In this episode from our centenary series exploring 100 ideas in genetics, we’re telling tales of sex and death, and exploring the very darkest side of genetics. We discover how Francis Galton’s eugenic ideas led to some of the worst atrocities of the 20th century, and ask how his legacy should be honoured today – if at all? Plus, the evolution of sex, and the total eradication of mosquitoes.
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A Research Fellow position is available at the Australian Regenerative Medicine Institute to study how body proportions are regulated during normal development and after perturbation of growth of the long bones. The focus of research is on the communication mechanisms that operate between organs to maintain body proportions during development, especially those involving the circulatory and nervous systems. The main animal model utilised in the research group is the mouse (embryos and pups) with the future goal of starting a zebrafish colony. The expected duration of the project is at least 2 years.
If you want to join a vibrant group and institute, and live in Melbourne, one of the most liveable cities in the world, follow the link!:
a doctoral qualification and/or progress towards a doctorate in the relevant discipline or a closely related field
Knowledge and Skills
Management of a complex mouse colony with multiple genetically modified strains.
Previous experience working with mouse placenta: structure, staining, histological techniques.
Proficiency with routine laboratory techniques including DNA cloning, histology, western blot, immunohistochemistry, tissue culture; use/maintenance of common laboratory apparatus, storage and handling of hazardous materials
Proficiency with 3D imaging and image analysis software (Imaris/ImageJ/CellProfiler).
Demonstrated outstanding work ethics and quality standards.
Ability to work and think independently, solve complex problems by using discretion, innovation and the exercise diagnostic skills and/or expertise.
Well-developed planning and organisational skills, with the ability to prioritise multiple tasks and set and meet deadlines. Excellent record keeping skills are a must.
Excellent written communication and verbal communication skills with proven ability to produce clear, succinct reports and documents
A demonstrated capacity to work in a collegial manner with other staff in the workplace
Demonstrated computer literacy and proficiency in the production of high level work using software such as Microsoft Office applications and specified University software programs, with the capability and willingness to learn new packages as appropriate
Experience in the following categories will be considered a plus: dissection of mouse limb/bones, maintenance of zebrafish colony, CRISPR-based functional screens, ex vivo tissue culture and management of (under)graduate students.
For more information, feel free to contact Alberto Rosello-Diez (alberto.rosellodiez@monash.edu).
Research technician position available for a project focused on functional technique development in annelid worms, with a conceptual focus on evo-devo and regeneration biology. This position is part of an NSF-funded project focused on developing approaches to test gene function in post-embryonic (juvenile and adult) stages of several annelid species. The project is a collaboration between Alexa Bely (U Maryland), Duygu Ozpolat (Marine Biological Laboratory, Woods Hole), and Elaine Seaver (U Florida, Whitney Marine Lab) and there are substantial interactions among the three lab groups.
This position is in the lab of Elaine Seaver, with primary focus on the annelid Capitella teleta. Position is for 1 year, with possibility of renewal for a second year, and with a start in Fall 2019.
Required qualifications: bachelor’s degree in biological sciences or related field, at least 1 year research experience in molecular or developmental biology, good fine-motor skills, ability to troubleshoot and persevere, effective time management and organizational skills, and team-oriented outlook. Preferred but not required: experience with microinjection, electroporation, and approaches to disrupt gene function.
Through this position, the successful candidate will gain valuable experience in the development of novel methods, will gain experience in both molecular and organismal research, and will be part of a highly interactive and supportive team.
Research technician position available for a project focused on functional technique development in annelid worms, with a conceptual focus on evo-devo and regeneration biology. This position is part of an NSF-funded project focused on developing approaches to test gene function in post-embryonic (juvenile and adult) stages of several annelid species. The project is a collaboration between Alexa Bely (U Maryland), Duygu Ozpolat (Marine Biological Laboratory, Woods Hole), and Elaine Seaver (U Florida, Whitney Marine Lab) and there are substantial interactions among the three lab groups.
This position is in the lab of Alexa Bely, with primary focus on the annelid Pristina leidyi. Position is for 1 year, with possibility of renewal for a second year, and with a start in Fall 2019.
Required qualifications: bachelor’s degree in biological sciences or related field, at least 1 year research experience in molecular or developmental biology, good fine-motor skills, ability to troubleshoot and persevere, effective time management and organizational skills, and team-oriented outlook. Preferred but not required: experience with microinjection, electroporation, and approaches to disrupt gene function.
Through this position, the successful candidate will gain valuable experience in the development of novel methods, will gain experience in both molecular and organismal research, and will be part of a highly interactive and supportive team.
Most bones in the vertebrate skeleton are made in the same way – endochondrial ossification – yet they display a variety of shapes and sizes. The question of how these unique bone morphologies, including the superstructures that protrude from their surfaces, arise during development is still unclear, and the subject of a new paper in Development. We caught up with first author Shai Eyal and his supervisor Elazar Zelzer, Professor in the Department of Molecular Genetics at the Weizmann Institute of Science in Rehovot, Israel, to find out more about the story.
Elazar Zelzer (L) and Shai Eyal (R).
Elazar, can you give us your scientific biography and the questions your lab is trying to answer?
EZ We walk, run and jump using the complex and ingenious musculoskeletal system. It is therefore puzzling that, although each of its components has been extensively studied, research on the musculoskeleton as an integrated system, and in particular on how it is assembled, has been scarce. My vision as a scientist is to demonstrate the centrality of molecular and biomechanical crossregulation between musculoskeletal tissues in the development and function of this system. In 2004, I established my independent research group at the Weizmann Institute. Over the years, I was joined by a group of extremely talented students. Together, we have made interesting discoveries about various aspects of musculoskeletal biology, including the regulatory interactions between the developing skeleton and its vasculature, the regulation of bone morphogenesis and joint formation by muscle-induced mechanical signals, and the development of secondary structures such as bone eminences and sesamoid bones.
One of the cornerstones of my philosophy as a mentor of young creative people is to allow team members space for individual expression. I believe that this has been key to our ability to develop new avenues of research. Indeed, we have recently opened a new and exciting direction by introducing the significance of proprioceptive signalling in skeletal regulation, specifically in spine alignment, bone regeneration and joint development.
And Shai, how did you come to work in the Zelzer lab, and what drives your research today?
SE I majored in Life Sciences at the Hebrew University of Jerusalem. During that time, I was exposed to various fields of biological research; however, the field that really caught my attention was developmental biology. When I think of how a single cellular unit, a fertilized egg, can develop into endless forms and shapes, it is like biological magic to me. Therefore, when I moved to the Weizmann Institute of Science, I looked for labs in which I could continue studying concepts in developmental biology. Looking into the research that was carried out by the Zelzer lab, I was very intrigued. Skeletogenesis, in particular how the skeletal and muscular systems are integrated, is a very interesting and largely unknown process. With Eli’s superb mentorship and the supportive lab atmosphere, I was able to highlight several key processes taking place during sesamoid bone formation and long bone morphogenesis. Today, my interest has shifted away from skeletogenesis, but I am still as hooked on developmental biology as I was when I began my research more than a decade ago.
Prior to your work, what was known about how each bone gets its distinctive shape?
EZ Although the generic mechanisms of long bone development, in particular elongation, have been extensively studied, far less was known about how each bone gets its distinctive shape. Nevertheless, several intriguing discoveries have been made in recent years. Muscle force was shown to regulate appositional growth, which forms the circumferential shape of the bone (Sharir et al., 2011), and bone superstructures were shown to develop modularly from a distinct population of Sox9 and scleraxis (Scx) double-positive progenitor cells under regulation of TGFβ and BMP signalling (Blitz et al., 2009, 2013). Lastly, the relative positions of superstructures along the bone shaft was found to be determined both by bone modelling and by a unique ratio between growth rates at the two bone ends (Stern et al., 2015).
SE The process of endochondral ossification was described a long time ago. In short, it is a process in which pools of Sox9-expressing chondroprogenitors condense into discrete cartilage templates, which later will ossify into mineralized bones. Until recently, the dogma in the field was that long bones acquire their specific morphologies at the stage of cartilage condensation, with some changes occurring after ossification by bone modelling. However, studies that were done by a former graduate student, Einat Blitz, challenged this dogma and presented evidence of a secondary population of chondroprogenitors that co-express both Sox9 and Scx.These cells are specified after the primary condensation stage and their addition forms superstructures along the bone shaft and thereby contributes to the final bone morphology. This finding served as our starting point and raised the question of how the addition of the Sox9+/Scx+cells to developing bones at specific sites is controlled.
Sagittal sections through the proximal humeri of limbs from control Prx1-Cre;Pbx1floxed mutant embryos, stained against SOX9 (red), COL2A1 (green) and DAPI (blue).
Can you give us the key results of the paper in a paragraph?
SE & EZ First, we show that the Sox9+/Scx+ progenitor cells contribute extensively to the formation of bone superstructures in mouse. Importantly, we show that they contribute not only to bone eminences, but also to condyles and sesamoid bones, which are auxiliary bones that are important for proper locomotion. Then, we show that the genetic programme that controls the patterning of these progenitors contains both global and regional regulatory modules, as Gli3 regulated this process globally, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 acted as either proximal or distal regulators. Finally, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in tandem. Together, these results demonstrate genetic regulation of superstructure patterning, thereby supporting the notion that long bone development is a modular process.
How do your findings contribute to the modular model of bone morphogenesis?
EZ Previously, we showed that bone superstructures originate from a dedicated pool of progenitors. Now, we expand the modular model of bone development by identifying part of the genetic programme that controls the patterning of these progenitors at specific locations along the bone. Furthermore, we show that modularity exists within the process of bone superstructure patterning, as some components of this genetic programme regulate all superstructures, whereas others regulate only proximal or distal superstructures. From an evolutionary perspective, this strategy allows changing the position of superstructures and, thereby, the locomotor abilities of the organism without rewriting the entire skeletogenic programme.
SE What allows modularity in long bone morphogenesis is the appearance of the secondary Sox9/Scx progenitors and the fact that they can be regulated independently from the sox9-expressing cells of the bone shaft. Our findings provide further evidence of the abundance and contribution of these progenitors and highlight several genetic components and programmes that regulate their patterning both globally and locally. These are missing from the present dogma of a rigid patterning programme executed by a single condensation step of a single population of progenitors, therefore changing our view of bone morphogenesis.
How do you think that Sox9+/Scx+ chondroprogenitors get to the right sites to form the superstructures?
SE & EZSox9+/Scx+ chondroprogenitors could get to their locations in one of two ways. The first option is their migration to specific condensation sites, where they will form superstructures. Alternatively, it is possible that the Sox9+/Scx+ chondroprogenitors are specified from cells that are already present at the designated superstructure formation sites. In the latter scenario, the mechanism would involve activation of a chondrogenic programme in selected cell subpopulations at specific spatiotemporal positions. Either way, it appears that the mechanism that decides where superstructure development should take place involves Gli3 and the Pbx and Hox genes.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
SE It is hard to choose a specific eureka moment. I think every experiment we performed presented some advancement toward our findings. Perhaps such a moment would be when we first put together the results from our Sox9/Col2A1-CreER;tdTomato 3D reconstructions and the SOX9/ScxGFP-labelled sections. It was then that we saw clearly for the first time how abundant these progenitors were. That was a very reassuring moment that gave me the confidence to move forward with studying the patterning of superstructures.
And what about the flipside: any moments of frustration or despair?
SE I think that working with a model organism, and especially with mutant or transgenic lines, can sometime be a source of frustration. For example, we had to postpone the revision of two papers twice by more than 6 months because we had to revive our double-mutant colonies sufficiently to carry out additional experiments that were requested by the reviewers. Another experiment required us to cross the compound HOX11 mutants to a ScxGFP reporter to follow the Sox9+/Scx+progenitors. The experiment was straightforward but the genetic ratio was against us and, after two years of trying, we had to submit the manuscript without these results.
So what next for you after this paper – I understand you are now in the US?
SE That is correct. I am now doing my postdoctorate in Professor David Traver’s lab at the University of California, San Diego (UCSD). The main areas researched in the lab are the formation and development of the haematopoietic stem cells and their niche in both juvenile and adult zebrafish. The advantage of working with zebrafish is that their embryonic development is rapid and the developing embryos are transparent. That gives us the opportunity to follow cellular behaviours over long periods of time with high-resolution imaging. Specifically, for my projects I am working on setting up a pipeline that will use cutting-edge labelling and imaging technologies to allow me to label and follow single cells and their cellular fates and lineages. That system will allow me to study heterogeneous populations within the blood system by following individual cells within such populations.
Where will this work take the Zelzer lab?
EZ We are interested in further understanding the process of superstructure development, both upstream and downstream to the
current study. We are investigating the unique identity of the progenitor cells that give rise to superstructures, focusing on their single cell transcriptome and chromatin landscape. At the same time, we are pursuing the molecular and biomechanical signals that regulate superstructure development. These studies will shed light not only on bone morphogenesis, but also on the connection of tendons to bones during musculoskeletal assembly. To achieve these goals, we would welcome new partners. We are always looking for curious students and postdocs on the quest for self-fulfilment, who are interested in musculoskeletal biology, organ shaping or the assembly of complex systems.
In my view, the field of developmental biology is currently at a crossroads. Pushing the field back to centre stage may require the integration of other fields such as physiology, engineering and mechanobiology. The musculoskeleton is the ideal model system for such integration. An example for that is the new direction we have recently taken in studying the regulatory effect of the proprioceptive system on skeletal development and function.
The field of developmental biology is currently at a crossroads
Finally, let’s move outside the lab – what do you like to do in your spare time in Rehovot and San Diego?
EZ I like mountain biking and practicing jujutsu.
SE I have moved to San Diego with my wife and two amazing children. Coming to San Diego opened up many new experiences for all of us. The city is beautiful and there are endless family attractions, especially in the larger area of Southern California. We also like to travel and camp on long weekends. For myself, I started rock climbing during my PhD in Israel and continue to climb in San Diego. In addition, as I am in California, it didn’t take long before I took up surfing, too. It is amazing to have the opportunity to start the day by surfing among sea lions and dolphins.
Scientists at the Federal University of Rio de Janeiro, Brazil, in collaboration with the Max Planck Institute of Molecular Cell Biology and Genetics, Germany, discovered that a single transcription factor drives retinal progenitor cells to reacquire the potency to generate Retinal ganglion cells. The transcription factor is Klf4, which became notorious for its role in reprogramming cells towards pluripotency. The novel findings open a new door to investigate regenerative therapies for retinal diseases that cause irreversible vision loss, such as Glaucoma. The research was published in the scientific journal Development at https://dev.biologists.org/content/early/2019/08/08/dev.176586.
Ganglion cells in the retina are the source of the optic nerve and are vital for seeing as this nerve sends the messages from the retina to the brain. Their degeneration in glaucoma leads millions of people to permanent vision impairment. Current treatments at best control the progression of the disease, but recovery of normal vision require not only the prevention of cell death but also regeneration of cells and their axons.
To address the latter, Mauricio Rocha-Martins, Mariana Silveira and a team of researchers investigated both the role of Klf4 in the normal development of ganglion cells, as well as its potential to coax retinal progenitor cells to generate these neurons.
Although an endogenous role of Klf4 in retinal development could not be confirmed, overexpression of this gene in retinal progenitors of newborn rats led to a switch in cell fate. Instead of giving birth mostly to photoreceptors (which sense light) the progenitors generated retinal ganglion cells, the projection neurons of the retina. Remarkably, less than two days after Klf4 overexpression, a molecular program was activated that is similar to that responsible for the generation of ganglion cells during retinal development.
“We literally cheered the first time we noticed that, instead of positioning on the photoreceptor layer, where most of those cells should be in normal conditions, this transcription factor led the progeny of progenitor cells to move to the region of the retinal ganglion cells, and project axons toward the optic nerve,” the authors said.
Retinas of control and KLF4+ 39h after in vitro electroporation, immunolabeled for GFP (green) and KLF4 (magenta); DAPI staining in blue
Future work will focus both on improving the protocol to guarantee that these induced ganglion cells acquire mature properties, but also to direct this strategy to the glial cells of the retina, of which the ability to regenerate retinal ganglion cells was lost in mammals through evolution, but remains in fish.
“We believe there is a long way towards actual therapy, and plenty to be understood, but our data indicate that the program to generate ganglion cells can be reactivated, which may open new directions for regenerative therapies,” said the authors. “This is a starting point,” they conclude.
This post was originally posted on the MRC Weatherall Institute of Molecular Medicine’s blog
Dominic Owens, PhD student in the De Bruijn group at MRC MHU, recounts how puzzling results and a fortuitous lab meeting uncovered unexpected outcomes of CRISPR editing and changed the direction of his research.
It’s May 2018 and I’m feeling nervous. I was presenting the latest results in my PhD research project at a large joint lab meeting involving several research groups. The last few weeks of experiments hadn’t gone the way I expected, and I had gotten some odd results that were puzzling. Unknown to me, from this lab meeting, I would be embarking on a yearlong journey of despair and eventual discovery which became central to my first publication, published in Nucleic Acids Research (Owens et al. 2019, https://doi.org/10.1093/nar/gkz459).
I had been using CRISPR-Cas9 to examine the function of ‘junk DNA’. So-called ‘junk DNA’ contains no genes but instead is increasingly understood to play important roles in regulating gene expression. The enhancers I study are believed to regulate blood stem cell generation, by activating a gene called Runx1 during embryonic development. This gene is required for blood stem cell generation and is frequently mutated in human leukaemia.
In mouse cells, I had been using CRISPR to remove specific enhancers, and wanted to see what the impact on blood cell generation would be. If removing these enhancers made a difference, then I could reveal unknown factors important for blood stem cell generation.
After using CRISPR to cut out this DNA, I needed to sequence short pieces around the cut sites to determine what kind of cut took place. As is common practice, I used PCR to amplify the DNA and Sanger sequencing to read it, over a region around 200 base pairs on either side of the target sites. After doing this, I often saw cells with what looked like both copies of the region deleted, right where we expected. However, when I tested the function of the cells, I saw inconsistent results. Even though the sequencing told me that the region was missing, some of the cells were behaving like it was still there.
Back to the lab meeting: a colleague who is very experienced with CRISPR from a different lab suggested that I could be seeing larger than expected deletions which meant that one of the PCR primer binding sites I relied on was no longer available. This would in effect prevent one copy of the region from being amplified and sequenced, and thus render PCR blind to such larger deletions. I can still remember that moment—the significance of what she said truly dawning on me. So, after the lab meeting, my lab mates and I discussed what to do next. We decided that I should look for larger deletions in all the cell lines we had made. To do this I used longer range PCR, with primers that bound to the DNA much further away from the CRISPR target sites. This way I would be able to detect any larger deletions, if they were there. And when I looked, I found them. In fact, no matter how far I looked, larger deletions were always there.
This left me desperately wanting answers to three questions: why are these larger deletions happening? Just how large can the deletions be? And can they be predicted? The only problem was: I was working in a lab of exceptional developmental biologists, not genome engineers! So, my supervisor and I reached out to two local experts – Lydia Teboul at MRC Harwell, an expert in CRISPR editing, and Peter McHugh at MRC WIMM, an expert in DNA repair. Both were generous with their time, helping me plan and perform the necessary experiments to answer the questions I was puzzled about.
Image credit: Martin Phelps
By sequencing many examples of larger deletions, I found that short regions of homology (microhomologies) were commonly found in places where the cut DNA was repaired back together. This suggested that a DNA repair pathway called microhomology-mediated end-joining was a likely culprit.
Next, I used a technique called droplet digital PCR that is incredibly useful for interpreting the outcomes of CRISPR experiments. The method uses an oil in water emulsion of droplets to perform 15,000 individual PCR reactions in a single tube. This is advantageous because it allows quantification of DNA with much greater accuracy and precision than comparable techniques. Taking advantage of this accuracy, my colleagues and I mapped deletions across thousands of base pairs of DNA in populations of cells targeted with CRISPR, without biases arising from the locations of longer-range PCR primers.
Modelling the effects of various potential predictors on the distribution of deletion sizes revealed the driving forces determining how large a deletion is likely to be. I found that the frequency of larger deletions was highly dependent on CRISPR’s cutting efficiency at a target cut site and the distance from it. This means that the closer to a cut site, the more likely a region is to be lost in a deletion. This makes sense of course, but this distance-dependency shows that the microhomology sequences often associated with the repaired breakpoints themselves cannot be used to predict the size of the deletions. This is in stark contrast to shorter CRISPR deletions, which have recently (Shen et al. 2018, Nature, and Allen et al. 2018, Nature Biotechnology) been shown to be highly predictable based on sequence characteristics such as microhomologies.
The work highlights important caveats about CRISPR that I wished I had known when embarking on this project. Larger deletions occur frequently after CRISPR editing and they will be easily missed if you aren’t looking out for them. Deletions can be any size when looking at individual cells or animals, but in general are much more common within 3 kilobases from a cut site. Even though extremely large deletions are rare, given a large enough population of cells, they should be expected to occur. In the case of gene-edited somatic cells for gene therapy, for example, a single rare oncogenic larger deletion could have disastrous health outcomes for recipient patients. In the future, inhibiting microhomology-mediated end-joining could help reduce this problem. Alternatively, gene-editing approaches that avoid DNA double-strand breaks altogether may be preferable.
I must admit, I was slightly disappointed when the questions I had set out to answer during my PhD didn’t quite work out. But I have also learned valuable lessons. It is often tempting to ignore outliers or repeat experiments that just look weird. But by staying curious and motivated to understand the things that don’t quite add up, you will understand the science more deeply, and maybe find your next big breakthrough!
We are increasing our investment in paediatric cancer research by launching two new significant funding calls. We would like to encourage proposals to investigate the following concepts amongst others-
the basis of tumour initiation and progression
novel models that would enhance pre-clinical research.
The Stand up to Cancer-Cancer Research UK Paediatric New Discoveries Challenge focuses on multidisciplinary, multi-institutional, transatlantic teams that want to pursue a step change in our understanding of the drivers of paediatric cancers and the development of novel or repurposed medicines, treatment strategies or technologies
If you have any questions then please don’t hesitate to contact Sheona Scales (Cancer Research UK Paediatric Lead) sheona.scales@cancer.org.uk .
A technician/lab manager position is available in the laboratory led by Ricardo Mallarino, Department of Molecular Biology, Princeton University (www.mallarinolab.org). The lab focuses on uncovering the genetic and developmental mechanisms by which form and structure are generated during vertebrate embryogenesis. We combine the study of emerging and traditional model organisms to explore questions relating to patterning and evolution of novelty in the mammalian skin. The lab uses a variety of approaches, including experimental embryology, genetics, genomics, imaging, and mathematical modelling to uncover gene function and understand mechanisms of evolutionary change.
Responsibilities
The successful candidate will manage essential operating procedures for the lab and work with the PI and other lab members to design and perform experiments. Specifically, the candidate will develop in vivo functional and genome editing approaches in non-traditional model species and perform cell-culture based enhancer screens. Other duties will include molecular cloning, histology, in situ hybridization, nucleic acid extraction and library preparation, tissue culture and media preparation, as well as lab maintenance, organization, safety and ordering.
Essential Qualifications
Bachelor’s or Master’s degree in the biological sciences plus previous laboratory experience is required. Previous experience in cell culture techniques and rodent model systems is necessary (colony management, genotyping and dissections). Basic molecular biology methods and computer literacy are essential. Must be highly motivated and have demonstrated ability to plan, coordinate and carry out independent research. Excellent organization skills, ability to communicate effectively and willingness to learn new techniques are necessary traits. Applicants should be willing to commit to the position for at least two/three years.
Preferred Qualifications
A strong background in molecular biology and/or genetics is preferred. Knowledge of rodent reproductive biology and surgical skills are a plus. Rank and salary are dependent upon qualifications and experience.
To apply for this position please submit a CV and a cover letter describing research interests to rmallarino@princeton.edu.
My name is Sandra Treffkorn, and I recently finished my PhD in the department of zoology lead by Georg Mayer at the University of Kassel, Germany. In our research group, we focus on studying the evolution of animal diversity by investigating two very interesting but largely understudied taxa, the Onychophora and the Tardigrada. Tardigrades, commonly known as “water bears”, are well known to scientists and non-scientists alike due to their ability to survive extreme environmental conditions including exposure to space [1-3]. In contrast to tardigrades, onychophorans comprise a less known animal group even among biologists. Hence, I will use this opportunity to introduce you to these exciting animals and give you a short overview of our research.
When we get asked what onychophorans are, we sometimes tend to describe them as worms with legs and a smooth velvety skin because this is basically what onychophorans look like (Fig. 1). This is also reflected in their common name “velvet worms”, which refers to the velvety appearance of their skin. But actually, they are not worms at all. Onychophorans are terrestrial invertebrates that belong to the Ecdysozoa – the clade of molting animals [4, 5]. Together with the tardigrades, they are the closest living relatives of the arthropods (spiders, myriapods, crustaceans and hexapods), which is the most abundant and diverse animal group on our planet. Compared to arthropods, however, the anatomy of onychophorans has changed little since the Early Cambrian period (~520 million years ago) [6], and they resemble fossil lobopodians of that time, which represent stem-group panarthropods (onychophorans + tardigrades + arthropods).
Figure 1: World map showing the distribution of Peripatidae (orange) and Peripatopsidae (blue), the two major taxa of Onychophora, and photographs of the onychophoran species Principapillatus hitoyensis (Peripatidae) from Costa Rica and Euperipatoides rowelli (Peripatopsidae) from Australia. World map modified from ref. [10]. Photograph of P. hitoyensis modified from ref. [18]; image of E. rowelli was taken by Ivo de Sena Oliveira.
The onychophoran body comprises a head, a uniform worm-shaped trunk bearing a variable number of unjointed limbs, and a limbless posterior anal cone (Fig. 1). The head is composed of three segments, the limbs of which have been modified into three pairs of specialized appendages: the antennae, the jaws, and the slime papillae [7, 8]. The slime papillae are used in a fascinating way to capture the prey: two jets of a sticky secretion are ejected by the slime papillae, which immediately immobilizes the prey (woodlice, crickets and other small invertebrates) [6, 9]. Thus, even though onychophorans are relatively slow, they are very effective hunters. However, the slime is not only used for prey capture but also for defense against predators [6, 9].
Onychophorans typically inhabit humid microhabitats such as leaf litter, soil and decaying logs in tropical and temperate forests. There are currently about 200 described species of Onychophora which are classified into two major groups, the Peripatidae and the Peripatopsidae (Fig. 1) [6, 10]. While the Peripatidae can be found in equatorial areas such as the Neotropics, West Africa and South East Asia, the distribution areas of the Peripatopsidae are mainly restricted to the southern hemisphere, including Chile, South Africa and Australasia (Fig. 1) [6, 10].
But why do we study onychophorans? What’s so interesting about them? The close phylogenetic relationship of onychophorans to two of the most extensively studied model organisms – the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster – makes them an attractive model for studying the evolution of animal diversity. Studies of onychophorans as an outgroup to arthropods can be used to polarize morphological and developmental characters of the arthropod ancestor. By extending this comparative approach to cycloneuralians (nematodes, priapulids, kinorhynchs and allies), we are able to make assumptions about the panarthropod ancestor, which in turn might help to clarify the ancestral characters in the ecdysozoan lineage when taking comparative data from spiralians and deuterostomes into account. Hence, onychophorans are a key taxon for understanding the evolution of animal diversity.
Our fields of research
In our lab, we study onychophorans in a wide variety of research fields, including taxonomy, phylogeography, population genetics, species conservation, biochemistry, physiology, behavior, evolutionary developmental biology, neuroanatomy and reproductive biology. Our major goal is to identify and understand the evolutionary changes that have taken place since the divergence of onychophorans from arthropods during the Cambrian radiation over 540 million years ago. Thus, even though working on an emerging model organism can be tricky, and we usually have to develop new methods and optimize existing protocols, the work is quite rewarding since we get the opportunity to look back in time and reconstruct evolutionary events that had taken place hundreds of millions of years ago.
A typical day in an onychophoran lab
Animal collection
For most of our work, we use the onychophoran species Euperipatoides rowelli from Australia, which is one of the most extensively studied onychophorans so far and has recently been established as a model for developmental studies (Figs 1; 2) [6]. Adults of E. rowelli are typically dark-blue colored with more or less prominent, reddish terracotta pattern (Figs 1; 2). Females reach up to 6 cm in length while males are usually smaller.
Figure 2: Before the lab work begins, there is the fun part. Members of our lab regularly set out for adventures Down Under to hunt onychophorans in the wild. The type locality of E. rowelli is in the Tallaganda State forest ([19]; top left), which is where we usually collect our animals. They are found in leaf litter (top right) as well as inside decaying logs (bottom left). After collecting the animals, we keep them in plastic jars with some earth and paper towels, in which they are transported to our lab in Germany (bottom right).
Before we can start our lab work, however, we actually have to go out and collect animals. Since E. rowelli lives in Australia but our lab is located in Germany, we are not able just to go out and collect animals from the wild whenever we need them. Thus, we have to go on collection trips from time to time to collect animals in Australia, which we then keep and breed in culture in our lab. We usually collect the animals in the Tallaganda State Forest situated about an hour’s drive southeast of Australia’s capital, Canberra (Fig. 2).
Like arthropods, onychophorans possess tracheae as respiratory organs. Unlike arthropods, however, they are not able to close their tracheal openings, which is why they prefer humid environments to avoid drying out (Fig. 2). Typically, onychophorans can be found in the humid leaf litter or under stones. The easiest way to collect them, however, is actually during dry periods when the leaf litter is too dry for the animals and they are all hiding in the decaying logs, which retain a decent level of humidity (Fig. 2). Instead of crawling around on the ground, turning every stone and searching the leaf litter for animals, we can just pry open the decaying logs and collect the animals inside. So, armed with crowbars, screwdrivers, shovels and other tools useful for prying open decaying wood, we head out and collect the animals, which we then put into plastic jars filled with some earth and damp paper towels to keep them safe for the journey around the world to their new home.
Maintenance in the lab
When back in Germany, the animals are cultured in plastic jars containing a layer of peat and damp paper towels, which we keep in climate cabinets under a constant temperature of 18 °C and 60% humidity (Fig. 3).
Figure 3: Back in the lab, we keep the animals in climate cabinets under specific light, temperature and humidity conditions (left). The animals are kept in plastic jars and plastic boxes with a layer of peat and damp paper towels (right).
The first thing to do after a collection trip is to separate the animals. We keep around six specimens – usually three males and three females – together in each jar. And then the most laborious part of our work begins. Of course, we have to take care of the animals to keep them happy and thriving. The paper towels have to be kept damp at all times in order to provide a nice humid environment (Fig. 4). However, the humidity promotes fungal growth, which can harm the animals. So, in addition to maintaining a constant humidity, we replace the paper towels once a week (Fig. 4). Every three weeks, the animals are fed with crickets, which we leave in the jars for two days. Afterwards, we transfer the onychophorans to new jars with fresh peat and paper towels, again to avoid fungal growth due to the decaying cricket remains. Taken together, animal care has to be done at least twice a week and takes several hours each time. This is especially laborious when a new batch of babies is born, which we separate from the parents into new containers. But with a lot of teamwork, it is doable.
Figure 4: In order to keep the animals happy and thriving, we have to take care of them, which includes changing the paper towels every week (top) and dampening them regularly (middle). Using this procedure, the animals can be kept in their jars for several years (bottom).
Collecting embryos
One major part of our research includes studying embryonic development of onychophorans. For this, we apply a variety of different methods, including in situ hybridization, immunohistochemistry, fluorescence and confocal microscopy, and scanning electron microscopy [11-14]. During my PhD, I specifically analyzed expression patterns of different developmental genes by using in situ hybridization. So, the next step for me was to obtain as many embryos as possible, which is much trickier than for the commonly used model organisms, such as D. melanogaster and C. elegans. In contrast to these model organisms, where embryos are available constantly throughout the year [15, 16], E. rowelli has an annual reproductive cycle with females usually giving birth between November and February under laboratory conditions [17]. Furthermore, the entire embryonic development takes place within the uterus of the mother [6]. In order to obtain embryos of different developmental stages for the experiments (Fig. 5), they have to be dissected from the females manually – usually between September and January – to cover all developmental stages. Thus, we have to plan ahead and get as many embryos as possible during this time, which we then fix and store for long term usage. But since each female typically bears 20 to 40 embryos of different developmental stages, it is possible to cover most stages by dissecting only a few females.
Figure 5: Summary of the seven developmental stages of E. rowelli according to Walker and Tait [17]. Anterior is left in all images. Stage I in ventral view. Stages II to IV in lateral view, dorsal is up; stage V in dorsal view; stages VI and VII in ventrolateral view. Scale bars: 500 μm.
To dissect the embryos, the female is first anaesthetized with chloroform vapor, the entire genital tract is then removed from the female, and the embryos are dissected from the uterus. The yolky embryos of E. rowelli are surrounded by two envelopes: an inner vitelline membrane and an outer chorion, which both persist until birth [6]. These envelopes are removed manually using fine forceps without damaging the embryo. This is especially difficult for the early developmental stages, which are extremely fragile, but with a bit of practice this becomes easier over time. After removing the envelopes, the embryos are fixed using different fixatives (depending on the experiments they are used for) and then stored until they are needed. Treated this way, the embryos can be stored for years.
Establishing a permanent culture of E. rowelli in the lab
Another problem with collecting embryos is that, thus far, we were unable to establish a stable culture in the lab that would reproduce over several generations. Newly collected females usually give birth for two or three years in culture and then stop reproducing. The F1 generation that is born in the lab from the females collected from the wild might also give birth in the lab, but beyond this, reproduction by the following generations has never been reported under laboratory conditions. So, in contrast to other model organisms, we need to collect new animals from the wild every couple of years for our research on embryonic development. One of our future goals is to establish a permanent culture of E. rowelli, which would reproduce in the lab by experimenting with different culturing conditions. However, due to the long gestation period of onychophorans, this is a long-term task. For now, our best option is to go on collection trips to Australia every once in a while.
I hope I could show you what a typical day in an onychophoran lab looks like. If you are interested in onychophorans, or are looking for further information about these fascinating animals, please feel free to check out our webpages: www.onychophora.com and http://www.uni-kassel.de/go/zoologie.
Mayer G, Franke FA, Treffkorn S, Gross V, Oliveira IS: In Evolutionary Developmental Biology of Invertebrates 3: Ecdysozoa I: Non- Tetraconata. Edited by Wanninger A. Wien: Springer; 2015: 53–98
Storch V, Ruhberg H: In Microscopic Anatomy of Invertebrates: Onychophora, Chilopoda, and Lesser Protostomata. Volume 12. Edited by Harrison FW, Rice ME. New York: Wiley-Liss; 1993: 11–56
Ruhberg H, Mayer G: Onychophora, Stummelfüßer. In Spezielle Zoologie Teil 1 Einzeller und Wirbellose Tiere. Edited by Westheide W, Rieger G. Berlin: Springer; 2013: 457–464
In this episode from our centenary series exploring 100 ideas in genetics, we’re telling tales of sex and death, and exploring the very darkest side of genetics. We discover how Francis Galton’s eugenic ideas led to some of the worst atrocities of the 20th century, and ask how his legacy should be honoured today – if at all? Plus, the evolution of sex, and the total eradication of mosquitoes.
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