In the latest episode of Genetics Unzipped we’re finding out how researchers are unlocking the information hidden within the human genome using new technologies like CRISPR gene editing and artificial intelligence with the aim of developing better medicines and getting them faster to the patients who need them.
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Recently we reported the unexpected ability of fish mutants to develop limb-like bones in their pectoral fins (Hawkins et al., 2021). However, the most critical element of the study—finding these mutants in the first place—receives relatively little attention in the paper. Here I describe our efforts to find these monsters lurking inside the unassuming zebrafish.
The nuts and bolts of fins and limbs
The transition from fins to limbs is a defining transformation in vertebrate history and has served as a pivotal study system for comparative anatomy, paleontology, biomechanics, and developmental biology (Clack, 2009). Insight from each of these fields has illuminated different facets of how a relatively simple ancestral fin evolved into the complex arms and legs of tetrapods. Modern fins and limbs look quite different from one another but revealed in the fossil record are intermediate forms that connect their disparate morphologies (Jessen, 1972; Shubin et al., 2006; Zhu and Yu, 2009). By comparing the gene programs active in fins and limbs, we can ask which patterning mechanisms are common to both appendages, which mechanisms are derived in each, and which could be responsible for the changes in form found in evolution.
Tetrapod limbs have many long bones that articulate end on end away from the shoulder with distinct regions such as the upper arm (humerus), forearm (radius and ulna), and hand (Figure 1). In contrast, the pectoral fins of teleost fishes have just four long bones set side by side (proximal radials) followed distally by some small nodular bones (distal radials) and the dermal fin rays (Arratia, 1999). Non-teleost ray-finned fishes such as the bowfin have a slightly more impressive endoskeleton with additional articulations in the posterior part of the fin. Paleontological evidence suggests that the common ancestor of ray-finned fishes and tetrapods had a pectoral configuration much like that of the bowfin (Jessen, 1972, Zhu and Yu, 2009). In this scenario, teleosts such as the zebrafish represent a reduction of the ancestral appendage skeleton, while limbs exhibit its impressive elaboration (Coates, 1995).
Figure 1. Pectoral appendage anatomy is variable across the bony fishes. From the ancestral configuration that exhibited moderate elaboration in the posterior fin (bottom), teleosts (left) simplified the endoskeleton such that it consists of a row of proximal radials followed by small nodular distal radials. In contrast, in the lineage leading to tetrapods (right), the fin was elaborated through the addition of distal long bones to form a limb with a three-part structure containing the upper arm (humerus), forearm (radius and ulna), and hand (wrist and digits). Anterior to left, distal to top in skeletal schematics.
Forming an impressive body of work spanning the last four decades, developmental geneticists across the globe have discovered and characterized the manifold genes and pathways that control the growth and patterning in the nascent limb, leading to a deep understanding of the signaling ligands, receptors, and transcription factors necessary to make a normal appendage (Capdevila and Izpisúa Belmonte, 2001). Surprisingly, despite the morphological differences, many of these key limb patterning pathways are also expressed in developing teleost fins and play analogous (or conserved) functional roles (Mercader, 2007). There are differences in the expression and signaling function of some of these important players, but on the whole fin buds and limb buds behave quite like one another, and there is not one clear genetic factor present in limbs and absent in fins that is sufficient to imbue ‘limb-ness.’
Fishing for fin mutants
While assessing the role of candidate limb genes in growing fins has yielded critical insights into fin development, my colleagues Katrin Henke, Matthew Harris, and I decided to investigate the genes that can modify the zebrafish fin pattern using forward genetics. In a forward genetics approach, mutations are made at random and the investigator screens through mutated animals to pick out individuals with an interesting phenotype (Patton and Zon, 2001). Once an interesting mutant is found, we then work backwards using genetic mapping to determine which gene was mutated to cause the phenotype. The beauty of forward screens is that they let the organism tell you which genes are important to the process you want to know about. Sometimes you find an allele of a known essential regulator that has been extensively characterized, other times you find a gene that hasn’t been studied at all.
Katrin conducted several prodigious screens with a focus on mutants that affect the formation of the adult skeleton, and isolated hundreds of novel mutants (Henke et al., 2017). One mutant she picked out because of its modified pigmentation and dysmorphic fin rays was of particular interest to me and Matthew. Upon observing the internal skeleton under the dissecting scope, Matthew saw that the fin endoskeleton was affected and suggested I take a look. Shockingly, instead of having just the four long bones set side by side, this mutant had additional long bones forming in the distal endoskeleton that articulated with the proximal elements with a joint analogous to an elbow (Figure 2). This mutant didn’t just have fins, but what it had were not quite limbs either: it grew “flimbs.” At the time my dissertation project was focused on craniofacial mutants, but after this discovery (and Katrin’s blessing) my focus shifted to this fascinating mutant with limb-like fins. All good mutants need a name, and my friend and Old Testament scholar Maria suggested rephaim, a race of biblical giants fabled to have extra digits on their hands and feet.
Figure 2. Novel long bones form in the distal endoskeleton of rephaim mutants. Top panels show the external appearance of wild-type and rephaim mutant fish. Middle panels contain micrographs of pectoral fins stained red for bone and blue for cartilage. The proximal radials are numbered 1 through 4, and the new bones in the mutant are indicated with an asterisk. Bottom panel schematizes the endoskeletons of wild-type and rephaim fins and indicates the position of the intermediate radials and novel joint in the rephaim mutant.
After receiving a name, mutants need to be mapped to determine which gene is affected. Genetic mapping of mutations used to be a long and involved process using chromosomal markers to track linkage and recombination. In early zebrafish screens, a mutant line would be crossed to a wild-type fish from a different genetic background, and PCR-based methods would be used to find variable genomic positions and track down regions of DNA that segregated with the mutant phenotype (Knapik et al., 1996). In the last 15 years, however, the advent of next-generation sequencing made it possible to sequence mutants and their wild-type siblings to quickly identify genomic regions that associate with the mutant phenotype. To map rephaim, we utilized a whole-exome mapping approach to determine genomic regions that likely contain the causative mutation (Bowen et al., 2012). The mapping data gave us two putative regions that could contain the rephaim mutation, one on chromosome 4 and one on chromosome 9. Chromosome 4 has a reputation as being a nightmare for mapping, replete with inversions and transposons, and the implicated interval didn’t contain any interesting limb patterning genes. I did not like chromosome 4. On the other hand, the interval on chromosome 9 contained the HoxD cluster, a battery of genes with critical roles in appendage patterning and particularly implicated in the differential patterning of fins and limbs (Sordino et al., 1995; Freitas et al., 2012; Woltering et al., 2014). Not only would a mutation in a HoxD gene fit my expectations of what could cause a phenotype like rephaim, it would make subsequent analysis of the mutant phenotype much easier and fit well within the existing fin-to-limb literature. I even thought I might finish my dissertation early.
This, however, was not to be. Linkage analysis definitively ruled out chromosome 9 and the HoxD cluster. The initial mapping signal that I had pinned my hopes on was due to a block of genetic homogeneity that was shared between mutants and wild-type siblings, meaning that both mutants and wild-type animals had the same alleles in this region and thus could not contain the causative mutation. Meanwhile, additional recombination analysis strengthened the association of rephaim with chromosome 4 and narrowed the linkage interval to a small window containing just one coding mutation in a gene called wiskott-aldrich syndrome protein like-b (waslb). Unlike my precious HoxD cluster, the waslb gene was not a known regulator of limb development, and everything I saw in the literature gave the impression that it was a “housekeeping” gene: ubiquitous expression, essential functions in actin metabolism, and involvement in myriad cellular pathways (Snapper and Rosen, 1999). Around this time, we were also mapping a second mutant with a similar phenotype, a fish called wanda (van Eeden et al., 1996; Haffter et al., 1996). The causative mutation for wanda mapped to the gene vav2, a similarly unexciting locus from a skeletal patterning perspective (Hornstein et al., 2004). I felt the path to my PhD lengthening in real time.
X marks the spot on a genetic treasure map
Nevertheless, all the mapping data pointed to waslb and vav2, so these genes demanded our attention. Mapping implicated these genes, but we still needed to experimentally confirm their role in the flimb phenotype. We used CRISPR-Cas9 to make loss-of-function alleles, but even homozygous null mutants had a wild-type phenotype (Figure 3). Next we tried injecting mutant mRNA into the embryo but saw no effect, likely due to the late appearance of the phenotype. In a final push to demonstrate the causative nature of the waslb and vav2 mutations, we used CRISPR to create frameshift lesions in cis to the candidate mutations and knockout the mutant alleles specifically. When the mutant copies of waslb and vav2 were removed, we rescued the phenotype and reverted the mutants to wild-type fin patterning. There was no doubt, mutations in waslb and vav2 cause the flimb phenotype. But this left us with a bigger question, how in the world are these genes changing skeletal patterning? I came up with an axiom to sooth myself: “if good science raises more questions than it answers, then the best science must raise only questions and answer nothing.”
Figure 3. Rescue experiments demonstrate that a mutation in waslb causes the rephaim phenotype. CRISPR-Cas9 was used to generate null alleles in wild-type and rephaim mutant waslb. Removing wild-type alleles (waslbΔ) had no effect on fin patterning. However, creating a frameshift and early stop upstream of the S265P mutation (waslbΔ+ reph) prevented the formation of intermediate radials and rescued wild-type patterning.
However, the wealth of limb patterning knowledge established by developmental geneticists was able to guide our inquiry. As mentioned earlier, Hox genes have critical functions in the patterning and growth of limb bones along the proximal-distal axis, and recent studies had revealed that Hox13 was required for the formation of the most distal structures in fins just like in limbs (Nakamura et al., 2016). We thought that the new bones in rephaim might also share this distal Hox13 regionality, and crossed rephaim into a Hox13-null genetic background. To our surprise, we found that loss of Hox13 actually enhanced the flimb phenotype and resulted in the formation of even more bones along the distal aspect of the endoskeleton (Figure 4). Hoxa13 is known to negatively regulate Hoxa11 expression in the limb (Kherdjemil et al., 2016), and we thought the enhanced phenotype might be the result of derepression of Hox11 genes. Around this time we also were analyzing limb-specific Wasl knockout mice, and observed limb defects similar to those seen in Hoxa11 mouse mutants. Intriguingly, Hox11 genes are also required for the normal development for the bones in the middle region of the limb, the radius and ulna (Davis et al., 1995).
Figure 4. Genetic interaction between rephaim and Hox genes suggests limb-like patterning mechanisms function in the fin. While loss of hoxa11a, hoxa11b, and hoxd11a has no effect on fin patterning in the wild-type background, removal of these genes prevents the formation of intermediate radials in rephaim mutants. In contrast, removing hoxa13a and hoxa13b from rephaim mutants enhances the phenotype and results in the formation of additional intermediate radials. The requirement of Hox11 genes is shared between intermediate radials and the limb forearm.
Following these clues, we generated null alleles of the hoxa11 and hoxd11 paralogs in the zebrafish. While loss of these genes had no effect on fin patterning in a wild-type background, we found that loss of hoxa11a and hoxa11b prevented the formation of the extra bones in rephaim mutants (Figure 4). Moreover, we generated knock-in hoxa11b reporter zebrafish and found that rephaim and wanda mutants cause the upregulation of hoxa11b expression. These results were quite interesting: even though the Hox11 paralogs are not required for normal fin patterning in zebrafish, they still possess the ability to specify the formation of an intermediate long bone position along the proximal-distal axis of the appendage skeleton. This suggests that the capacity to specify ‘middle’ and ‘distal’ regions is not unique to limbs, but was present in the common ancestor of ray- and lobe-finned fishes. Although not expressed in teleosts, this developmental potential has been retained in a latent state and can be redeployed by simple perturbations.
Wasl, Hox, and Beyond
This forces me to wonder what other latent limb patterning mechanisms that might reside in the developing fin bud, and how waslb is able to activate at least some of them. The mechanistic connection between waslb, vav2, and Hox regulation is an open question. In part, we know that waslb mediates the formation of F-actin foci that colocalize with Hox-positive cells in the distal fin (Hawkins et al., 2021). Given its roles in cell motility it could be that waslb effects the migration of these cells, but there are many other possibilities. Wasl also directly regulates transcription (Wu et al., 2006), modulates Wnt (Lyubimova et al., 2010) and TGFB (Lefever et al., 2010) signaling, and is directly involved in the colinear activation of the HoxB cluster (Ferrai et al., 2009). Then again, there could be another pathway that we do not yet understand. I will go out on a flimb here and say the zebrafish still has more to tell us about these mechanisms…as long as we are willing to trust the mapping.
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On Wednesday 14 April Development welcomed three researchers with interests in developmental neurobiology to our seventh Development presents… webinar.
Below you’ll find each of the talks, plus a Q&A chaired by Development Editor François Guillemot. The next #DevPres webinar will be held on 12 May 2021, and chaired by Paola Arlotta – subscribe to our mailing list for updates.
Iva Kelava (LMB Cambridge) – ‘Sex hormones and the human developing brain’
Wael El-Nachef (UCLA) – ‘Schwann cell precursor contribution to the enteric nervous system in post-embryonic zebrafish’
Wael’s paper was published last year in Development (you can also find a link to an interview we did with Wael and Marianne Bronner at the bottom of the abstract)
Stéphane Nedelec (Institut du Fer à Moulin) – ‘Dynamic extrinsic pacing of the HOX clock in human axial progenitors control motor neuron subtype specification’
Stéphane’s paper – featuring co-first authors Vincent Mouilleau and Célia Vaslin – was recently published in Development (you can also find a link to an interview we did with Vincent, Célia and Stéphane at the bottom of the abstract).
Our seventh profile in the series features Dorotea Fracchiolla, who works as a Project Manager in Frankfurt and is also a scientific illustrator.
Where are you originally from and what do you work on now?
I’m originally from Ruvo di Puglia, a town in the province of Bari, Puglia, South Italy. I worked as a Postdoctoral fellow in the field of in vitro reconstitution of Autophagy at the University of Vienna in Austria. In January 2021 I started working at the Max Planck Institute for Biophysics in Frankfurt (Germany) as a Project Manager of an International research collaboration that aims at studying Parkinson’s Disease pathogenesis. In April last year I started my project as a scientific illustrator and founded Art&Science, which gave me the chance to work with many scientists worldwide on art projects in different fields.
Has science always been an important part of your life?
I’ve always been a curious person and with some difficulty in finding THE subject to study. At school I liked everything and was always excited to start reading about new things. In this sense I think I’ve always been a scientist in everyday life. Later on, the actual scientific training provided me with material to feed my curiosity and exercise critical thinking.
And what about art?
Drawing, painting, crafting and trying out new materials were my preferred activities as a child/teenager. Soon enough I started photography, too. All these activities had an aspect in common: sensing. In other words, exploring the world around me, experiencing the new, interacting.
What or who are your artistic influences?
During my studies I have always been attracted by the personality and life of Leonardo da Vinci, an artist and a scientist in one person. As for him, my place of inspiration is Nature. In the beginning, I used to draw things at a macroscopic scale that I had observed while exploring, then I ended up depicting the molecular mechanisms of nano machines like those scientists study in labs. When it comes to style, I think I cannot underplay the influence on me of my place of origin, in its light and bright colors. One example: here in Puglia there is a tradition to make colored clay whistles as local art pieces. Among others, I have recently built a clay model of a set of proteins that participate in the formation of autophagosomes in cells. I think it grossly resembles in style these artcrafts.
How do you make your art?
For me, making art comes after creating an idea in my mind. The creation cannot precede the study and understanding of the subject. I start from reading and while doing so I put together images in my head that finally make up the puzzle. If something is not clear I keep reading until the full picture is completed. My motto is: if you can draw it, you understood it in the first place.
Does your art influence your science at all, or are they separate worlds?
In my opinion, art and science are tightly connected and rely on each other. This is why I named my activity as an illustrator ‘Art&Science’. Very often, while studying a new topic, I tend to first visualize things and make schematics of concepts. Simplification is my approach to understanding. That’s when the art comes into play: it depicts the core message and makes it simple for the eye to grasp.
“Art depicts the core message and makes it simple for the eye to grasp”
Tell us about the work you’ve shared with us.
One project turns around the topic of autophagosome formation. I started drawing these pieces during my PhD when I learnt hardcore biochemistry in the Laboratory of Prof. Sascha Martens at the University of Vienna while studying autophagy in his lab. The entire team was focused on getting recombinant purified components of the autophagy machinery and studying their properties in vitro. The goal was to understand how the different proteins work together to build autophagosomes. Scientific research needs to be coupled to a certain degree of imagination in order for questions to arise. I always liked to visualize things and my preferred way to convey a message is drawing.
Supported by structural biology data, I started off sketching how I thought the molecular machinery looks like, and that’s how the “Autophagosome Biogenesis” project arose.
Clay model, top view
Clay model, top view, close up
Clay model, side view
Clay model, side view 2
Clay model, front view
I first made the drawing and then I created a 3D clay model as shown in the gallery above.
This model was then used as a basis for a stop motion animation (for more information about this movie see my website).
Next is my Ub-p62-cargo illustration: like a sticky sugar muffin that gets stuck in its paper wrap, ubiquitin-tagged misfolded protein aggregates are tightly attached to LC3-positive isolation membranes via p62 during selective autophagy in human cells. I created this for the following publication: https://elifesciences.org/articles/08941
Finally, this gallery shows a lab life series done with acquerells. I created this one during lockdown as well because I was a bit nostalgic for the lab.
What are you thinking of working on next?
Like everyone, I have a dream in the drawer: I would love to illustrate a book, a short collection of recent scientific discoveries made into figures for students. The idea comes not only from the fun I’d have in engaging with such a project but also from my belief that if you make something interesting for students, they will enjoy studying it. At school, drawing things was my way to make things simple for myself – when things are simple it is easier to understand them, and in turn appreciate them.
We’re looking for new people to feature in this series throughout the year – whatever kind of art you do, from sculpture to embroidery to music to drawing, if you want to share it with the community just email thenode@biologists.com (nominations are also welcome!).
We are looking for researchers to participate in the special issue “Maternal-Fetal Crosstalk Impacts on Offspring Development” in Frontiers in Cell and Developmental Biology journal.
In this episode we’re taking a look at the history of gene editing, from the early days of restriction enzymes in the 1960s through to the CRISPR revolution and the very latest base editing techniques.
But while these tools are undeniably powerful and hold great promise for treating disease, with great power comes great responsibility: what are the acceptable limits of genome engineering in humans, and will we see more CRISPRd babies in the future?
If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip
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The seventh webinar in our Development presents… series will be chaired by Development Editor, François Guillemot (The Francis Crick Institute), who has brought together three exciting talks on the development of the nervous system.
Wednesday 14 April 2021 – 17:00 BST (GMT+1)
Iva Kelava (Postdoc in Madeline Lancaster’s lab at the Laboratory of Molecular Biology) ‘Sex hormones and the human developing brain’
Stéphane Nedelec (from the Institut du Fer à moulin) ‘Dynamic extrinsic pacing of the HOX clock in human axial progenitors control motor neuron subtype specification’
The webinar will be held in Remo, our browser-based conferencing platform – after the talks you’ll have the chance to meet the speakers and other participants at virtual conference tables. If you can’t make it on the day, talks will be available to watch for a couple of weeks after the event; details will be posted on the Node or you can sign up to our mailing list for email alerts.
Feel free to share this poster with your colleagues:
In mucociliary epithelia, such as the mammalian airway epithelium or the embryonic epidermis of Xenopus tadpoles, the correct balance between multiciliated cells (MCCs) and secretory cells provides the functional basis for removal of particles and pathogens to prevent infections and to maintain organismal oxygenation (Walentek and Quigley, 2017).
While the rest of the Walentek lab is working on how mucociliary epithelia are established during development, I am the “black sheep” of the lab, because I wanted to know how this tissue is remodeled to become a non-ciliated epithelium during metamorphosis. Indeed, mucociliary epithelial remodeling and MCC loss are observed in human chronic lung disease as well as during metamorphosis of the Xenopus epidermis. However, it remained unresolved how and why MCCs were lost in Xenopus, and how the process compares to observations made in mouse models of airway inflammation and human cells from chronic airway disease patients. By addressing this question, we hoped to find the underlying molecular mechanism for MCC loss in Xenopus, and to establish a new model to study mucociliary remodeling in the vertebrates.
Thus, I started my PhD on the loss of MCCs during Xenopus tadpole development. I found it especially interesting that everyone in the field was aware of this loss, but no one really looked at it in detail before to see how and when this was precisely happening. The first paper that I found on this topic dated from 1988 (Smith et al, 1988), where the authors describe a loss of MCCs from areas around the developing lateral line. Additionally, another group described MCCs with reduced ciliation that were positive for mucus staining in advanced tadpole stages (Nishikawa et al., 1992). But then, I could not find further work that would explain these phenomena. It remained unclear how the complete loss of MCCs from the tadpole epidermis was accomplished and why MCCs were lost in the first place. Therefore, I got interested in the case and I was hoping to resolve this mystery like a detective.
At the start of my investigations, I first established the time line of MCC loss during Xenopuslaevis tadpole development by immunofluorescence confocal- and scanning electron-microscopy.Being new to the field of Xenopus epidermis biology, my first discovery was to see how amazing normal MCCs look, with their hundreds of motile cilia and their dense apical F-actin network. Then, I started to observe how their morphology changed over time of epidermal remodeling. During these studies, I found that MCCs were lost during a first “local” phase in areas where lateral line neuromasts (NMs) would emerge. A bit later, MCCs were also lost everywhere else in the epidermis. This suggested to me that there could be two distinct mechanisms for MCC loss, depending on the location and the timing. So, I set out to investigate both processes in more detail.
MCCs undergo lateral line-induced apoptosis
Investigating the relationship between MCC loss and neuromast (NM) development seemed like a good start, because it confirmed previous findings by Smith et al. in other frog species and demonstrated a conservation of this phenomenon.
Figure 1 : MCCs are lost locally around the neuromasts of the lateral line. A)MCC stained for acetylated-α-tubulin and actin. MCC express low level of p27::GFP. B) Neuromast of the lateral line labeled by p27::GFP and stained for acetylated-α-tubulin and actin.
To understand the temporalrelationship between the migration of the lateral line primordium, NM deposition and the loss of MCCs, I started to transplant fluorescently labeled lateral line primordium cells into non-fluorescent hosts, and to use a transgenic reporter line (p27::GFP) (Rubbini et al., 2015) which expresses GFP in the lateral line primordium and NMs. Interestingly, I found that MCCs are still present while the primordium is migrating, but are lost when NMs emerge through intercalation in the epithelium (Figure 1). In parallel, I conducted immunofluorescent staining, confocal microscopy and analyzed scanning EM images, which showed that MCCs could be shed from the epithelium, suggesting removal through apoptosis. Therefore, I stained tadpoles with an anti-cleaved Caspase 3 antibody and performed TUNEL assays that showed signals exclusively in MCCs. This confirmed that MCCs over the lateral line were lost via apoptosis. Thus, we hypothesized that emergence of neuromasts induces loss of MCCs via shedding-apoptosis.
As we like to do in the lab, I first performed an easy and fast experiment to provide a proof-of-concept for our hypothesis that NM emergence is really the cause of local MCC loss. For that, I simply ablated the anterior part of the embryo where the lateral line primordium originates from and from where primordial cells migrate out in various directions to populate head, trunk and tail with NMs. This experiment confirmed that MCCs were not locally lost in absence of NM deposition.
But how did NMs induce this loss of MCCs? Looking into the literature, we realized that NMs are signaling centers that express Notch ligands. During specification, MCCs are inhibited by Notch signaling, and mature MCCs retain some level of Notch receptor expression, which means that they could also respond to Notch signaling changes. This led us to hypothesize that high Notch signaling from NMs could signal to MCCs and induce apoptosis. It did not take too long to find out which ligands are expressed in NMs, because fellow graduate student Magdalena Brislinger in the lab is working on Notch signaling and has analyzed the expression of all Notch ligands and receptors throughout early Xenopus development. On her beautiful images of sectioned tadpoles stained for Notch ligand expression, we found that the lateral line primordium and NMs express jag1 at high levels and induce hes1 expression in the overlying epithelial cells. This validated that NMs are Notch signaling centers that communicate with epidermal cells. By incubating the embryos in DAPT, which inhibits Notch signaling, and by performing Caspase 3 and TUNEL assays, I could show that MCC apoptosis and loss over the lateral line were suppressed in absence of Notch activation, confirming that Notch signaling is required for MCC loss via apoptosis.
The majority of MCCs coordinately trans-differentiates into Goblet secretory cells
Figure 2: MCCs trans-differentiate into a mucous-secretory goblet cells. Left: Normal MCC stained for acetylated-α-tubulin (grey), PNA (magenta) and actin (green). Right: Trans-differentiating MCC stained for acetylated-α-tubulin (grey), PNA (magenta) and actin (green) shows reduce ciliation and acetylation as well as mucin production and apical actin remodeling.
But my investigations were not finished yet! Broad epidermal TUNEL staining was missing from areas farther away from the lateral line, which made us think that an alternative mode of MCC removal was used there. To find out what was going on, I stained tadpoles throughout the time of global MCC loss to visualize MCC cilia, to identify secretory cell types via mucus staining, and for F-actin to outline cell borders and to assess cell morphology. Interestingly, confocal microscopy on these samples revealed altered apical F-actin morphology in a subset of MCCs, which also stained positive for mucus. I will always remember the moment when I found those cells and, still new to the Xenopus field, I ask Peter naively if it was normal to see some MCCs with mucus, and he got all excited about the finding (Figure 2).Quantification of this dataset showed that while the overall number of MCCs decreased over time, the proportions of mucus-positive MCCs increased. This suggested MCC to goblet cell trans-differentiation as an additional mechanism for MCC removal in the Xenopus epidermis.Subsequently, we also found that mesoderm-derived intermediate Notch signaling levels cause MCC to goblet cell trans-differentiation, but only when thyroid hormone was produced, which elevated Jak/STAT signaling that has an anti-apoptotic effect and is required to allow MCCs to undergo this transition- probably by making them more resistant against stress. Based on these findings, a key aspect of the paper became the dual role of Notch in MCC apoptosis and cell fate change. We (and the reviewers of our paper) thought that a genetic manipulation of Notch signaling, which could induce both behaviors in young MCCs, would strongly support our statement. Thus, I wanted to use a Notch gain-of-function approach to manipulate MCCs specifically. So, I generated a construct that expresses constitutive active Notch intracellular domain NICD fused to GFP under the control of a MCC-specific promoter. The cloning seemed easy but not if you consider the unexpected magic of cloning. After struggling for weeks to have this construct ready and perform my last experiments for this paper, I finally succeeded to generate the construct and open a bottle of Champaign to celebrate my success. After injecting the construct, I could see nuclear GFP in MCCs, but importantly, a significant proportion of GFP-positive cells showed goblet cell morphology, demonstrating that Notch signaling activation in MCCs can trigger fate change. Additionally, TUNEL assays showed the induction of apoptosis in early stage tadpoles. Together, these experiments provide evidence that ectopic Notch signaling can induce apoptosis as well as cell fate conversion in MCCs.
MCCs retract cilia and loose basal body components
Figure 3: Trans-differentiating MCCs remodel basal body distribution and composition. Confocal micrograph of a normal MCC and a trans-differentating MCC reveals disorganized basal bodies (Centrin4-CFP, grey), cilia de-acetylation (Ac.-α-tubulin, green), F-actin remodeling (Actin, green) and reduce levels of basal body distal appendages proteins (mCherry-Cep164, magenta), actin interactors (FAK-RFP, magenta), and rootlet components (Clamp-RFP, magenta).
We also found that trans-differentiation is initiated through loss of ciliary gene expression, including foxj1 (a master transcription factor for motile cilia maintenance) and pcm1 (a protein that protects cilia and basal bodies from degradation). At the cellular level, we could observe altered proteostasis, cilia retraction, basal body elimination (Figure 3, Figure 4G) as well as initiation of mucus production and secretion. Some of these changes resembled processes observed during primary cilia retraction, which is initiated in cycling cells when they re-enter the cell cycle to divide. So, I wondered if MCCs that trans-differentiate and become goblet cells could they also re-enter the cell cycle and divide again? I found that trans-differentiating MCCs also lost expression of the cell cycle inhibitor p27, supporting the idea that MCCs could re-enter the cell cycle, and presence of a hybrid cilium (Liu et al, 2020) could suggest that MCCs retrain a parental centriole that could serve as a base for mitotic division (Figure 4E). Therefore, I tried to follow individual cells using live-cell imaging and various techniques to label individual MCCs before trans-differentiation, including photo-convertible proteins, MCC-specific fluorescent labeling, etc. However, either the constructs turned out to be toxic to the cells, or the labeling could not be restricted to individual MCCs, thus, I could not exclude the possibility that I followed co-converted goblet cells, or I lost the cells during imaging. So, setting up this experiment properly would require a transgenic line, which takes a long time to establish in the Xenopus laevis system. Sadly, due to these technical limitations, we were not able to provide genetic tracing data in this paper. But we are looking forward to find out more about the cellular behaviors of MCCs during and after the trans-differentiation process in the future. This will fill an extremely important gap in our understanding of tissue remodeling and MCC loss!
Figure 4: Cilia and basal body structure visualised by electron microscopy. A) Transversal section and transmission electron microscopy of a motile cilium and its associated basal body. Motile cilia of MCCs are composed microtubules in a 9+2 configuration, a transition zone and a basal body. B-F) Parallel sections and transmission electron microscopy of a cilium (B), basal bodies with one (D) or two (E) basal feet and rootlet (F). G) Trans-differentiating MCCs are enriched in electron-dense structures corresponding to lysosomes.
So, in summary, our work describes two modes for MCC loss during vertebrate development, the signaling regulation of these processes, and demonstrates that even cells with extreme differentiation features can undergo direct fate conversion (Tasca et al.,2021). In addition to our scientific findings, this project was an amazing experience for me personally. It is a fantastic feeling to know that I could unravel, in large parts, a decades-long mystery, and to generate insights into the molecular processes of mucociliary tissue remodeling. I also enjoyed the scientific investigation, the collaboration with group members as well as with the Mitchell lab, and with Martin Helmstädter from the group of Gerd Walz in our department, who provided the beautiful electron microscopy images for the paper.
References
Liu, Z., et al., Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells. Dev Cell, 2020. 55(2): p. 224-236 e6. DOI: 10.1016/j.devcel.2020.09.016
Nishikawa, S., J. Hirata, and F. Sasaki, Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis. Histochemistry, 1992. 98(6): p. 355-8. DOI: 10.1007/BF00271070
Rubbini, D., et al., Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells. J Neurosci, 2015. 35(47): p. 15752-66. DOI: 10.1523/JNEUROSCI.1099-15.2015
Smith, S.C., M.J. Lannoo, and J.B. Armstrong, Lateral-line neuromast development in Ambystoma mexicanum and a comparison with Rana pipiens. J Morphol, 1988. 198(3): p. 367-379. DOI: 10.1002/jmor.1051980310
Walentek, P. and I.K. Quigley, What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia. Genesis, 2017. 55(1-2). DOI: 10.1002/dvg.23001
Tasca, A., et al., Notch signaling induces either apoptosis or cell fate change in multiciliated cells during mucociliary tissue remodeling. Dev Cell, 2021. 56(4): p. 525-539 e6. DOI: 10.1016/j.devcel.2020.12.005
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