retrograde labeling of neurons, including live neurons in situ and in vitro. In the present experiments, DiIC18(3) was used to label motoneurons in the spinal cords and sensory neurons in the dorsal root ganglia of embryonic rats. When ... More
Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes.
AuthorsSpink CH, Yeager MD, Feigenson GW
JournalBiochim Biophys Acta
PubMed ID2317494
'Gel-fluid partition coefficients, Kp, were measured for a series of indocarbocyanine dyes in multilamellar lipid vesicles. The dyes examined had alkyl chain lengths from 12 to 22 carbons. Fluorescence quenching by a spin-labeled phosphatidylcholine-enriched fluid phase created a large difference in quantum yield for indocarbocyanine fluorescence between fluid and gel ... More
Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers.
AuthorsPackard BS, Wolf DE
JournalBiochemistry
PubMed ID4074686
'The fluorescence lifetimes for the 1,1''-dialkyl-3,3,3'',3''-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not ... More
Changes in the organization of the sea urchin egg plasma membrane upon fertilization: indications from the lateral diffusion rates of lipid-soluble fluorescent dyes.
AuthorsWolf DE, Kinsey W, Lennarz W, Edidin M
JournalDev Biol
PubMed ID7461282
Changes in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: indications from the lateral diffusion rates of fluorescent lipid analogs.
AuthorsWolf DE, Edidin M, Handyside AH
JournalDev Biol
PubMed ID7250512
Calorimetric investigation of the phase partitioning of the fluorescent carbocyanine probes in phosphatidylcholine bilayers.
AuthorsEthier MF, Wolf DE, Melchior DL
JournalBiochemistry
PubMed ID6838845
Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell.
AuthorsStruck DK, Pagano RE
JournalJ Biol Chem
PubMed ID7372642
Forward plasma membrane flow in growing nerve processes.
AuthorsPopov S, Brown A, Poo MM
JournalScience
PubMed ID7678471
Nerve growth requires addition of new plasma membrane material, which is generally believed to occur at the growth cone. Local incorporation of a fluorescent lipid analog into the plasma membrane of fast-growing Xenopus neurites revealed an anterograde bulk membrane flow that correlated with neurite elongation. The rate of membrane flow ... More
The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation.
AuthorsChernomordik LV, Frolov VA, Leikina E, Bronk P, Zimmerberg J
JournalJ Cell Biol
PubMed ID9508770
The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes ... More
Assessment of fluorochromes for cellular structure and function studies by flow cytometry.
AuthorsPetit JM, Denis-Gay M, Ratinaud MH
JournalBiol Cell
PubMed ID7693118
Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, ... More
Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN) was identified by positional cloning. The predicted protein is 1432 amino acids in ... More
Thermodynamics of transfer of indocarbocyanines from gel to fluid phases of phospholipid bilayers.
AuthorsSpink CH, Clouser D, O'Neil J
JournalBiochim Biophys Acta
PubMed ID8155672
Application of the regular solution model to thermal transition data obtained by differential scanning calorimetry has allowed the determination of partition coefficients, Kp, and the thermodynamics of transfer of a series of indocarbocyanine solutes between the gel and fluid phases of phospholipid bilayers. The indocarbocyanines with alkyl chain lengths of ... More
Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes.
AuthorsSchwille P, Korlach J, Webb WW
JournalCytometry
PubMed ID10404965
We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in ... More
Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa.
AuthorsWolf DE, Lipscomb AC, Maynard VM
JournalBiochemistry
PubMed ID2835079
In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique ... More
Differential Ca(2+) signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture.
Physiological studies on functionally identified myenteric neurons are scarce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca(2+) concentration ([Ca(2+)](i)) signaling to study excitatory neurotransmitter responsiveness of myenteric motor neurons. 1, 1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea ... More
Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.
The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering ... More
Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes.
AuthorsTerasaki M, Reese TS
JournalJ Cell Sci
PubMed ID1378452
The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the ... More
Endocytic recycling compartments altered in cisplatin-resistant cancer cells.
AuthorsLiang XJ, Mukherjee S, Shen DW, Maxfield FR, Gottesman MM
JournalCancer Res
PubMed ID16489040
The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling ... More
Selectivity of fluorescent lipid analogues for lipid domains.
AuthorsKlausner RD, Wolf DE
JournalBiochemistry
PubMed ID7470460
We have examined the phase partition preferences of the even chain length (n = 10-22) diacyl-3'3'-indocarbo-cyanine iodides (Cn diI) incorporated in disaturated lecithin (PC) vesicles. Two parameters were used to determine this phase preference: (i) the direction of shift of the phase transition temperature (Tm) induced by the dyes and ... More
Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents.
AuthorsFeder TJ, Chang EY, Holowka D, Webb WW
JournalJ Cell Physiol
PubMed ID8263030
The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)-receptor complexes on the surface ... More
Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane.
AuthorsVrljic M, Nishimura SY, Moerner WE, McConnell HM
JournalBiophys J
PubMed ID15516525
Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native ... More
Partition of membrane probes in a gel/fluid two-component lipid system: a fluorescence resonance energy transfer study.
AuthorsLoura LM, Fedorov A, Prieto M
JournalBiochim Biophys Acta
PubMed ID10930513
A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanol amine, and a short-tailed FRET acceptor, 1,1'-didodecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC12(3)), were shown to prefer the fluid ... More
Characteristics of mucosally projecting myenteric neurones in the guinea-pig proximal colon.
AuthorsNeunlist M, Dobreva G, Schemann M
JournalJ Physiol
PubMed ID10332100
1. Using retrograde tracing with 1,1'-didodecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) in combination with electrophysiological and immunohistochemical techniques we determined the properties of the putative intrinsic primary afferent myenteric neurones with mucosal projections in the guinea-pig proximal colon. 2. Eighty-four out of eighty-five DiI-labelled myenteric neurones were AH neurones with a late ... More
Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function.
A number of supravital fluorochromes are available to study leucocyte functions in vitro and in vivo. The fluorescein ester most widely used, fluorescein diacetate, has the disadvantage of rapid cellular efflux, whereas more recently developed fluorescent probes do not exhibit this inconvenient trait. However, their effect on cellular functions has ... More
Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation.
AuthorsMcCurdy LH, Graham BS
JournalJ Virol
PubMed ID12525608
The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of ... More
Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails.
AuthorsMukherjee S, Soe TT, Maxfield FR
JournalJ Cell Biol
PubMed ID10087269
To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in ... More
The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains.
AuthorsScher MG, Bloch RJ
JournalExp Cell Res
PubMed ID2055278
We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to ... More
Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.
AuthorsSchootemeijer A, Van Beekhuizen AE, Tertoolen LG, De Laat SW, Akkerman JW
JournalEur J Biochem
PubMed ID7925356
The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) ... More
Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy.
AuthorsCrane JM, Kiessling V, Tamm LK
JournalLangmuir
PubMed ID15697284
There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta ... More
Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones.
AuthorsPfannkuche H, Schemann M, Gäbel G
JournalCell Tissue Res
PubMed ID12195290
The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways ... More
Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity.
AuthorsButler PJ, Norwich G, Weinbaum S, Chien S
JournalAm J Physiol Cell Physiol
PubMed ID11245613
Blood flow-associated shear stress may modulate cellular processes through its action on the plasma membrane. We quantified the spatial and temporal aspects of the effects of shear stress (tau) on the lipid fluidity of 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16)(13)]-stained plasma membranes of bovine aortic endothelial cells in a flow chamber. A confocal ... More
Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording.
AuthorsGelperin A, Flores J
JournalJ Neurosci Methods
PubMed ID9128173
A versatile technique for dye application in living tissue is described, which results in labeling of viable cells from which electrophysiological or optical recordings can be obtained. The dye-coated surface of a glass microelectrode tip is used to apply anatomical tracers or calcium sensitive probes with spatial precision. A total ... More
Morphological identification of thoracolumbar spinal afferent nerve endings in mouse uterus.
Authors
JournalJ Comp Neurol
PubMed ID33190293
Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence.
Authors
JournalCell Rep
PubMed ID34320359
A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division.
Authors
JournalNat Commun
PubMed ID34836941
The Ciliopathy Gene Ftm/Rpgrip1l Controls Mouse Forebrain Patterning via Region-Specific Modulation of Hedgehog/Gli Signaling.
Authors
JournalJ Neurosci
PubMed ID30692221
The role of primary cilia in corpus callosum formation is mediated by production of the Gli3 repressor.