Heart development is a complex process involving numerous cell types and different morphogenetic events to make an organ that begins its vital function long before it reaches its final, intricate shape. That this process is error prone is borne out by the high prevalence of congenital heart defects in humans. For de la Cruz, a Cuban cardiac embryologist who spent most of her career in Mexico City, a precondition for understanding the aetiology of congenital defects was a comprehensive description of normal heart development. She was particularly preoccupied with the question of where the various parts of the final (or ‘definitive’) heart mapped onto earlier developmental stages.
Her 1989 paper begins with a nod to an earlier age: the descriptive work of Carl Davis, who in the 1920s used human embryo samples from the Carnegie Collection to infer the lineage of the compartments of the heart. Davis had described one particular embryo, #3709, the type specimen for stage 9, at which point the heart is a straight, symmetrical tube, yet to undergo looping and formation of the chambers (the same embryohas recently been reconstructed in 3D).
Davis inferred that each of the regions of the straight tube heart is the primordium of a definitive cardiac cavity; that is, by stage 9, the atria and ventricles of the heart are mapped out onto the straight tube heart, and subsequent development involves morphogenesis of these pre-patterned regions.
For de la Cruz, decades later, purely descriptive embryology on fixed and sectioned samples was inadequate to really test lineage, as there was no means of following regions in the same heart. She thus turned to in vivo labelling, which allowed the “study of the cardiac zones up to their anatomical expression in the mature heart in a continuous and uninterrupted sequence”. Of course she required a non-human model, and used the easily accessible eggs of leghorn chickens (like mammals, birds have a four-chambered heart). Regions of the heart could be injected with iron oxide particles, and the embryo left to develop either in vitro or in ovo until the desired stage. Over the decade preceding the paper, she and others had gathered evidence which questioned the existence of Davis’ primitive cardiac cavities; for instance, the primordial atria were not apparent in the heart tube tissue, but only later in the loop stage heart.
Excerpt from Figure 2 showing labelled regions in St. 9 hearts, and where they ended up in St. 12. de la Cruz, et al. 1989. J Anat. 165:121-131. Reproduced with permission of Wiley.
The 1989 paper was her latest attempt to map out where the regions of the heart tube ended up, and would she hoped “allow us finally to discard the term ‘primitive cardiac cavities’.” To start with, the straight tube heart was labelled in two caudal regions (a and b in the figure excerpt), and then the label was observed in the loop heart stage. Label a, at the left border of the heart tube, ended up in the left border of the loop stage heart, consistent with previous descriptive embryology. But label b showed something different: cells at the tube’s midline ended up at the right border of the loop, which dismisses a simple ‘one to one’ correlation between the regions of the tube and the loop. A final caudal label, at venous edge of the tube, ended up in the border between the left ventricle and left aorta in the mature heart. These labelling experiments were complemented by SEM and histology to compare the morphologies of the straight tube heart with later stages.
This paper, along with de la Cruz’s previous and later work, demonstrated that the straight tube heart does not contain single primordia for each of the definitive cavities. As the initial heart tube only contributes to a subset of the final heart (mainly the left ventricle), the rest of the heart must be added during later development. This work predated the molecular definition of additional regions of the mesoderm that give rise to the heart (the so-called second heart field) by more than a decade, but appears not to have been widely appreciated, perhaps due to the methods employed. It could however be argued that the power of the method is its simplicity: label a cell or set of cells and see where they end up, and deduce from there how the final organ is built. Indeed lineage tracing is still being used to great effect to this day.
Thoughts from the field:
Benoit Bruneau (Gladstone Institute for Cardiovascular Disease)
“Victoria de la Cruz used somewhat crude methods to map out where segments of the early heart tube end up in the more developed heart, and the results were not what people might have expected based on preconceived notions. Her work went largely ignored as people examined gene expression patterns and made erroneous conclusions about the chambers of the heart being already patterned and present as primordia in the linear heart tube. The discovery of the second heart field brought back to the forefront her work, which suggested that there might be an additional source of heart cells.”
“The chick embryo is easily accessible and has been used extensively for fate mapping studies. This paper by Maria Victoria de la Cruz is a prime example of classic mapping experiments that contributed to changing the thinking in the field. Her work using labeling of the linear heart tube with iron oxide particles, indicated that new heart segments are added successively, in particular to generate outflow myocardium. She also concluded that precursors for the right and left primitive atria are not yet present in the early straight heart tube but become incorporated later during loop stages. This was not fully appreciated until the origins of secondarily added cell populations were discovered; in the chick using essentially similar approaches, and in mouse using genetic labelling.”
“This 1989 paper was an important step in a series of cell labeling studies from Maria Victoria de la Cruz and colleagues that demonstrated the dynamic nature of early heart development. Her purely embryological approach was initially underappreciated yet set the scene for many of the molecular and genetic studies underway today. MV de la Cruz was one of the first to realise that there was a myocardium-forming region outside the linear heart tube, a critical advance in emergence of the second heart field model of vertebrate heart development. Indeed, the concepts that certain parts of the heart are late added components and that primitive cardiac regions only contribute to parts of the definitive cardiac chambers stem from this work and have had significant impact on our understanding of congenital heart defects. Her book Living Morphogenesis of the Heart is highly recommended further reading for those interested in the major embryological questions, including heart tube growth, septation, coronary development and the mechanisms underlying anomalous cardiac development, that continue to drive the field today”
A little more reading
I learned a little about Maria de la Cruz’s life from two obituaries (1, 2)
This post is part of a series on forgotten classics of developmental biology. You can read the introduction to the series here and read other posts in this series here.
We are seeking an enthusiastic and outstanding postdoctoral researcher to join a multidisciplinary team led by Prof. Chris Thompson at the University of Manchester. You will use single cell RNA sequencing to identify groups of heterogeneously expressed genes within normal populations of cells, and study the role of these genes in cell fate choice.
Cell fate choice and proportioning are typically considered to be ordered, robust and reproducible. However, noise and stochasticity can lead to heterogeneous gene network activity. Consequently, it has been proposed that gene networks may be ‘wired’ to buffer these fluctuations. Alternatively, heterogeneity may be functionally important to prime cells or increase the spectrum of differentiation capabilities. Addressing these questions represents one of the greatest challenges in Developmental and Stem Cell Biology. However, to date it has been impossible to follow entire gene network behaviour in individual cells, or to follow their temporal changes in activity in individual cells as cells commit to differentiation along different lineages. Single cell gene expression analysis, together with novel computational reconstruction of gene network dynamics provides this opportunity.
This work builds upon our recent finding (Chattwood et al, eLife 2014) that the interplay between dynamic heterogeneity in Ras-GTPase activity and nutritional status is required for normal lineage priming and robust running of an ultradian cell fate oscillator. Computational approaches will be used to identify putative genes involved in lineage priming and cell fate choice. In addition, the role played by these genes will be tested in the lab through the analysis of gene knockout strains, live cell imaging and molecular genetics.
Candidates with extensive experience of using either computational approaches or wet lab approaches to understand the molecular basis and gene networks will be considered.
You should currently hold or be about to obtain a PhD in a relevant field.
The post funded by the Wellcome Trust and is available for up to 3 years.
In 2009, FaceBase was launched in response to the need for more comprehensive analysis of craniofacial development: with so much craniofacial data being generated, there is a danger of relevant datasets being buried in the avalanche of genomic and other data. FaceBase is a curated, one-stop shop for facial development and research offering the community input and access to datasets that can bring their research to the next level. After the first 5 years (known as FaceBase 1) resulting in almost 600 datasets and over 100 publications, the next phase of FaceBase (FaceBase 2) began in August 2014 with a new Hub that developed an updated data model allowing for more data integration and faceted searches with a new server interface. The FaceBase website (http://www.facebase.org/) continues to be a resource for the community.
What is FaceBase? A collaborative NIDCR-funded consortium to generate data in support of advancing research into craniofacial development and malformation.
Serves as a community resource by generating large datasets of a variety of types and making them available to the wider research community at http://www.facebase.org/.
Emphasizes a comprehensive and multidisciplinary approach to understanding the developmental processes that create the face.
Spotlights high-throughput genetic, molecular, biological, imaging and computational techniques.
Facilitates cooperation and collaboration between projects and beyond.
Find datasets by various filters: organism (e.g., human, mouse, zebrafish), investigator, data type, various developmental ages, mutation, genotype, gene, chromosome and much more.
New data are now available from the FaceBase 2 projects as well as continued uploads of FaceBase 1 datasets
What you’ll find:
Global and specific gene expression patterns
Regulatory elements and sequencing
Anatomical and molecular atlases
Human normative facial data and other phenotypes
Genetics of craniofacial development
Repositories of animal models and of human samples and data
Software tools and animal models for analyzing and testing and integrating these data
Resources for researchers who may be new to the craniofacial field
Finding the data you need:
Start your search by clicking the Data Browser button on the homepage or in the top navigation. The video below provides a tutorial on the site’s search functions. There is also extensive documentation on the website.
3D skull anatomy:
Here is a video which highlights every bone in the craniofacial region and provides internal and external views of the skull as well as measurements of selected bones.
FaceBase 1 projects (2009-2014) and their contributions:
Contributed MR Image data of normal and a mouse model of cleft palate (tgfß KO) presented as orthogonal sections through fetal embryo heads (stages 13.5-18.5).
Mapped on a genome-wide scale distant-acting gene regulatory sequences (enhancers) involved in craniofacial development and examined the precise activity pattern of individual enhancers in transgenic mouse assays.
Used laser capture micro dissection to isolate specific compartments of the developing face, which were then used for gene expression profiling with microarrays and/or RNA-seq.
Produced software – CranioGUI – that can be used by the craniofacial research community to help analyze facial abnormalities. The software works on 3D head meshes and can be used for multiple conditions.
FaceBase 2 projects (2014-present) and data currently being deposited:
Advance the use of zebrafish in the study of skull development, and facilitate comparative studies with mammals that will advance treatment options in human patients.
Characterize epigenetic landscapes and transcriptomes of human and chimpanzee cranial neural crest cells and analyze candidate human-specific craniofacial enhancer activity in vivo.
Develop and maintain an infrastructure that will store, represent, and serve craniofacial data to the research community and develop tools for visualizing, integrating, annotating, linking and analyzing the data
Characterize the gene regulatory landscape of craniofacial development using epigenomic profiling of developmental mouse and human facial tissues, coupled to characterization of craniofacial enhancers in transgenic mice.
Develop a genomics analysis interface that makes analysis of pertinent genomics data available to FaceBase users without releasing the individual level data.
Generate comprehensive gene expression atlases of the major and functionally important craniofacial sutures of the mouse.
References:
The FaceBase Consortium: a comprehensive resource for craniofacial researchers.Brinkley JF, Fisher S, Harris MP, Holmes G, Hooper JE, Jabs EW, Jones KL, Kesselman C, Klein OD, Maas RL, Marazita ML, Selleri L, Spritz RA, van Bakel H, Visel A, Williams TJ, Wysocka J, FaceBase Consortium, Chai Y. Development 2016 143:2677-2688. doi: 10.1242/dev.135434
The FaceBase Consortium: a comprehensive program to facilitate craniofacial research. Hochheiser H, Aronow BJ, Artinger K, Beaty TH, Brinkley JF, Chai Y, Clouthier D, Cunningham ML, Dixon M, Donahue LR, Fraser SE, Hallgrimsson B, Iwata J, Klein O, Marazita ML, Murray JC, Murray S, de Villena FP, Postlethwait J, Potter S, Shapiro L, Spritz R, Visel A, Weinberg SM, Trainor PA. Dev Biol. 2011 355(2):175-82. doi: 10.1016/j.ydbio.2011.02.033
In this new series, we interview the people behind some of the most exciting recent papers in developmental biology and related fields, to give context to the work and find out how the story came together.
To inaugurate the series, we start with a paper that came out recently in Cell, and uncovered a mechanism for how nuclear pore complexes are inserted into the nuclear envelope in early Drosophila development.
We hear from five of the people behind the paper, all of whom are based at the EMBL in Heidelberg: lead author Bernhard Hampoelz, his PI Martin Beck, and their EM collaborators Yannick Schwab (PI and head of the EM facility), Nicole Schrieber and Paolo Ronchi.
They gave us their perspectives on this collaborative, multi-disciplinary project.
Introducing the players, from left to right: Nicole, Bernhard, Yannick, Paulo and Martin
We’ll start with Martin: can you tell us the brief history of the Beck lab, and what key questions the group is trying to answer?
MB I have a mixed training in structural biology (PhD with Wolfgang Baumeister at the MPI Martinsried) and systems biology (postdoc with Ruedi Aebersold at ETH Zurich). I had already worked on nuclear pores as a student and when I started my own laboratory at EMBL in 2010, I felt that my training positioned me well to attempt to understand nuclear pore complex architecture. We have combined electron microscopic with mass spectrometric approaches to structurally analyse nuclear pores in situ.
I was always intrigued by two biological aspects of this: i) that understanding the assembly pathways of large macromolecular machines can help us to understand their architecture because nature essentially has broken down the problem into smaller pieces for us that are more feasible to approach. And ii), that in order to understand the function of large macromolecular machines in situ, one needs to understand how their structure is spatiotemporally modulated, e.g. across cell types or the cell cycle.
From my perspective, the beauty of Bernhard’s project is that it brings all of this together to elucidate a new phenomenon. It builds on the methodological strength and quantifies a compositional variation of nuclear pores across space and time to discover an unanticipated way to get a nuclear pore complex (NPC) in to the nuclear envelope.
And Bernhard, how did you come to join the Beck lab? Am I right in thinking you brought flies to the lab?
BH Exactly – I think flies were not very popular in Martin’s group before I entered. Also my background was perhaps unusual for the lab, since I came from developmental biology. When working at the IBDM with Thomas Lecuit, my focus was on nuclear morphology and its developmental control. Thomas gave me a lot of freedom in investigating things that popped up and it happened that I observed annulate lamellae (AL; stacked cytoplasmic membranes that are a subset of the ER and decorated with NPCs) insertion into the nuclear envelope by imaging. Aware of the potential of this finding, I knew that in order to nail this down mechanistically I had to convince somebody that works on NPCs at the ultrastructural level to give me the chance to pursue this project. I came to EMBL as a visiting scientist and soon presented my findings to Martin. I am very glad that Martin, although the project was not in the direct focus of his lab at that time, agreed on supporting me to continue this work in his group.
“I knew that in order to nail this down mechanistically I had to convince somebody that works on NPCs at the ultrastructural level to give me the chance to pursue this project.”
So Yannick, you’re neighbours in Heidelberg, but did you know Martin well beforehand? How did you get involved in Bernhard’s project?
YS Martin and I work on the same floor at EMBL. In fact, I head the electron microscopy core facility (EMCF) which is sharing the space where Martin’s Unit (Structural and Computational Biology) has its set of cryo electron microscopes. Therefore, we see each other very often. Even though Martin’s group is focused on structural biology, they do not hesitate to cross the border towards cell biology which is the field of expertise of the facility. Whilst Bernhard had already solid background in EM and was already an advanced user, his project required advanced expertise both in volume EM and in Correlative Light Electron Microscopy (CLEM). For this, he teamed up with some of the EMCF staff (Pedro Machado, Paolo Ronchi and Rachel Mellwig) and with Nicole Schieber a specialist in Focussed Ion Beam Scanning Electron Microscopy (FIB-SEM) from my team. My involvement in this project was mostly at the level of organizing this collaborative work and setting priorities when the last set of experiment had to be done.
Finally, Nicole and Paulo: how were you recruited to this story?
NSI started with the Schwab team when it was just beginning as the research technician. My background has had a strong focus on Electron Microscopy for the past 9 years since my undergraduate degree got me hooked at the University of Queensland, Australia. For the past 3 years here at the EMBL I have shifted more towards the 3D EM techniques, especially FIB-SEM.
My role in the lab means that I try to connect the team to the facility and on some occasions I can step in to help with projects that I either find interesting or see I can add some expertise. For this story, my colleagues Rachel and Pedro from the EMCF were already working on this project together with Bernhard and I had been following its progress in our regular meetings. They had managed to solve some difficulties with the EM sample preparation and we all quickly realised that FIB-SEM would add to the three dimensional picture of the story. Since this would be a demanding task for the FIB-SEM I put my hand up to acquire the data and really enjoyed the challenge as well as being able to team up and work more closely with my colleagues than we normally would.
PRI have been working in the EMCF for 2 years after a postdoc experience at EMBL, working on membrane trafficking. Since I’ve been here, I have always been very keen on pushing CLEM methods in the facility and Bernhard’s project was a great opportunity. But it was a big challenge as well.
I got an email from Yannick asking whether I would be interested in helping Bernhard with a CLEM experiment. I knew his project and I had always found it very interesting, but it was on short notice (it was for the revision of the paper) and I was on holiday! The project required to adapt a high accuracy CLEM method that had been previously developed on yeast cells to the Drosophila embryos. Luckily, I had been setting up the best conditions to treat Drosophila ovaries for another collaboration and thought that Bernhard’s system was similar enough. Therefore I used the same protocol and, for once, everything worked smoothly at the first attempt.
Where did the interest in nuclear pores come from, Bernhard? And what was the key problem you wanted to address with this paper?
BH I have to admit that my interest in NPCs came by accident. In Thomas Lecuit’s group I worked on nuclear morphology and used fluorescently labelled Nucleoporins as means to outline the nucleus in imaging. Naively I realized that these Nups do not only label the nuclear envelope (NE) but also foci in the cytoplasm and I learned about AL, which have been known for decades actually. Curious about their function, I imaged them live and saw that they insert into the NE. Puzzled by the fact that this has not been observed before I started to think how to further develop this project.
A dense network of roads feeds into the Boulevard Périphérique, which surrounds the city of Paris. Major junctions, some of which are depicted in this image, bridge the Périphérique and link the city to the metropolitan region. Likewise Nuclear Pore Complexes (NPCs) pierce the nuclear envelope (NE) and control transport between the cell nucleus and the cytoplasm. The NE is continuous with the surrounding ER network that feeds nuclear expansion during interphase. Credit: Bernhard Hamploez.
What makes the early Drosophila embryo an ideal model for the question of nuclear pore insertion? What are the model’s challenges?
BH The fly embryo offers a couple of advantages. The most intriguing is probably its ease to do live imaging. Basically you just glue an embryo expressing your fluorescently labelled protein of interest onto a coverslip and start to image. Moreover the embryo is susceptible to injection of drugs or genetic manipulation that could interfere with your process of interest. Conceptually, I like that it allows you to put a cell biological question into the context of animal development. A challenge is the short cell cycles, especially for EM – we sectioned numerous embryos that happened to be frozen during mitosis and were thus useless for our purpose.
“Basically you just glue an embryo expressing your fluorescently labelled protein of interest onto a coverslip and start to image.”
Can you briefly describe what you found out about the relationship between annulate lamellae, nuclear pore complexes, and the nuclear envelope?
BH AL are sub-compartments of the endoplasmic reticulum that contain stockpiled NPCs. Because AL are in particular enriched in oocytes and early embryos across species, they were always considered as storage pools of maternally derived NPC material. Whether they could somehow contribute to the NPC pool at the nuclear envelope had remained elusive. Our study proves that AL are indeed inserting into the nuclear envelope when the nucleus expands during interphase in Drosophila embryos at the blastoderm stage. This can work because unlike in differentiated cells NPCs in the early fly embryo are laterally mobile within the NE and thus can redistribute. We reveal that NPCs at AL are pore scaffolds that only mature to the full NPC complement once inserted into the nuclear envelope. And, based on EM, we suggest a topological model how such insertion could happen.
So what makes a nuclear pore at the AL different from one in the nuclear envelope?
MB Its composition. Certain nucleoporin subcomplexes are missing in AL. This includes for example the Nup214 complex that is important for mRNA export out of the nucleus in differentiated cells but also for some import pathways that might play already a role in the early embryo. We believe that it is assembled into AL-originated pores only after they inserted into the nuclear envelope.
And why was EM necessary? I understand three different techniques were used (CLEM, FIB-SEM and tomography): were any of them a particular challenge?
YS EM in general is a challenging set of techniques. From the sample preparation to the image analysis, specific skills and dedicated protocols are required. Fortunately, the EMCF has accumulated a great deal of experience since its creation more than 10 years ago. Benefiting from a good integration within the research scene, it has, among others, developed specific methods for preparing Drosophila embryos (by high pressure freezing and freeze substitution) and for imaging them with 3D EM (tomography and FIBSEM).
NS FIB-SEM is a difficult technique that requires a lot of patience but is extremely rewarding in the results you can obtain. For this project one of the main difficulties came after finding the correct stage of the embryo and an interesting event by TEM thin sections. We then wanted to immediately image this same embryo in the FIB-SEM by looking at the block face itself and use the focussed ion beam (FIB) to ablate very thin slices to allow consecutive images that build the 3D data. During standard preparations for FIB-SEM you have a buffer where you can allow the sample to stabilise during the imaging so that the slice thickness is consistent, and can also test the imaging conditions to get the best possible resolution. In this case we didn’t have such luxuries and this was a challenge especially since the event we were looking for required the best possible resolution. The other challenge came from remounting the sample from how it was sectioned for TEM to the stub for FIB-SEM, here we were dealing with a piece of resin containing the sample that was very small (in the range of several hundred microns) and the orientation of this block was critical.
PR The CLEM method we used was developed by the Briggs and Kaksonen groups at EMBL to study endocytosis on yeast and mammalian cell cultures. Their work has made this technique very popular in the community and therefore our facility in the last 2 years has dealt with a number of projects using this method, adapting it to different organisms and different subcellular structures. Bernhard’s case was more challenging because we had to look for embryos of the right developmental stage and in the right stage of the cell cycle (interphase). After preparing the samples for EM, we therefore sectioned a few embryos and inspect them by conventional EM to identify the ones that displayed interphase nuclei, before proceeding with the CLEM workflow.
What was the key insight given by EM?
YS EM techniques enabled us to visualize for real what a bundle of other techniques could only suggest. For example, live fluorescence imaging strongly suggested the physical connection, continuity, between the AL and the nuclear envelope. Thanks to 3D EM (tomography and FIB-SEM) this continuity appeared obvious. FIB-SEM furthermore demonstrated the organization of the ER sheets relative to the nuclear envelope, an observation that led us to propose the model exposed in the paper. It was very important as well to demonstrate that the very dynamic fluorescent patches observed in vivo were indeed AL. CLEM clearly confirmed this.
“EM techniques enabled us to visualize for real what a bundle of other techniques could only suggest.”
Does the mechanism of AL-NPC insertion change during development, and is this important?
BH Yes, indeed. Insertion of entire AL is a maternal program and declines starting with zygotic induction. One reason might be simply the temporal constraint. AL insertion happens fast and could overcome the slow kinetics of classical interphase NPC assembly, as we know it from tissue culture cells. In the prolonged interphase 14 of Drosophila embryogenesis, AL diminish from the nuclear layer at the embryos’ cortex. The vast abundance of AL in oocytes and early embryos of many animal species also argues that they have a general role in the earliest stages of development.
Did you have a single ‘eureka!’ moment when everything came together or you got a particularly stunning result?
BH I would say two of them: First the photoconversion experiments where I could see that converted Nucleoporins distributed from an extranuclear spot into the nuclear envelope and there dissipated laterally. This really proved to me that AL do insert. And secondly when we could reveal insertion ultrastructurally in the FIB-SEM analysis.
Is there a loose end or surprising result in the paper you would particularly like to get to the bottom of?
MB Various experiments described in our paper show that nuclear envelope organisation before the start of transcription is very different from well-studied conditions in differentiated cells. That overexpression of the lamin B receptor is sufficient to make nuclei in the early embryo look as their counterparts at later stages I personally found very surprising.
And what are you working on now?
BH As always many questions emerge: for example how do the NE openings that seem critical for AL insertion form? How are they stabilised? To get a handle on this on a molecular level is my next goal.
Martin and Yannick, any planned future collaborations?
MB YES!
YS Yes definitely. This collaboration was one of these great and rewarding moments when we witness how the expertise from a service facility helps our colleagues to progress in their science. Martin’s and Bernhard’s enthusiasm and collaborative spirit successfully engaged everyone in this fantastic story. I can tell you they have plenty more, as exciting as this one!
“This collaboration was one of these great and rewarding moments when we witness how the expertise from a service facility helps our colleagues to progress in their science.”
Bernhard Hampoelz, Marie-Therese Mackmull, Pedro Machado, Paolo Ronchi, Khanh Huy Bui5, Nicole Schieber, Rachel Santarella-Mellwig, Aleksandar Necakov, Amparo Andrés-Pons, Jean Marc Philippe, Thomas Lecuit, Yannick Schwab, Martin Beck. Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development. 2016. Cell. Volume 166, Issue 3, p664–678
You can catch up with the latest People behind the Papers here
A postdoctoral researcher position is available in the Plageman lab in the College of Optometry at the Ohio State University to elucidate mechanisms of vertebrate epithelial morphogenesis. The project will utilize the invaginating lens placode of the mouse as a model in combination with live-fluorescent microscopy. Candidates should have a Ph.D., a strong publication record and ideally have experience with mouse genetics, immunofluorescent imaging, and a background in molecular biology, biochemistry, embryology, and/or cell biology. Expertise in other model systems are also welcome. To apply, follow the instructions at the following link: https://www.jobsatosu.com/postings/72726
More information about the lab can be found at: https://u.osu.edu/plageman.3/ and inquiries about the position can be made directly to: plageman.3@osu.edu
New embryonic-lethal knockout mouse lines are now available on the DMDD database.
If you haven’t previously taken a look at our data (or even if you have) now would be a good time to explore our website. We’ve added new embryo phenotype data and HREM images for many knockout lines, taking our total dataset to more than 4 million images of 550 embryos. We also have placental histology images and phenotypes available for over 100 mutant lines.
This post explores some of the phenotypes observed in the new data, and highlights new lines that could be relevant for clinicians researching rare diseases and developmental disorders. But there isn’t enough space here to include every interesting feature of the data – the best thing to do is to explore it yourself.
Embryos from the line Adamts3 display both subcutaneous edema and bifid ureter. A bifid ureter is the most common malformation of the urinary system, [1] in which there is a duplex kidney drain into separate ureters. This observation highlights the incredible resolution of HREM images, which allow detailed phenotypes to be scored for each embryo.
Bifid ureter (left side) observed in an Adamts3 mutant embryo. The red arrows highlight a single ureter on the right side, but two branches on the left side.
Embryos from the line H13 suffered from severe abnormalities in heart morphology, and had an abnormal heart position within the body. The stomach situs was also inverted, as shown in the image below. Note that severely malformed embryos often have different tissue characteristics, which can result in reduced image clarity.
Comparison between a H13 mutant embryo (left) and its wild-type litter-mate (right). The yellow arrows indicate situs invertus of the stomach.
Embryos from the line Brd2 exhibited a profound ventricular septal defect, as shown in the video below.
PLACENTAL PHENOTYPES
Our placental image and phenotype dataset is growing rapidly and now contains more than 100 lines.
H13 knockout placentas were smaller than their wild-type counterparts and showed reduced vascularisation in the placental labyrinth, the region of the placenta that allows nutrient and gas exchange between the mother and the developing embryo.
A comparison of the placenta from a H13 mutant embryo and that of its wild-type litter-mate.
Vascularisation of the labyrinth is crucial to allow the embryo to receive the oxygen and nutrients needed for normal development. This is just one example, but many more placental phenotypes are available on our website.
LINKS TO CLINICAL STUDIES
Systematic knockout mouse screens can offer a wealth of information about the genetic basis of rare diseases. Many DMDD lines have human orthologues known to be associated with developmental disorders, and the nature of our study means that it would not be possible to derive equivalent systematic data from human patients.
New knockout lines of potential clinical interest include:
1 Department of General Medicine, The Ipswich Hospital, Ipswich, UK. 2 Department of Radiology, Barking, Havering and Redbridge NHS Trust, Romford, Essex, UK.
A postdoctoral position is available for a bioinformatician to investigate the mechanisms by which microRNAs, transcription factors and developmental signals control skeletal and cardiac muscle differentiation. The post is based in the School of Biological Sciences at the University of East Anglia, Norwich, in the group headed by Professor Andrea Münsterberg. Their research aims to dissect genome-wide processes that regulate the cell fate choice of progenitor cells in early embryos.
more details here:
http://www.jobs.ac.uk/job/AOB513/senior-research-associate-bioinformatician/
Steroid hormones have crucial roles in regulating a broad range of biological processes in most multicellular organisms. They are produced in specialized endocrine organs and act as ligands for the nuclear receptor family of transcription factors. In mammals, sex steroid hormones, such as estrogen and testosterone, regulate sex maturation, reproductive physiology and behavior in both sexes. In insects, the major insect steroid hormones are ecdysteroids, including the most biologically active form 20-hydroxyecdysone (20E). Ecdysteroids are called the “molting hormones”, as they are well-known for coordinating developmental transitions, such as molting and metamorphosis. In contrast, little attention has been paid to “post-developmental” roles of ecdysteroids until recently. However, studies in the past several years have demonstrated that ecdysteroids are also present in adult tissues and regulate diverse biological processes such as reproduction, learning, memory, stress resistance, and lifespan (Uryu et al., 2015).
In 2010, Ables and Drummond-Barbosa at Johns Hopkins University beautifully demonstrated that ecdysone signaling directly controls female germline stem cells (GSCs) (Figure 1), which give rise to mature eggs in adults of the fruit fly Drosophila melanogaster (Ables and Drummond-Barbosa, 2010). Interestingly, their data showed that ecdysteroid signaling components including EcR and its downstream gene cascades regulate GSC maintenance with intrinsic chromatin remodeling factors. In addition, some other important studies have also showed that ecdysteroid signaling is required for GSC regulation (König et al., 2011; Morris and Spradling, 2012). I was an undergraduate student at the time, and very impressed by these works, because their findings supported a link between systemic steroid hormones and adult stem cell self-renewal. On the other hand, I realized that all of these works did not examine whether ecdysteroid biosynthesis is regulated by any environmental factors in the ovaries. It seems to me that this question is fundamental, as ecdysteroid biosynthesis during the larval stages is influenced by external environmental conditions (Niwa and Niwa, 2014a). In addition, I also wondered whether and how ecdysteroid “biosynthesis” in the adult ovaries contributed to the GSC regulation.
Figure 1. Germline stem cells (GSCs) give rise to mature eggs
In 2012, I joined Dr. Ryusuke Niwa’s laboratory at the University of Tsukuba and wanted to tackle my questions stated above using Drosophila. Ryusuke and I spent several months thinking about the direction of our research, and eventually focused on the stimulating effect of mating. The reason why is because it has been known that mating dramatically induces behavioral changes, increased egg laying and decreased receptivity in mated females (Kubli, 2003; Wolfner, 2009). To ask whether mating may affect ecdysteroid biosynthesis, we first measured ovarian ecdysteroid levels in virgin and mated females. I was very glad that our expectation was correct, as we found that mated females showed higher ovarian ecdysteroid levels compared to virgin females in our recent PLoS Genetics paper(Ameku and Niwa, 2016). Moreover, we have confirmed that the post-mating ovarian ecdysteroid production is induced by a peptide from male’s seminal fluid called Sex Peptide, and its specific receptor Sex Peptide receptor, is expressed in the female neurons in the same way as the canonical post mating response in mated females (Ameku and Niwa, 2016).
The next question we raised was about the function of mating-induced ecdysteroid biosynthesis. Taking previous studies into consideration, naturally we decided to investigate the relationship between biosynthesis and the regulation of GSC activity. We indeed found that mating stimulates GSC proliferation in mated females. We concluded that this mating-induced GSC proliferation requires ovarian ecdysteroid production, as GSC proliferation did not occur when we inhibited ecdysteroid production in the ovary by knocking down genes required for ecdysteroid biosynthesis (Niwa and Niwa, 2014b) (Figure 2). Interestingly, our data revealed that ovarian ecdysteroid biosynthesis is regulated by Sex Peptide signaling and mediates mating-induced GSC proliferation. This is the first study to show that GSC is under the control of a characterized neuroendocrine system in response to the mating stimulus (Ameku and Niwa, 2016).
Figure 2. The roles of ecdysteroid during developmental and post-developmental stages
Currently, we are investigating how neuronal input is transmitted to reproductive tissue to regulate GSC activity, which is required for reproductive success. What I have learned from this study is the importance of focusing on biological questions at different levels, such as behavior, neuron, endocrine and stem cells. I hope I will become a researcher who can see things from various perspectives after completing my Ph.D., by making use of my experiences in carrying out this work.
My research interest is the evolution of multicellularity. How did cells ‘learn’ to communicate with each other to build a structure that is more complex than its parts and shows new emergent behaviour? Which and how many new genes would be required to transform a unicellular ancestor into a well-organised multicellular structure? The Nature Communications Article from our lab that was published recently (1) sets out to answer some of those questions.
Most of us will be familiar with the type of multicellularity, which evolved in animals, some fungi and plants: cells derived from a zygote continue to divide, but remain attached to each other and eventually differentiate into the different cell types that form the tissues of the organism. All cells are genetically identical and altruistic differentiation into somatic cells that support the propagation of the germline has no cost to them. A quite different case is the colonial multicellularity of social amoebas, which we are studying in our lab. Here, individual cells that can be genetically distinct come together and create a multicellular fruiting structure, consisting of spores and stalk cells. The stalk cells have to altruistically die to support spores. When the amoebae are not genetically identical, conflicts of interest can arise, such as cheating by avoiding stalk cell differentiation. Just like multicellularity by adhesion, colonial multicellularity evolved multiple times (2,3), suggesting that colonial organisms have devised mechanisms to deal with genetic conflict.
The Dictyostelid social amoebas can be subdivided into four major groups, which differ in the size and shape of fruiting bodies, the presence of an intermediate migratory form, the “slug” and the number of cell types, which is largest in group 4. In addition, group 4 species pre-differentiate amoebas into the correct ratio of prespore, prestalk and the other supporting cell types, whereas in groups 1-3 all amoebas first differentiate into prespore cells that then only locally dedifferentiate into stalk cells (Figure 1).
Figure 1. Schematic of life cycle complexity of the Dictyostelid test species. Dictyostelium fasciculatum (DF), Polysphondylium pallidum (PP) and Dictyostelium lacteum (DL) form multiple fruiting bodies directly from the aggregate. All cells first differentiate into prespore cells and then form the stalk by dedifferentiation of prespore cells at the tip. Dictostelium discoideum (DD) and Dictyostelium purpureum (DP) form single fruiting bodies from aggregates and display an intermediate migratory “slug” in which cells pre-differentiate into prestalk and prespore cells. During fruiting body formation, two more cell types emerge which support the stalk and spore mass. 1: aggregate, 2: early sorogen (slug), 3: migrating slug, 4: mid-culminant, 5: fruiting body. Light red: prespore; dark red: prespore/spore; light blue: prestalk; dark blue: prestalk/stalk; green: basal disc or/ supporter; yellow: upper and lower cup.
In Dictyostelia, cell proliferation is entirely separated from multicellular development and we can therefore loosely define “multicellularity genes” as genes that are essential for multicellular development, but not for cell proliferation. We wanted to know to what extent such genes were already present in the unicellular ancestors of Dictyostelia and how such genes changed or appeared in the course of Dictyostelid evolution to increase the morphological and behavioural complexity of the organisms. To achieve this we sequenced three genomes that represented groups 1, 2 and 3 of Dictyostelia. The genome of two group 4 species, D. discoideum and D.purpureum were already available as well as the genomes of three unicellular Amoebozoa. Additionally we investigated how expression of all genes in these genomes is regulated during their development by high throughput RNA sequencing. This allowed us to trace gene evolution across the whole phylogenetic tree of social amoebas and their unicellular Amoebozoan relatives. From previous studies, 385 genes in D.discoideum were known to produce a defect in multicellular development when disrupted. We found that 305 of these genes were already present in unicellular Amoebozoa. The majority is conserved in all Dictyostelia regarding the conservation of domains and expression regulation. However, 80% of those genes, which are mainly cytosolic and nuclear proteins and protein kinases, are already present in their unicellular relatives. Eighty genes were unique to Dictyostelia and this set was enriched in plasma membrane and secreted or extracellularly exposed proteins, G-protein coupled receptors and sensor histidine kinases. Also, a set of 37 proteins that were only conserved in group 4 or groups 3 and 4 were highly enriched in plasma membrane and secreted or exposed proteins (Figure 2).
Figure 2. Signal peptide (SigP) and transmembrane (TM) domains. Proteins in the 305 and 80 sets, as well as 37 proteins with limited conservation within Dictyostelia (green) were analysed with Phobius63 for transmembrane domains and signal peptides. Percentages of proteins with either SigP or TM domains, or with both are presented.
For conserved genes, we also investigated whether their developmental regulation and their protein functional domains were conserved. If not, we scored how such changes were distributed across the Dictyostelium phylogeny. Logically, one expects such changes to be greater when the species are evolutionary more distant from each other. In case of functional domains, the changes were mostly scattered across the phylogeny (Figure 3), suggesting that changes in protein function did not contribute greatly to changes in phenotypic complexity. However, changes in developmental regulation occurred much more frequently between group 4 on one hand and groups 1-3 on the other, than between branches I and II that are evolutionary more distant. Because group 4 species are also phenotypically most distinctive (Figure 1), this indicates that phenotypic innovation in group 4 was more likely to be caused by changes in gene expression than changes in protein function. Finally, investigating the closest relatives of the 385 genes in species outside the Amoebozoa, we found that a relatively large percentage had closest homologs in bacteria. Further scrutiny identified four genes that were only present in Dictyostelia and bacteria and likely entered Dictyostelia by lateral gene transfer. Three of these genes synthesise three out of the five non-peptide signals that induce cell differentiation in D.discoideum: c-di-GMP, DIF-1 and discadenine (4-7).
Figure 3. Phylogenetic distribution of conserved features and outgroup homologs. A. Phylogenetic tree of Dictyostelida and unicellular amoebozoan species with sequenced genomes, as inferred from 30 concatenated proteins by Bayesian inference. B. Gene expression. Phylogenetic distribution of numbers of developmentally essential genes (DEG) with different patterns of conservation of gene expression. (4)(123) signifies that developmental expression was different between group 4 and groups 1-3, while (I)(II) signifies different expression between branches I and II. C. Protein domain architecture. Phylogenetic distribution of DEG with different patterns of functional domain conservation. D. Origin of outgroup homologs. Numbers of DEG (out of 385) without no homologs outside Amoebozoa, or with outgroup homologs in each of the eukaryote kingdoms or in prokaryotes.
In conclusion, it seems that innovation to multicellularity largely relied on repurposing of existing genes that were already present in the unicellular ancestor. Conversely, genes encoding exposed and secreted proteins with likely roles in adhesion and cell communication and the sensors to detect these signals appeared only in the multicellular forms, with genes for some novel signal molecules being acquired directly from bacteria. Furthermore, changes in gene regulation appear to have been more important for evolution of phenotypic complexity than changes in gene function.
Du, Q., et al., The Evolution of Aggregative Multicellularity and Cell-Cell Communication in the Dictyostelia. Journal of molecular biology, 2015. 427(23): p. 3722-33.
Schilde, C. and P. Schaap, The Amoebozoa. Methods in molecular biology, 2013. 983: p. 1-15.
Abe, H., et al., Structure of discadenine, a spore germination inhibitor from the cellular slime mold. Tetrahedron Letters, 1976. 17(42): p. 3807-3810.
Chen, Z.H. and P. Schaap, The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium. Nature, 2012. 488(7413): p. 680-3.
Neumann, C.S., C.T. Walsh, and R.R. Kay, A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1. Proceedings of the National Academy of Sciences of the United States of America, 2010. 107(13): p. 5798-803.
Saito, T., A. Kato, and R.R. Kay, DIF-1 induces the basal disc of the Dictyostelium fruiting body. Developmental biology, 2008. 317(2): p. 444-53.
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