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Spatial and Temporal Expression Pattern

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Welcome to the course of Plant Developmental Biology.So, in from previous class we are continuing reverse genetics based approaches.So, if you recall previous lectures.So, we have finished identification and isolation of gene for functional study, we have studiedalready analysis of expression pattern particularly global analysis or the expression patternwhere differential expressed genes has been already identified.In this class we will more focus on the spatial expression pattern analysis.So, this is very important in context with the developmental biology, because developmentof any organs or any tissue is a context dependent process.And if you recall our few of the very previous lectures or very one or first or second lecture,we have discussed that, cell-cell communication is one major driving factor in case of thedevelopment.So, if you want to study function of a particular gene through reverse genetic based approach,the first and very important thing what you have to do you have to very precisely studywhat is the expression pattern particularly it’s spatial and temporal expression pattern.Spatial means which tissue, which cells or which organs it is expressed and temporalmeans at what stage of the development it is started expressing.So, there are several ways to study to analyse expression particularly spatial and temporal,one way is RNA in situ hybridization.Here you can do in whole mount issue or tissue sections.So, basically whole mount issue you can take a full organ or full structure and then youcan probe that with a specific gene specific probe and then identify where is a particulargene expressed?In tissue section what we do you first fix the section and then make a cross sectionsof the tissues, 8 micron thickness; thick tissue or 10 micron thick cross sections andthen you probe that cross section with the gene specific probe.So, this method you can use to identify RNA messenger RNA which means that you can identifythe transcriptional activity of a gene in a tissue specific manner.Second thing you can study is the protein in situ hybridization, where you can lookwhere a particular protein is present.So, there are other way to study temporal and spatial gene expression pattern whichis promoter reporter assay and promoter trapping, we will discuss one by one.So, first we will take RNA in situ hybridization.So, as I said that here you can do in two either you can do whole mount in situ hybridizationor you can do on the section, but what are some steps which you have to follow?So, the first major step of RNA in situ hybridization is tissue preparation.So, you collect the sample depending on what tissue or what organs you are going to study,you can collect root, stem, inflorescence, flowers depending on your interest.Then first important thing is that you have to fix the tissues.This is very important to terminate any ongoing biological reactions or bio chemical reactions.There are various fixatives available to fix the plant tissue and then these two processesare common in both whole mount in situ as well as in situ on the cross sections or longitudinalsection, but if you want to do on the sections then another thing what you have to do?You have to take this tissue and embed in embedding media this is very important becauseyou want to make a very thin section, which is 8 micron or 10 micron section and the embeddingmedia more preferably what we are using is a paraffin.So, there is a process of the dehydration this is important we do through ethanol seriesto remove the water and then you infiltrate this with the paraffin.And then once your paraffin blocks are ready, you take this samples you make a cross sectioncollect this sections on poly lysine, Poly-L-lysine coated slide, this is very important becauseyou want your section to stick on the slide and it should not fall off.Then another important thing is that you have to generate RNA probe.As you can see here this is RNA-RNA in situ hybridization which means that you are detectingmessenger RNA for a particular gene and using RNA as a probe.So, if you have to make probe you will have antisense RNA as a probe because this antisenseRNA will go and hybridize with the sense RNA.But as a control you will also make sense probe to basically check what is the backgroundsignal.And how you make this probes maybe I will describe in detail, there are two ways oneis that you can do PCR based probe synthesis or you can clone a gene specific fragmentin a vector and then use that as a template to generate the probe.Since we are generating RNA probe the process which involves is in vitro transcription andhere we are using either T7 RNA polymerase or T3 RNA polymerase depending on which promoteris available and then you can use labelled NTP.So, there are two way of doing it one the initial way where people used to make probethey used to make radioactive labelled probe and now the because of lot of issues withthe radio activity, we are moving more towards the non radioactive labelled probe.And in this probe synthesis what happens either you take one of the NTP as isotope labelledor you can use some of the chemical synthesized NTP.For example, this is biotin labelled UTP, DIG or digoxigenin label UTP and then thisprobe depending on what is the size of your gene, you can use them and you can hydrolysethem.So, what you do basically?You take your gene specific fragment you clone in a cloning vector, the important thing isthat your cloning vector should have T3 and T7 promoter or you can use SP6 promoter dependingon the vector available.And then if your gene specific fragment is in the sense orientation, if you have to generateantisense probe.You will have to use the T 3 promoter and T 3 RNA polymerase and then for doing thiswhat you do you linearize this vector from here and then use T 3 RNA polymerase to transcribethis region as a antisense probe.But while transcribing this region you are adding UTP which is tagged with digoxigeninwhich is called in DIG-UTP and then if this probe synthesis or if the size of probe ishigher than what you go you prefer to hydrolyse it and make a probe size of roughly 75 to100 nucleotide long.This is important because if size of probe is very high it will not properly penetratethe tissues at the time of hybridization.So, for example, this is a gel picture which is showing how to generate the probe.So, this is a linearized plasmid; so, you have plasmid with your gene specific fragmentcloned in it, then you have done in vitro transcription and you have generated the probesthis is RNA probe.But this probe size is quite high then we have hydrolysed this probe and we have madea small hydrolysed probe.Now, this hydrolysed probe is ready for hybridization.For hybridization what we do?Before going for the hybridization there are some treatment with the tissues, and one veryimportant treatment there is a proteinase K treatment this is very important to partiallydigest the tissue to allow better probe penetration.And then once you have treated your tissues then you use this RNA probe and do hybridizationusually 16 to 20 hours at 50 to 55 degree centigrade then once your hybridization isfinished it is very important for you to remove unbound probes otherwise they will also givesbackground signal.So, what you what we do to remove them one treatment we do which is RNase treatment typicallywe are using here RNase A. RNase A which specifically degrades single stranded unbound RNA.So, this will remove all unbound RNA from the tissues then we do stringent washing toremove even if there is something un-hybridised probes.This is very important before going for the detection of the signal, if you do not dothis properly then you are going to have lot of background signals.Then the process goes once you have already sections, you have already probed with theantisense RNA probes, then you use antibody.So, typically we use Anti-DIG antibody.So, you know that our antisense RNA probe is tagged with DIG.Now we are using a antibody which recognise digoxigenin.So, this Anti-DIG antibodies we are using and then depending on what method or whatway you are going to detect it, if you want to do the colorimetric assay then what weuse DIG anti DIG antibodies which has alkaline phosphatase conjugates.But if you want to detect through the fluorescent base detection, then you can use anti DIGantibody with some of the fluorescent tagged protein for example, Rhodamine.So, basically you have now treated slides with the antibody then you have to wash toremove unbound antibodies, then go for the detection.Again the method of detection will depends on what kind of probes what kind of antibodyyou have used.So, if you have used radioactive probe then you can detect the signal by autoradiography,if you have used antiDIG antibody which has alkaline phosphatase or other colour basedenzymes then you can do the colour reaction, but if you have fluorescent tagged antibody,then you can do the fluorescent microscopy to detect the signal.I will give you some of the examples.So, this is a case of whole mount in situ and one of the gene which is rice transcriptionfactor MADS2 was detected or analysed for its expression pattern using nonradioactiveDIG-UTP labelled antisense RNA probe and this was detected by fluorescence microscope.So, just to quickly know that this is the organ where we want to see the expression,this organ is called lodicule it is a small rice flower organ you can see here very clearlythat this is the organ here, and we want to see that how MADS2 express across the organs.So, we have basically taken this entire lodicule and we have done the whole mount in situ hybridizationusing antisense probe against the MADS2 and then detected using anti rhodamine labelledantibody.And you can clearly see here that with this in situ method we can tell that that expressionof MADS2 is asymmetrical distributed.If this is a lodicule you can see that tips and the peripheral region of the lodiculehave more MADS2 signal as compared to the proximal and basal region of this.So, this analysis basically not only allowing us to check the expression in a particularorgan, but it is also allowing us to check if the transcript is asymmetrically distributed.Similarly, if you take this example.So, here basically these were done on the longitudinal sections of the panicle.So, this is again rice panicle, and rice panicle is like inflorescence it has different florets,florets at a different developmental stages and this upper panel if you look they hasbeen labelled with antisense RNA probe for another gene which is called MADS1, but itis radio labelled probe.So, you can clearly see the expression is throughout at early stage and then later stageit is more restricted to the lemma and palea and expression in inner whorl organs are disappearing.So, this is a typical example of radioactive label RNA in situ hybridization on section.On the other hand if you look this OsMGH3.OsMGH3 is a downstream gene which is regulated by MADS1 and what happens if you see hereat this stage.So, this is done by non radioactive labelled antisense RNA probe and if you look at thisstage you can see that very similar to the MADS 1 it shows the high level of expressionthroughout the floral primordia.But in the later stage when the floral organs are organogenesis are already started youcan see the expression in lemma and palea is decreased, but the inner whorl organs arehaving high level of expression.So, this way; this method is allowing us to study where exactly a particular gene is expressed.So, this was example of RNA RNA in situ hybridization, but we all know that RNA in most of the casesthe functional molecule is protein not the RNA.Of course, there are a lot of RNAs which are functioning without translated into a protein,but in general cases or typically RNAs are undergoing the process of translation andthey are making the protein.So, if you want to detect the protein in a tissue or if you want to detect the spatialand temporal localization the protein, you can perform protein in situ hybridizationwhich is also called immunohistochemistry and what you can do this is a typical exampleof a whole mount.Here this is quite similar with the RNA in situ hybridization except the detection methodis slightly different.So, here also you collect the tissue then do the fixation, then after fixation you aredoing the membrane permeabilization and then you can add the antibody.So, there are two possibility if you want to detect a protein against which you alreadyhave antibody then you can use that antibody, if you do not have antibody against a particularprotein of interest then you can tag the protein, you can tag your protein with some tag.For example, you can add HA tag, MYC tag or any kind of tag and then you can use antibodyagainst the tag to detect the protein localization.And then there are two way, if you have a primary antibody already tagged with the withthe conjugates if it is already conjugated, you can just use primary antibody else youcan first detect with the primary antibody.And then you can use secondary antibody which is against the primary antibody and then youcan do, you can visualise the signal.Signal visualisation you can do in different way you can in fact, co stain with differentdye.So, for example, if you take this example here a protein which is PIN1 protein veryimportant Axin transporter protein has been seen for its is localisation and you can seethe green signal is for PIN protein.And then another molecule or another protein which is basically tagged with the red signalit is ATPase so basically this is going to mark the membrane protein and then DAPI whichis blue in colour DAPI basically binds with the DNA.So, it is going to stay in the nucleus.So, this all three together has been used to basically check the localisation of PINprotein.So, if you look here; So, this is may be enlarged version of it and you can clearly say thatthis is the cell boundary and in the cell boundary the PIN protein is very specificallylocalised or asymmetrically localised on one side of the cell membrane, but it look thisside of membrane the protein localisation is very small.So, this is I am taking just one example, but there are plenty of examples availablewhere you can detect this protein by protein in situ hybridization or immune immune-histochemistryok.So, next method which is being used to monitor temporal and spatial gene expression is promoterreporter assay, very commonly used.So, in if you recall one of our previous lectures.So, we have discussed that there could be two types of promoter reporter assay.You can either make a transcriptional fusion construct or make a translational fusion construct,what is difference between them?In a transcriptional fusion construct so, you only take the promoter or the upstreamsequence of your gene.So, if this is your gene you only take the promoter region and this construct basicallywill tell you the transcriptional or promoter activity wherever it is available.And then you use a kind of reporter gene here, reporter gene either you can use GUS kindof gene or you can use GFP, RFP any kind of reporter gene you can use here.Then if you generate this kind of construct it is called transcriptional fusion construct,and this construct basically will tell you where is the promoter activity domain.But this alone cannot be used because in plants most of the time it has been seen that promotersare not very localised, there are some cis regulatory elements which are essential forthe promoter activity and they are even found within the gene sometime in the introns.So, it is always challenging to choose that how much region or how much segment of theDNA to be taken for the promoter activity.So, if you take a fragment let us assume if you are taking 3 Kb upstream sequence fromthe start transcriptional start side, then you will clone it and you will see a expressionpattern, but if you want to validate it or if you want to be very sure that actuallythis is the promoter activity you will have to do this analysis together with the in situhybridization.So, in situ hybridization just now we have discussed, there you are going and you arenot doing anything you are just detecting the endogenous expression pattern of a geneor of a RNA.And then you are making the transcriptional fusion construct and when you are using thistranscriptional fusion construct you see where is the expression then you can compare.If your promoter activity or if your promoter reporter activity is same as the in situ hybridization,then you can say that the promoter element or the promoter region which you have selected,it is complete and it has all the cis regulatory elements which is part of this promoter orwhich is required by this promoter.In translational fusion construct what we do that, this is more important because itwill tell domains of the expression as well as sub cellular protein localisation.So, here what we do?We basically take the desired promoter and then we take the gene as well, but we fusethis gene with some of the reporter.So, basically what we are making here, we are mutating the stop codon of the gene ofinterest and then in frame we are fusing with some of the reporter and then what happensthat?This and if you use this construct this is called translational fusion construct.So, this will tell first thing that, where ever is the promoter activity it will reportand second thing is that what is the subcellular localisation of your protein.So, because your protein is now tagged with the GUS and GFP so, wherever your proteinwill move it will report the reporter will report its presence, but here there is oneissue.The issue is that since you are making a fusion protein there is a possibility that this fusionmight inactivate your proteins.So, your protein might not be in its the proper confirmation and then if you report its localisationit is always questionable that the localisation is because of the artefact or it is the truelocalisation pattern.So, this has to be validated this you can validate it, how you can do you can validateyour construct by doing the complementation which means that, if you take mutant of thisgene of interest, where you see some phenotype and then take the translational fusion constructand put back to the mutant.If the phenotype of mutant is complemented by this construct, which means that your fusionprotein is functional and you can rely on its the localisation pattern.So, I will give you now few very important examples where transcriptional and translationalfusion construct has been successfully used to reveal a very very important information.For example, if you look this SHORTROOT gene.So, SHORTROOT gene it is very important for root development in Arabidopsis and if youdo just RNA in situ hybridization or if you generate a transcriptional fusion construct.So, this is endogenous RNA detection by RNA in situ hybridization and here you have takenSHORTROOT promoter and fuse in here GFP here GUS.In all this case if you look the expression domain of SHORTROOT protein what you see thatthis gene is expressed in the vascular issue.So, if you look this is a longitudinal view of the growing Arabidopsis root this is endodermis.So, if you see this in endodermis you do not see any signal, but the tissue after endodermislike pericycle, xylem, phloem, procambium they have the activity this is same you cansee here and this is also you can see when you make when you do in situ hybridizationhere in the root.So, this tells that the SHORTROOT promoter is active in the vascular tissue, but whenyou make a translational fusion, where you are using same SHORTROOT promoter, but nowSHORTROOT protein has been translationally fused with the GFP.What you observe that apart from the expression domain which is in the vascular tissue, youalso see that protein is also present in the endodermis and in endodermis it is gettinglocalised to the nucleus, and this has a lot of importance maybe in some of the futureclasses, we will discuss what is the functional significance of this.So, how this is possible?If your promoter is active here transcriptions is going on only in the vascular tissue, howcome your protein is present in the tissue where transcription is not happening?So, this gives an information that there might be a possibility that your protein is gettingsynthesised in the vascular tissue, then from vascular tissue it is migrating to the neighbouringcells which is endodermis in this case.And this is only possible if you study both transcriptional as well as translational fusionconstruct for the gene.Similar kind of study has been done in shoot apical meristem of Arabidopsis thaliana, andhere the a very important protein which is called WUSCHEL.WUSCHEL is important to regulate and to maintain the meristematic activity, you will see inthe next class, but here what I wanted to show if you look the in situ hybridizationpattern of the WUSCHEL.You will always see that this WUSCHEL is expressed somewhere here in this domain, but this domainwhich is L 1 and L 2 layer, this is inflorescence meristem this is floral meristem.In all the cases you can see that L 1 and L 2 layer they do not have RNA which meansthat transcription of WUSCHEL is not happening in L 1 and L 2 layer.But when you take and when you look the protein localisation of WUSCHEL then what you seehere.So, this is WUSCHEL promoter driving GFP fused with the WUSCHEL protein, you can see thatin L 1 and L 2 layer both the layers has the protein.So, transcriptionally it is here, RNA is here and then the protein is also present in theregion where transcription the active transcription for WUSCHEL is not happening.So, this tells that this protein might be moving from one cell to another cell ok.The final method what is being used to study temporal and spatial expression is promotertrapping or enhancer trapping or gene trapping, what is this trapping?This is very important method and what you can do there are different types of trappingyou can do.This is typical genomic DNA and then for example, you want to identify a gene with a very particularor with a defined expression pattern how you can do?One way of it doing in enhancer trapping here we will focus more on the promoter trapping,I think in enhancer trapping we have already discussed in one of the previous class.But what will happen if you have to trap a promoter of your interest, if you want toidentify a gene which has let us assume expression in leaf or if it is expressed only in thepetals and you want to identify that kind of gene for the reverse genetic based studyhow can you do?So, basically you can generate a construct in this construct what happen that, you puta reporter gene, but in the reporter gene just promoter less reporter gene, and put,transfer make a construct in the T DNA and use this T DNA to raise the transgenic plant.So, basically what is happening this T DNA will randomly get inserted in the genome,but if this T DNA got inserted let us assume in gene of interest which is expressed inthe petal and if let us assume that if this is a promoter, this is the gene and here letassume if your construct got inserted.Then now it has got a promoter activity from here of the gene which is like petal expressionand then you have a reporter gene here.So, basically you are randomly integrating a reporter in the genome and trying to identifya transgenic line, where your reporter has been integrated in the downstream of a promoterwhich is active in a particular tissue.And then you can basically isolate them, similarly you can do the gene trapping; you can do theenhancer trapping in enhancer trapping what basically we do?We take a minimal promoter and then reporters.So, promoter minimal promoter as well as reporter and then try to trap the enhancer, here weare trying to trap the promoter.And you can see some of the example that this has been very successfully used to identifya lot of genes whose expressions are in a very restricted manner for example, this geneis expressed in the leaf this is expressed in the trichome then this is expressed inthe in the root apical meristem similarly here if you look the lateral primordias arecoming lateral root primordias are coming.So, basically then then since you know the T DNA sequence you can use some of the molecularbiology based technique to map the gene where is the site of integration, then you can identifythe gene for functional study.Yeah here is one very good example, where where the genes or the lines has been identifiedwhich show either petal or stamen and or some of them are expressed both in petal and stamen.So, you can see they has been generated.So, large number of gene trap line has been generated and if you look these lines theyare having a different expression pattern.Some of them are specifically expressed in the petal or specifically expressed in thestamen some of them has been expressed both in petals and stamens, then you can go mapthis gene and then identity and take this gene for the further functional study.So, if I recap what we have discussed today.So, today we have finished the analysis of expression pattern particularly we have takenwhat are the ways and to study the spatial and temporal expression pattern of a genein plant.And particularly these are very very important when you are studying development of a particularorgan or if you are identifying some putative regulator of a developmental process, andif you want to study their specific function in the process of development.So, I will stop here, in next class we will discuss functional characterization of a gene.So, once you have identified a gene, how you will take it for further functional characterization.Thank you very much.