Learn more about Amazon Prime. This simple book will help you recognize and tune-in to your potential. It can be opened to any page for a sound-bite of insight. People say it's like having a good friend by their side. Fine Tuning is the art of changing with the times. Instead of getting mired down with regrets, habits and stress, you learn to tune in to keep your mind open to the flow of living. Fine Tuning is using the natural gifts of our senses to connect with what we want, need and must deal with, in order to get results in our lives that make it "feel right" in the present.
Of course the vast majority of my training group will take the safe million. It would therefore be interesting to know whether a Dll1 phenotype also phenocopies the Fng and Jag2 results. To ignite law of attraction to work in the real world, one thing you may need to do is to create multiple channels of how you do things. He has to discover who he really is and what his superpowers can be. The holes in a cartridge body, meant to accept the screws which affix the cartridge to the headshell, generally come in two flavors:
Thinking with your senses connects with your passion for being alive. It reduces stress, helps you know yourself better and guides you to make connections that are rewarding. Open to any page and fine tune with simple reflections and insights that put you in sync with yourself. Fine Tuning, Connecting with Your Inner Power is full of quotes, insights and suggestions that open your mind to tune in to your intuition. This book will guide you through changes so that you come out feeling good. Have you ever had a feeling that someone was watching you and turned around to find it true?
Have you ever had a feeling that something was terribly wrong, and it was? The advantages are endless! Read Fine Tuning and feel the buzz. It's proven to bring satisfaction and peace of mind. Prior to playing, the musician tightens the bow by turning a screw to pull the frog the part of the bow under the hand back, and increase the tension of the hair.
Rosin is applied by the player to make the hairs sticky. Bows need to be re-haired periodically. Baroque style — cello bows were much thicker and were formed with a larger outward arch when compared to modern cello bows. The inward arch of a modern cello bow produces greater tension, which in turn gives off a louder sound. The cello bow has also been used to play electric guitars. Jimmy Page pioneered its application on tracks such as " Dazed and Confused ". This curved bow BACH. Bow is a convex curved bow which, unlike the ordinary bow, renders possible polyphonic playing on the various strings of the instrument.
The solo repertoire for violin and cello by J. Bow is particularly suited to it: When a string is bowed or plucked, it vibrates and moves the air around it, producing sound waves. Because the string is quite thin, not much air is moved by the string itself, and consequently if the string was not mounted on a hollow body, the sound would be weak.
In acoustic stringed instruments such as the cello, this lack of volume is solved by mounting the vibrating string on a larger hollow wooden body. The vibrations are transmitted to the larger body, which can move more air and produce a louder sound. Tightening a string stiffens it by increasing both the outward forces along its length and the net forces it experiences during a distortion.
Shortening a string stiffens it by increasing its curvature during a distortion and subjecting it to larger net forces. Shortening the string also reduces its mass, but does not alter the mass per unit length, and it is the latter ratio rather than the total mass which governs the frequency. Thus shortening a string increases the frequency, and thus the pitch.
Editorial Reviews. About the Author. Jane Bernard lives in New York City. Degrees in Fine Tuning: Connecting With Your Inner Power 2nd Edition (Fine Tuning Series Book 1) - Kindle edition by Jane Bernard. Download it once and read it on. Fine Tuning: Connecting With Your Inner Power 2nd Edition [Jane Bernard] on This simple book will help you recognize and tune-in to your potential. exclusive access to music, movies, TV shows, original audio series, and Kindle books. .. with Kindle Unlimited to also enjoy access to over 1 million more titles $ to.
This is a prime reason why the different strings on all string instruments have different fundamental pitches, with the lightest strings having the highest pitches. The wood resonance appears to be split into two frequencies by the driving force of the sounding string. These two periodic resonances beat with each other.
This wolf tone must be eliminated or significantly reduced for the cello to play the nearby notes with a pleasant tone. This can be accomplished by modifying the cello front plate, attaching a wolf eliminator a metal cylinder or a rubber cylinder encased in metal , or moving the sound post. When a string is bowed or plucked to produce a note, the fundamental note is accompanied by higher frequency overtones. Each sound has a particular recipe of frequencies that combine to make the total sound.
Playing the cello is done while seated with the instrument supported on the floor by the endpin. The left hand fingertips stop the strings on the fingerboard, determining the pitch of the fingered note. The right hand plucks or bows the strings to sound the notes. The left hand fingertips stop the strings along their length, determining the pitch of each fingered note. Stopping the string closer to the bridge results in higher-pitched sound, because the vibrating string length has been shortened.
In the neck positions which use just less than half of the fingerboard, nearest the top of the instrument , the thumb rests on the back of the neck; in thumb position a general name for notes on the remainder of the fingerboard the thumb usually rests alongside the fingers on the string and the side of the thumb is used to play notes. The fingers are normally held curved with each knuckle bent, with the fingertips in contact with the string.
If a finger is required on two or more strings at once to play perfect fifths in double stops or chords it is used flat. In slower, or more expressive playing, the contact point can move slightly away from the nail to the pad of the finger, allowing a fuller vibrato. Vibrato is a small oscillation in the pitch of a note, usually considered expressive.
Harmonics played on the cello fall into two classes; natural and artificial. Natural harmonics are produced by lightly touching but not depressing the string with the finger at certain places, and then bowing or, rarely, plucking the string. For example, the halfway point of the string will produce a harmonic that is one octave above the unfingered open string.
Natural harmonics only produce notes that are part of the harmonic series on a particular string. Artificial harmonics also called false harmonics or stopped harmonics , in which the player depresses the string fully with one finger while touching the same string lightly with another finger, can produce any note above middle C. Glissando Italian for "sliding" is an effect played by sliding the finger up or down the fingerboard without releasing the string.
This causes the pitch to rise and fall smoothly, without separate, discernible steps. In cello playing, the bow is much like the breath of a wind instrument player. Arguably, it is the major determinant in the expressiveness of the playing. The right hand holds the bow and controls the duration and character of the notes. The bow is drawn across the strings roughly halfway between the end of the fingerboard and the bridge, in a direction perpendicular to the strings. The bow is held with all five fingers of the right hand, the thumb opposite the fingers and closer to the cellist's body.
Tone production and volume of sound depend on a combination of several factors. The three most important ones are: Double stops involve the playing of two notes at the same time. Two strings are fingered simultaneously, and the bow is drawn so as to sound them both at once. In pizzicato playing, the string is plucked directly with the fingers or thumb. Pizzicato is often abbreviated as "Pizz. Position of the hand is slightly over the finger board and away from the bridge. A player using the col legno technique strikes or rubs the strings with the wood of the bow rather than the hair.
In spiccato playing, the strings are not "drawn" by the bow hair but struck by it, while still retaining some horizontal motion, to generate a more percussive, crisp sound. In staccato , the player moves the bow a small distance and stops it on the string, making a short sound, the rest of the written duration being taken up by silence. Legato is a technique where the notes are smoothly connected without accents or breaks. It is noted by a slur curved line above or below — depending on their position on the staff — the notes of the passage that is to be played legato.
Sul ponticello "on the bridge" refers to bowing closer to the bridge, while sul tasto "on the fingerboard" calls for bowing nearer the end of the fingerboard. Sul tasto produces a more flute-like sound, with more emphasis on the fundamental frequency of the note, and softer overtones. The smaller cellos are identical to standard cellos in construction, range, and usage, but are simply scaled-down for the benefit of children and shorter adults.
Cellos made before approximately tended to be considerably larger than those made and commonly played today. Around , changes in string-making technology made it possible to play lower-pitched notes on shorter strings. The cellos of Stradivari , for example, can be clearly divided into two models: This later model is the design most commonly used by modern luthiers. The new size offered fuller tonal projection and greater range of expression. The instrument in this form was able to contribute to more pieces musically and offered the possibility of greater physical dexterity for the player to develop technique.
Cellos are made by luthiers , specialists in building and repairing stringed instruments, ranging from guitars to violins. The following luthiers are notable for the cellos they have produced:. A person who plays the cello is called a cellist. For a list of notable cellists, see the list of cellists and Category: Careers in cello vary widely by genre and by region or country.
Most cellists earn their living from a mixture of performance and teaching jobs. The first step to getting most performance jobs is by playing at an audition. In some styles of music, cellists may be asked to sight read printed music or perform standard repertoire with an ensemble. In classical music, cellists audition for playing jobs in orchestras and for admission into university or Conservatory programs or degrees.
At a classical cello audition, the performer typically plays a movement from a Bach suite or a movement from a concerto and a variety of excerpts from the orchestral literature. Orchestral auditions are typically held in front of a panel that includes the conductor , the Concertmaster , the Principal cellist and other principal players. The most promising candidates are invited to return for a second or third round of auditions, which allows the conductor and the panel to compare the best candidates.
Performers may be asked to sight read orchestral music. The final stage of the audition process in some orchestras is a test week , in which the performer plays with the orchestra for a week or two, which allows the conductor and principal players to see if the individual can function well in an actual performance setting. Performance jobs include playing as a freelancer in small groups, playing in a chamber music group, large ensembles, or performing solo music, either live onstage or as a session player for radio or TV broadcasts or for recordings; and working as the employee of an orchestra, big band, or recording studio.
Many cello players find extra work by substituting "subbing" for cellists who are double-booked or ill. It is hard for many cello players to be able to find full-time, full-year work at a single job. About the closest that a cellist can come to this is in the case of those who win an audition at a professional orchestra. Even full-time orchestra jobs do not usually last for the entire year. When the orchestra stops playing which is often in the summer , orchestral cellists have to find other work, either as a teacher or coach, or in another group. Teaching work for cellists includes giving private lessons in the home or at colleges and universities; coaching cellists who are preparing for recordings or auditions; doing group coaching at music camps or for youth ensembles; and working as a high school music teacher.
Due to the limited number of full-time orchestral jobs, many classical cellists are not able to find full-time work with a single orchestra. Some cellists increase their employ-ability by learning several different styles, such as folk or pop. In some cases, cellists supplement their performing and teaching income with other related music jobs, such as working as a stringed instrument repairer luthier ; as a contractor who hires musicians for orchestras or big bands, composing music, songwriting, conducting, or organizing festivals e.
Specific instruments are famous or become famous for a variety of reasons. An instrument's notability may arise from its age, the fame of its maker, its physical appearance, its acoustic properties, and its use by notable performers. The most famous instruments are generally known for all of these things.
The most highly prized instruments are now collector's items, and are priced beyond the reach of most musicians. These instruments are typically owned by some kind of organization or investment group, which may loan the instrument to a notable performer. For example, the Davidov Stradivarius , which is currently in the possession of one of the most widely known living cellists, Yo-Yo Ma , is actually owned by the Vuitton Foundation. From Wikipedia, the free encyclopedia. This article is about the stringed musical instrument.
For other uses, see Cello disambiguation. Cello, front and side view. The endpin at the bottom is retracted or removed for easier storage and transportation, and adjusted for height in accordance to the player. This section does not cite any sources. Please help improve this section by adding citations to reliable sources. The following individual involved in review of your submission has agreed to reveal his identity: Fernando Giraldez Reviewer 2.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. This is an interesting study that addresses the role of Fringe family proteins in regulating Notch signalling in the developing organ of Corti of the mammalian inner ear.
Notch signalling is known to pattern the organ of Corti, and Fringe proteins are also known for their role in fine-tuning Notch signalling in other systems. The new finding here is that regulation of Notch signalling by Fringe plays a role in the generation of inner hair cells and their associated supporting cells, the inner phalangeal cells, in the cochlear duct. A surprising result is that a partial loss of Notch signalling results in both supernumerary hair cells and supernumerary supporting cells. This differs from the classical model for lateral inhibition in the developing cochlea, where a disruption of Notch signalling is predicted to result in supernumerary hair cells at the expense of supporting cells.
The data are clear and well-presented, with careful and appropriate quantitation, use of controls and statistical analysis. All three reviewers were positive towards the work and found it interesting, novel and potentially important. They praise the careful approach and high quality of the data presented. However, there was consensus that the manuscript needed revision in places, especially with respect to the focus on cis-inhibition. The revisions must therefore include:. Further experimental support for the claim that Notch signalling activity levels differ both over time and in different cell types in the developing cochlear duct is required see comments from reviewers 2 and 3.
If this cannot be provided, the argument for cis-inhibition should be presented as an untested model and the overall focus on cis-inhibition in the manuscript should be toned down. It would therefore be interesting to know whether a Dll1 phenotype also phenocopies the Fng and Jag2 results. I appreciate that this may be beyond the scope of the current study, but would the authors predict a similar or a different result? Can the authors provide more information as to why they do not think this is the case?
Are there more cells overall, or are they packed in a different way due to altered shape of the organ? For example, the following statement is made in the Introduction: If Fng increases cis-inhibition between N and Dl later in the aforementioned paragraph , given the information presented, that should lead to a decrease in Notch signalling activity in the Notch-expressing cell. I appreciate that the terms 'lateral inhibition' and 'cis-inhibition' are now entrenched in the literature, but I, for one, find the explanations above contradictory and feel that more clarification is needed.
If the authors can make sure that their text is completely unambiguous that would be helpful. If Fng potentiates Dl-N cis-inhibitory interaction, but attenuates Jag-N cis-inhibitory interaction, the dotted arrow from Fng to Jag-N in the green cell should be drawn as an inhibitory line - , not an arrow, and the cis-inhibitory interactions between Dl and N should be stronger thicker lines in the green cell than those in the white cell, whereas the diagram shows the opposite. These apparent discrepancies make it hard to follow the argument.
If the sentence refers to the neighbouring cells, the 'these' is ambiguous, and the sentence does not follow logically from the previous one. If the authors mean that the green cells have responded to hair cell-inducing signals, but have not yet received instruction to undergo final differentiation into hair cells as in C , this needs clarifying further. The paper by Basch et al. The main and certainly interesting aspect of the work is the disclosure of a very specific function of Fng proteins in cochlear development and the exploration of their association with Notch signalling.
The experiments are extremely careful and beautifully presented, and there is no doubt about the consistency of the various phenotypes and treatments. Data are of high quality and involve a variety of approaches including lineage tracing, phenotypic analysis, organ culture, etc. The authors propose an interesting model that involves Notch activity levels and cis- and trans- modes of ligand operation to accommodate the results and with those of the literature. However, it is here where the work requires some improvement. Although the expression patterns are overlapping, they are quite dissimilar.
This suggests that modulation of Notch is different in the two cell types. Could it be that Lfng and Mfng have different effects on different ligands? Is it possible that Lfng maintains a low and constant Notch activity and Mfng favours Dll signalling? However, the correlation with Notch signalling levels remains obscure. Jag1 is expressed all throughout prosensory development and in supporting cells, but Jag2 is expressed along with Dll1 only in hair cells. How this is matched?
Those are interesting experiments and show that almost the complete sensory epithelium derives from Lfng expressing progenitors probably also from Jag1-positive ones. However, genetic tracing shows the common origin of the sensory epithelium, but not much about the restricted expression. In this view, the genetic tracing of Manic cells would have been of interest, because it seems that the uniqueness of the effects may come from the co-expression. That is the main line of the argument along the paper.
But from here, I would expect the authors to consider how this could happen by showing the effects of Fng on ligands or on Notch activity would have been informative. Neither it is clear to me whether the levels of Notch affect the expression of modulators or vice versa. We do not really know how Notch operates outside lateral inhibition and all possibilities should remain open.
But nevertheless, the different conditions generated by the experiments in the paper open some questions about what are the levels of Notch in the different regions of the sensory epithelium. In this sense, conditions are not strictly comparable. Can this be assessed specifically for Fng mutants? Please comment on that. And it shows the differential sensitivity of inner and outer hair cells to Notch. This is in good correspondence with MAMl and Pofut experiments. But what about in Fng mutants? The experiments say little about cis-inhibition.
Is there a good evidence for cis-inhibition in ear sensory cells? The results from Daudet's lab indicate rather the opposite for Dll1. The argument on cis-inhibition continues in the second and third paragraphs of the Discussion, quoting LeBon as if an ear paper…: However, the experiments do not provide any evidence of that occurring in the ear…. In summary, the results are indeed sound and of general interest, but as to the model, it is unclear that the function of Fng on Notch and cis-inhibition are sufficiently substantiated.
Leaving aside cis-inhibition, which actually I see out of the paper, the model still needs a link between Fng, ligands and Notch in the cochlea. Something that may give a hint of what are the effects of Fng on Notch, Notch ligands or Fng. The main evidence for the model is correlative, the parallel between phenotypes. The main question, in my view, is what is the connection between both. The organ of Corti is characterized by strikingly precise rows of hair cells, although how this precision is established is not known. This manuscript by Basch et al.
The authors show that Lfng and Mng are initially precisely positioned at the location of the first inner hair cell row. In their absence, multiple rows form, along with a duplication of supporting cells. By comparing the results of minor reductions in Notch signaling to more robust loss of Notch, the authors suggest that establishment of the initial row of hair cells is inherently different in that it requires lower levels of Notch signaling, and is therefore more susceptible to mild reductions in Notch signaling.
Overall the data is well-performed and presented, and the authors analyze quite a number of different Notch mutants to determine differences.
The authors put forward an interesting hypothesis as to why the hair cell genesis is initially restricted to a single row. However, it is not clearly established that a lower level of Notch signaling is required for boundary formation — while milder defects in Notch signaling may first lead to an expansion of the sensory domain, it may be that the non-sensory regions or supporting cells outside the hair cell regions are simply more sensitive to loss of Notch signaling, and thus boundary formation is lost first.
The authors suggest that cis-inhibition mediated by Lfng and Mfng expression results in moderate levels of Notch activation in nearby Kolliker's cells Figure 6B , and that this is important for setting up the boundary. However, in this case the prediction would be that deleting both fringes would lead to higher levels of Notch, as cis-inhibition would be largely relieved. However, this does not seem to be the case, as new hair cells develop, suggesting Notch activity is reduced in their absence. Since the model indicates that moderate levels of Notch activity are important for setting up this boundary, it would be important to show directly how levels of Notch are regulated at the boundary.
It is not clear that severe loss of Notch signaling shows an inherently different phenotype than milder forms.
Is it not more likely that the severe loss obscures the initial milder phenotype? After all, there is a dramatic loss of boundary formation in the severe cases of loss of Notch signaling. While additional supporting cells are present in the milder cases, these may be induced by the excess hair cells, which themselves also convert to hair cells in the case of more severe reductions in Notch. If the regulation of inner hair cell formation to a single row is not via lateral inhibition, what type of signaling is it?
It would seem that even at the proposed lower levels the cell expressing the ligand is inhibiting nearby cells in Kolliker's organ from adopting the hair cell fate, and therefore this would fit the definition of lateral inhibition see Figure 6B. The authors show that Lfng likely initially marks the inner hair cell using fate-mapping, although given that they have reporters for both Lfng and Atoh1-it seems relatively straightforward to show that these both overlap during differentiation, further strengthening the hypothesis that Lfng and Mfng mark the inner hair cell boundary. It would also be interesting to look at the onset of these factors-Does Lfng mark the inner hair cell prior to Atoh1?
The suggestion that Lfng modulates Jag1 levels initially Figure 6A has not been demonstrated previously-is there any evidence of this from the Lfng loss of function? If not this part of the model should be modified.
It may be helpful to explain how we came to think about cis-inhibition of Notch signaling in our study. We had spent a number of years characterizing the cochlear phenotypes of a number of different Notch mutants. In mutant after mutant, we kept observing a duplication of inner hair cells AND inner phalangeal cells, which did not fit in with the standard lateral inhibition model of Notch signaling that distinguishes between hair cells and supporting cells. The common thread in all our mutants was that they reduced, but did not completely abolish, Notch signaling.
We were also able to confirm this phenotype in culture by quantitatively reducing Notch signaling with small doses of different Notch inhibitors. Our results are consistent with a model in which a column of differentiating progenitor cells that express Atoh1, Jag1, Jag2 and Dll1 is induced at the boundary of the future organ of Corti. Thus, deleting Notch ligands, or genes that are required for receiving Notch signals reduces this inhibitory signal and causes a second column of cells to form, duplicating both the inner hair cells and the inner phalangeal cells.
In the course of this work, we realized that this column of boundary cells was transiently marked by expression of Lfng and Mfng. We verified this by fate mapping with our Lfng-CreER mice. We have now also examined activated Notch protein levels and find reduced Notch signaling in these double mutants. Our Lfng and Mfng expression data and double knockout results are therefore consistent with Lfng and Mfng acting in this column of cells to promote Notch signaling from this column to adjacent cells.
In principle, there are two mechanisms by which Fringe proteins can regulate the amount of Notch signal delivered by a cell. One is direct glycosylation of Notch ligands by Fringe, rather than Notch receptors. Although this has been shown to occur in vitro, there is very little published data on the biological relevance of these modifications, and what data there is suggests that Fringe modification of the ligands is not necessary for their function.
The second mechanism is the regulation of cis-inhibition by Fringe proteins between Notch receptors and ligands in the same cell, which reduces the activity of both receptor and ligand. The best evidence for cis-inhibition and the role of Fringe proteins in this mechanism comes from Drosophila genetics and cell line studies rather than from in vivo vertebrate studies, but the phenomenon is nevertheless well accepted in the literature. There is no reliable marker to detect cis-inhibition, as it is simply an attenuation of Notch signaling under certain circumstances.
We therefore stress that we are invoking cis-inhibition as an explanation for our experimental data, rather than observing it directly. We have tried to explain the phenomenon of cis-inhibition more clearly in the revised manuscript. Finally, in recognition of the fact that much of our data on loss of function mutants does not involve Fringe activity directly, we have revised the title of the paper to focus more on Notch signaling and less on Fringe function.
We have now added data showing how levels of Notch signaling change in Lfng;Mfng double knockouts and how this is reflected by changes in cell fate only at the border of the organ of Corti. We hope that this data, together with the demonstration from multiple mouse knockouts and culture experiments that different regions of the cochlea are differentially affected by reduction of Notch signaling demonstrates that Notch activity differs in different regions of the cochlea. We would emphasize that there is no specific method of detecting cis-inhibition, other than observing changes in the strength of Notch signaling after manipulations that are predicted to increase or reduce cis-inhibition.
We have shown that Lfng, Atoh1 and Mfng co-localize transiently in the cochlea and that some Lfng-expressing cells go on to form inner hair cells by using Lfng-CreER mice. These events happen before more mature hair cell markers such as Myosin7a are expressed, and so we feel this is the best data we can propose in support of this argument. We discuss this issue further in our response to reviewer 3, below.
We have revised the manuscript to try and resolve this confusion. Current evidence strongly suggests that Jag1 acts like Serrate in flies: Fringe modification tends to attenuate Jag1 signaling. This was shown most recently and in greatest detail by a recent cell line study in eLife:. However, there are almost no published data on the effects of Fringe proteins on Jag2 signaling. Van de Walle et al. Although we cited this for the sake of completeness, the potentiation of Jag2 signaling by Lfng observed by the authors in this study, although statistically significant, was extremely small — an increase in signaling of just 1.
We have therefore decided to remove this citation from the paper. Moreover, unpublished data recently communicated to us by the Elowitz lab the authors of the eLife paper above suggests that Lfng and Mfng modification of Notch receptors attenuates both Jag1 and Jag2 signaling. The published work of Kiernan et al. However, as the reviewer notes, we do not have access to the Dll1 conditional allele, and so we cannot perform this study in a timely fashion. Nevertheless, we agree that it is an excellent experiment, and we have included our prediction about the Dll1 conditional phenotype in the revision.
Our data on the expression of Lfng and Mfng shown in Figure 1 suggests that Mfng appears in hair cell progenitors at approximately the same time as Atoh1. Thus, there is only one time and place at which the expression of these two Fringe proteins occurs in the same cell type — exactly at the boundary of the organ of Corti as the first Atoh1-expressing hair cell progenitors. It is here that we observe the phenotype reported in our paper. We therefore believe that Lfng and Mfng have redundant functions at this boundary, such that loss of either Fringe gene does not alter the patterning of the boundary.
We do not know why knockouts of either gene alone show no obvious patterning phenotype in other regions of the organ of Corti, but presumably this is because changing Notch signaling by small amounts has no effect elsewhere. That said, it should be noted that we have not examined adult Lfng or Mfng mutant mice, and it is possible that they have subtle phenotypes, such as premature hair cell loss or abnormal hearing thresholds.
We have tried to emphasize these points better in the revised manuscript. We noted in the text that work by Gridley and Kelley shows that mutation of Lfng can rescue the inner hair cell phenotype of Jag2 mutants, but not the outer hair cell phenotype. This again reinforces the unique sensitivity of the boundary region to Notch signaling.
Our results predict that Mfng mutants would rescue Jag2 mutants in a similar manner to Lfng mutants, and we have added this prediction to the text. The reason we believe the column of future inner hair cells and inner phalangeal cells is inhibiting their neighbors though the Notch pathway is that when we reduce but not abolish Notch signaling, either by deleting Notch ligands or Notch signaling partners we see an extra row of inner hair cells and inner phalangeal cells. This seems to represent an inhibitory, rather than an inductive interaction.
We have observed no significant differences in cochlear length in any of our mutants. We do see more organ of Corti cells in all our mutants, but this is due to the extra row of inner hair cells and inner phalangeal cells seen when we reduce Notch signaling. We also observe some lateral inhibition defects in the Notch mutants that change the proportion of hair cells and supporting cells, some of which have been previously published e.
Jag1 heterozygotes and Jag2 nulls. To clarify one point for the reviewer in the opening sentence of point 5 , lateral inhibition is the inhibition of cell fate that is delivered from one cell to a neighbor. Thus, neurons deploy Notch ligands to inhibit neural progenitors from adopting a neuronal fate, hair cells deploy Notch ligands to inhibit supporting cells from adopting a hair cell fate and so forth. Cis-inhibition, on the other hand, is a protein-protein interaction that occurs when a Notch receptor and a ligand are expressed in the same cell and interact with each other. This interaction reduces the amount of free receptor and ligand in the cell, thus lowering the ability of the cell to both send and receive Notch signals.
The amount of cis-inhibition that occurs between receptors and ligands in a cell can be regulated by Fringe: As the reviewer points out, the concept and terminology of cis-inhibition are now embedded in the literature for better or worse. Nevertheless, we are sympathetic to the potential for confusion and have rewritten these parts of the manuscript to try to clarify this issue. As mentioned in point 1 above, we have now tried to clarify what is known about the effects of Fringe on Jag1, Jag2 and Dll1 and we have revised Figure 6 accordingly.
We hope the revision makes this clearer. We have modified Figure 6 in response to comments from the reviewers, and we have also revised the legend and description of the figure to make it clearer. We have also added another figure Figure 7 to try and better explain the mutant phenotypes. To date, the only strong quantitative evidence for the function of Fringe modifications on Notch signaling comes from cell line culture systems, such as the one used by the Elowitz group in their eLife paper.
In that paper, they found no difference between the effect of Lfng and Mfng on Notch signaling, although they did find that Rfng behaved a little differently to the other two Fringe proteins. Moreover, there are only a few documented cases — including our study — where multiple Fringe knockouts have a more severe phenotype than Lfng alone. So, while it is possible that Lfng and Mfng have different but related functions in the cochlea, the tools and markers we currently have at our disposal cannot reveal this. We show this small reduction can be achieved in multiple ways, including by removing one copy of Jag1.
In these cases, the genes we are knocking out are expressed broadly, but the effects are confined to the future inner hair cell region. Our conclusion is that this region is most sensitive to changes in Notch signaling. We agree that Mfng-Cre mice would have been a more specific way to show that inner phalangeal cells and inner hair cells derived from a common Mfng-expressing progenitor. We hope this addresses this point. Although this is entirely consistent with our conclusion that the levels of Notch signaling at this border are quite low and sensitive to small changes, it makes it very hard to detect quantitative changes, especially since in situ hybridization itself is not very quantitative.
The argument on cis-inhibition continues in the second and third paragraphs of the Discussion, quoting LeBon as if an ear paper: However, the experiments do not provide any evidence of that occurring in the ear. We have rewritten these parts of the Discussion to clarify that cis-inhibition is predicted to occur in the cochlea when Notch ligands and receptors are expressed in the same cell.
As mentioned above, the majority of evidence for cis-inhibition comes from both mammalian cell lines and Drosophila genetics. The process of cis-inhibition cannot be visualized by a unique marker; rather it is inferred by a evidence for co-expression of receptor and ligand in the same cell and b evidence for intermediate levels of Notch signaling that can be increased or decreased by manipulating levels of receptor, ligand or Fringe proteins in the cell. The Daudet paper of is based on over-expression experiments and it is possible that the residual Notch signaling was sufficient to induce the rather sensitive Hes5-dsEGFP reporter in their experiments.
We have now added data showing that Lfng;Mfng double mutants have significantly reduced Notch signaling at the time and place where the boundary of the organ of Corti forms. We hope this adds weight to the idea that Lfng and Mfng activity are together necessary to promote Notch signaling from the cells that transiently co-express them. As mentioned above, we have now provided data showing that Lfng;Mfng double knockouts have reduced Notch signaling in the border region of the future organ of Corti. This is exactly what we believe is happening in our Notch1 mutants and previously published Notch1 mutants — there is a duplication of inner hair cell and inner phalangeal cell progenitors at the boundary of the organ of Corti, but the loss of Notch1 causes the inner phalangeal cell progenitors to form hair cells.
We have stated this more explicitly in the revision. We do believe the signal is inhibitory, but it is clearly different from conventional Notch mediated lateral inhibition, both qualitatively we see a different loss-of-function phenotype and quantitatively it is sensitive to changes in Notch signaling that do not affect the choice between hair cells and supporting cells. We felt that referring to this phenomenon was lateral inhibition would be confusing. The reason we chose to use the Lfng-CreER mice to show that inner hair cells derive from Lfng-expressing progenitors rather than other methods is that a fluorescent protein reporters can persist after the gene is switched off; b this is also true of protein reporters such as Atoh1-GFP fusion mice we have used in the past and c in situ hybridization probes either fluorescent or DIG-labeled each have different sensitivities as can be seen in Figure 1—figure supplement 1 , making it hard to determine the precise temporal order of expression if expression patterns are changing rapidly as they do in the developing cochlea.
We recently showed that Mfng is a candidate Atoh1 target gene Cai et al. We were not suggesting that Lfng modulates Jag1 levels , but rather Jag1 activity , which has been well-documented in the past in vivo and in vitro. We have now tried to clarify this in the text.