INTERVIEW

The people behind the papers – Elena Popa and Abigail Tucker

While many vertebrates have multiple sets of teeth over their lifetime,some, like humans, have just a single set of replacement teeth(diphydonty), while others, like mice, manage with a single set(monophydonty). This diversity raises both evolutionary questions –

how did different tooth replacement strategies evolve? – anddevelopmental ones – what mechanisms prevent replacement teethin animals that have lost them? A new paper in this issue ofDevelopment tackles these questions with a molecular analysis ofmouse tooth development. We caught up with first author Elena Popaand her supervisor Abigail Tucker, Professor of Development andEvolution at King’s College London, to find out more about the work.

Abigail, can you give us your scientific biography and thequestions your lab is trying to answer?AT My lab is interested in how bodies are formed duringdevelopment, both from a clinical perspective of understandingbirth defects, but also from the point of view of understanding howevolution has shaped our bodies. I started out investigating tadpoletail development for my PhD with Jonathan Slack and thenswapped ends and moved to the head for my first postdoc with PaulSharpe. Here, I investigated how the face and dentition arepatterned. This is where I first encountered tooth development, andalthough I have moved on to study the cranial neural crest, the jaw,cranial glands and ear, I have always kept some experiments goingto understand more about the tooth. Teeth are often the only thingleft preserved in the fossil record, so they have a central importanceto our understanding of evolution. There are still lots ofunanswered questions, such as what regulates tooth number,tooth size and tooth shape? It’s a great model for understandingepithelial-mesenchymal interactions, as both tissues are integral tothe formation of the final tooth but take it in turns to play theleading role.

Elena, how did you come to join the Tucker lab and whatdrives your research?EP Developmental biology was by far my favourite subject duringmy undergraduate years at Royal Holloway. I didn’t know it wasgoing to be tooth development in particular back then, but as soon asI read about Professor Abigail Tucker’s research I was completelycaptivated. Her lab provided the opportunity to perform acomparative study of molecular interactions and developmentalprocesses that allow or disrupt tooth replacement in awide variety ofvertebrates. Why can’t we have more than two sets of teeth butsnakes can? This was essentially the question that drove my researchin the Tucker lab.

What initially led you to tryand ‘reawaken’ tooth replacementin the mouse?EPWhy the potential for tooth replacement varies so much acrossvertebrates is an intriguing question. Having performed an in-

depth study of the development of the dental lamina in manydifferent animal models, we suspected that this structure retains thecapacity to give rise to a subsequent generation of teeth, even in themolar region of the mouse (the mouse only has one generation ofteeth and molars, in general, do not replace). The dental laminanext to the first molar can be seen protruding at E16.5 butdisappears soon after birth, and has been termed the rudimentarysuccessional dental lamina (RSDL). We compared the RSDL withthe successional lamina of the minipig (which gives rise to asecond generation tooth), and found that both expressed theepithelial stem cell marker Sox2; however, Wnt activity was onlypresent in the minipig lamina. Knowing that stimulating Wntsignalling by means of different transgenic lines leads to theformation of supernumerary teeth, we based our experimentaldesign on these comparisons, aiming to recapitulate toothreplacement in the mouse.

Can you give us the key results of the paper in a paragraph?EP & ATOur results show that, although the mouse normally doesnot form a second replacement set of teeth, it still has the potential todo so if given the right signals. Stimulation of Wnt signalling in therudimentary replacement lamina in transgenic mice or isolation ofthe lamina in culture both led to the formation of a new tooth. Westarted by showing that the RSDL exhibits molecular similarities tothe competent dental lamina in a diphyodont mammal and retainsodontogenic capacity, which we were able to reawaken byselectively inducing Sox2+ cells to activate canonical Wnt/β-catenin signalling.Wewere able to confirm the dental identity of thestructures that arose from the RSDL in the mutant mice byperforming in situ hybridization for genes known to be expressedduring normal tooth development. The mutant RSDL was alsohighly proliferative and gave rise to multiple ectopic teeth, many ofwhich were complex in shape and mineralised after transplantationin kidney capsule. We also uncovered an inhibitory relationshipbetweenWnt signalling and Sox2, where ectopic stimulation ofWntsignalling leads to downregulation of Sox2 expression.

Elena Popa (L) and Abigail Tucker (R).

Centre for Craniofacial and Regenerative Biology, Department of CraniofacialDevelopment and Stem Cell Biology, King’s College London, London SE1 9RT, UK.E-mail: abigail.tucker@kcl.ac.uk

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© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev176313. doi:10.1242/dev.176313

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When you remove the first-generation tooth, this frees theRSDL to form a tooth bud. How is this potential inhibited inthe context of normal development?EP&AT Tooth number has previously been shown to be controlledby a balance between activators and inhibitors, creating aninhibitory cascade. For example, in many mammals three molarsform at the back of the mouth by serial addition from a single molarplacode. If the primordium for the subsequent molars is separatedfrom the murine first molar in culture, the second molar initiatesdevelopment faster and grows to a larger size than if left intact. Thefirst molar therefore appears to be controlling the development ofthe next tooth in the series. In the shrew, the first generation of teethinitiate but then regress and are replaced by the permanent set ofteeth. Here, again, it has been suggested that early formation of thepermanent teeth might inhibit the development of the first set:timing and spatial arrangement of tooth germs is therefore clearlyimportant in the control of final tooth number.In our paper, we show that the RSDL has the potential to form a

tooth and speculate that the adjacent molar sends a Wnt-inhibitorysignal to the surrounding dental tissue. This then prevents Wntactivity in the RSDL, and leads to its regression. This is relevant tohuman tooth replacement, as structures similar to the RSDL havebeen identified next to the permanent teeth during development. Innormal development of our dentition, therefore, the permanent toothmay inhibit the generation of a third set of teeth.

Wnt signalling leads to a reduction in Sox2 in the dentalepithelium but not associated epithelia – what makes thisrelationship context dependent?AT The context-dependent nature of the relationship between Wntsand Sox2 was very striking. This fits, however, with the literature,which has shown similar context-dependent interactions. Forexample, in the airway submucosa, Sox2 has both an inductiveand a repressive effect on Wnt signalling that is dependent on thepresence of other factors, whereas in the lungWnts inhibit Sox2 butonly at early stages of development. In the tooth, the repression ofSox2 by Wnts might be dependent on other factors with dynamic

temporal and spatial expression patterns. It will be very intriguing towork out what these factors might be.

Do you know of any evolutionary scenarios wheremonophyodonty transitioned to diphyodonty, and if so doesthis involve a similar revitalisation to that you havediscovered in the mouse?EP&AT Throughout evolution, the general trend is one where animalsreduce the number of tooth generations in favour of more-complex toothshapes and better occlusion. As in themouse, there is often evidence of arudimentary structure, which points to this reduction in number. Forexample,wehave shown that in thediphyodont fruit bat, the canine showsevidence of a third generation as it displayed a vestigial structurehomologous to the mouse RSDL next to the second-generation tooth. Innature, there are rare cases proposed where teeth have been lost and thenreappear, e.g. in the frogGastrotheca guentheri, where teeth are found onthe lower jaw but are absent in all other frogs. This would suggest thatrudimentary tooth structures can be reawakened not just in the lab.

Did the research include any particular result or eurekamoment that has stuck with you?AT For me the eureka moment was when we generated a tooth germfrom the RSDL by simply cutting off the main tooth. Really it’s asimple experiment but has a key message, which is that the reason amouse doesn’t have a second set of teeth is that the first generationof teeth inhibits this from happening. This has importantconsequences, as it means that if this inhibition could be lifted anextra set of teeth might be possible.

And what about the flipside: any moments of frustrationor despair?EP For my PhD I worked with a lot of non-model organisms (bats,geckos, guinea pigs, opossum) in addition to the minipig and mouseshown here. These samples were always much more difficult toobtain and every time you wanted to look at gene expression itmeant cloning, so things took much longer than expected. Inaddition, the anti-Sox2 antibody has been a particularly tricky towork with across species. Considering that it is at the core of myresearch, it became frustrating when it simply refused to work. Afterwhat felt like hundreds of failed attempts, finally being able to seeSox2 staining under the microscope felt like a huge relief!

After what felt like hundreds of failedattempts, finally being able to see Sox2staining under the microscope felt like ahuge relief!

So what next for you after this paper?EP I have loved my time in science and particularly in the Tuckerlab, where I had the opportunity to learn a great deal and diversifymy wet lab skillset beyond what I could have ever hoped for. I’venow shifted my focus to science media production, observing anddocumenting scientific discoveries from behind a camera lens.

Where will this work take the Tucker lab?AT For me the next question is what signals from the first tooth stopthe second generation forming? Our results predict that such signalsprevent canonical Wnt signalling from being activated in the RSDL.Expression patterns predict possible roles for dickkopf 2 and dickkopf3 that could be tested in culture or in vivo. Another avenue is the

New tooth formation from the RSDL, showing Sox9 expression.

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vestibular lamina. This is a really understudied structure but appears tohave the potential to form teeth when stimulated by Wnt signalling.I am really interested in the relationship between the dental lamina andvestibular lamina, as these two epithelial structures are united duringearly development, forming from the same placode. What signalsdetermine whether you become a dental lamina and form teeth, or avestibular lamina and form the cheek ridge, is next on my list.

Finally, let’s move outside the lab – what do you like to do inyour spare time in London?EB I think the UK is an incredibly beautiful country, so I tend tospend most of my free time travelling and exploring its fantastic

landscapes. My favourite places to go are Cornwall and the LakeDistrict. I also have a characterful little Whippet puppy, who takesup a lot of my time at the moment!

AT I commute into London from Kent where I live with myhusband, children, cat, bearded dragon and five snakes. I loveLondon, having grown up there, but have become a convert to thecountryside. I love cooking, eating and travel, and am writing thisfrom the Atacama desert in Chile.

ReferencePopa, E. M., Buchtova, M. and Tucker, A. S. (2019) Revitalising the rudimentary

replacement dentition in the mouse. Development 146, dev171363.

INTERVIEW Development (2019) 146, dev176313. doi:10.1242/dev.176313

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