The growth factor Fgf8a has been suggested to act as a morphogen during zebrafish gastrulation, spreading from a localized source to form a concentration gradient and impart positional information to cells along a tissue field. In a new paper in Development, Michael Brand and colleagues directly visualize the endogenous Fgf8a gradient in the developing zebrafish embryo. We caught up with the first author Rohit Krishnan Harish, and his PhD supervisor Michael Brand, Professor at the Center for Regenerative Therapies (CRTD) at TU Dresden.

Michael Brand (L) and Rohit Krishnan Harish (R)

Michael, can you give us your scientific biography and the questions your lab is trying to answer?

MB: I have always been interested in biology, but I can date my professional fascination with developmental biology quite clearly to my fifth semester at the university, when I read Ed Lewis's (1978) review about the Bithorax Complex of homeotic genes, with the beautiful logic it provided in analysing genetic control of the insect body plan. This has ‘anchored’ my interest to use genetics (and later molecular biology) to understand the complex phenomena of development and regeneration of the nervous system, first in flies and then in zebrafish as a vertebrate model. Work on the Fgf8a mutant and some others that I described as a postdoc in the Tübingen large-scale screen for ENU-induced zebrafish mutants (see the Zebrafish Issue of Development from 1996) led us to ask how organizers and morphogens control embryonic development of the nervous system – including the subject of the current paper. The classic Spemann/Mangold organizer experiments in the 1920s illustrated the power of the organizer concept, but this idea did not catch on for later steps of patterning the nervous system. Such organizers seemed to me, however, in the modern molecular era, to be a great way to potentially explain the much less hard-wired (less determined) neural development that can be observed in vertebrates, compared with the relatively hard-wired fly and worm development. In a very unexpected chance observation, work on this Fgf8a mutant and the midbrain-hindbrain boundary organizer also led us to discover the plasticity and neurogenesis in the adult zebrafish nervous system, which we then found to form the basis for its fascinating ability to regenerate (Kroehne et al., 2011) – something that we lack as mammals and which we now try to understand mechanistically in my lab.

Rohit, how did you come to work in Michael's lab and what drives your research today?

RKH: Well, I have to primarily thank my previous supervisors for this, Girish Ratnaparkhi at IISER (Pune, India) and Stefano De Renzis at EMBL (Heidelberg, Germany), for nurturing my passion for developmental biology and paving the path for my scientific aspirations. Stefano encouraged me to apply to the Dresden International PhD programme, knowing very well about the excellent scientific community in Dresden. As someone who was very keen to learn how a homogenous population of cells in the embryo develops to an adult organism, I must admit that Michael was initially not my primary choice at the time of application, because the lab was focusing mainly on adult neurogenesis and regeneration in zebrafish, and the project on Fgf8a was not advertised. I met him for the first time during my poster session as part of the interviews, during which we had a very fruitful chat about my Master's thesis project (on using optogenetics to modulate Notch signalling in Drosophila). We met again the next day, and that was when he told me about his other project on Fgf8a and how the group now had a knock-in line that would ideally allow one to visualize the molecule from its endogenous locus in real-time. I remember feeling so mesmerized at this point, at the prospect of monitoring a morphogen in real-time in a vertebrate species and also at how the advancement of CRISPR/Cas9 had eased the process of genome editing. The passion with which Michael was explaining the work and the rapport that I was having with him immediately struck me, and once I had spoken to the other lab members too, I didn't need much convincing to join the group. It's been 7 years since then – I am currently doing my postdoc with Anna Kicheva at the Institute of Science and Technology Austria, and I am still driven by the same urge – to understand how the various morphogenetic programs in the embryo are coordinated in time and space.

I remember feeling so mesmerized at this point, at the prospect of monitoring a morphogen in real-time in a vertebrate species and also at how the advancement of CRISPR/Cas9 had eased the process of genome editing

What was known about Fgf8a functioning as a morphogen before your work?

MB: Fgfs in general, and Fgf8a in particular, are known to be involved in many inductive interactions and patterning events during development, for instance during mesoderm, brain and limb development. Our own experiments on Fgf8a started with my favourite mutant from the Tübingen screen, which lacked a cerebellum (I hence named it ‘acerebellar’) and which my fledgeling-lab later showed to be a Fgf8a loss-of-function mutant (Brand et al., 1996; Reifers et al., 1998). We found that Fgf8a was acting in the neural primordium, and from our work and that of several other labs, it was clear that the concentrations of Fgf8a, for instance when used in Fgf8a protein bead implants or after Fgf8a mRNA injection experiments, are very important. Following the initial discovery of bFgf as a mesoderm inducer by Marc Kirschner's lab at UCSF, Fgfs were of course always thought to somehow work extracellularly, as ligands of receptor tyrosine kinases. The problem was, how would they be able to move over long extracellular distances to elicit their effects and generate information? This problem became especially pressing to answer when lipid adducts were discovered on other morphogens, for instance in the Hh and the Wnt families, and when cytonemes were discovered – how could such lipidated molecules even diffuse?

Following the initial surge of interest in Lewis Wolpert's French Flag model, free diffusion was, for quite some time in our field, no longer thought to be capable of generating the postulated graded concentration of a morphogen in extracellular space; proteins such as secreted GFP fill those spaces up in no time, for instance, and don't generate a gradient. And yet, from our work on the midbrain-hindbrain organizer in the forming neural plate in zebrafish, and the work of Salvador Martinez and Gail Martin in chick, we had evidence that the signal-receiving cells probably see a signal acting somehow at a distance, and in a graded manner, suggesting, but not proving, gradient and diffusion as a possible mechanism. I recall a talk where I reported the limiting role that endocytosis plays in the target cells for Fgf8a signal propagation (Scholpp and Brand, 2004), which I gave at an EMBO workshop in Heidelberg in 2004: Lewis Wolpert was sitting in the front row, and shouted right after my talk, ‘But you are not going to want me to believe again in diffusion, are you!’. This was before we were able to directly measure Fgf8a diffusion in living embryos at the single molecule level, using fluorescence correlation spectroscopy (FCS). Wanting to look directly at single-molecule diffusion of Fgf8a was the fortuitous consequence of me having to take, as a graduate student, an (excellent!) biophysics class at Harvard with Jim Wang, where we discussed Fick's diffusion law, and of my later learning about FCS as a suitable imaging method. So we used FCS for the first time in a living embryo and measured diffusion constants, concentrations and binding affinities of Fgf8a-GFP protein fusions, and came up with the morphogen-source-sink model for Fgf8a that way, which we described in Yu et al. (2009). The problem was, however, that we had to use mRNA injections to generate small clones of Fgf8a-GFP expressing cells as a source – a standard technique in zebrafish embryos, really. Although we tried our best in terms of calibrating the amounts injected and their biological effects, we were of course not looking at endogenous sources of Fgf8a or at the endogenous protein. We could not exclude the possibility that, for instance, the secretory pathway might be oversaturated, which could cause the protein to take a different extracellular route to convey its information. The ability to knock-in GFP into the endogenous Fgf8a locus, which we worked with in the present paper, then overcame this problem – in fact, this knock-in line was a very nice birthday present that my group gave me when knock-in techniques started to work in zebrafish!

Can you give us the key findings of the paper in a paragraph?

RKH: By integrating the powerful tool of genome editing using CRISPR/Cas9 with sensitive in vivo imaging and single-molecule FCS, we have directly monitored the distribution of endogenous Fgf8a in the developing neural plate of zebrafish gastrula. To our knowledge, it is the first time that an endogenous morphogen (morphogen produced from its endogenous locus) has been visualized in a living vertebrate. We show that Fgf8a, produced at the embryonic margin, propagates by diffusion via the extracellular space and forms a graded distribution towards the animal pole. Interestingly, although we find that the majority of molecules in the extracellular space are fast diffusing, a minor fraction is slow moving and/or relatively immobile, due to their interaction with heparan sulphate proteoglycans (HSPGs). Therefore, by overlaying the Fgf8a gradient curve with expression profiles of its downstream targets, we determine the input-output relationship of Fgf8a-mediated patterning, which supports the role of Fgf8a as a morphogen. Manipulating the extracellular input of Fgf8a is then found to alter the signalling output, thus providing functional proof for the activity of endogenous Fgf8a as a morphogen. Finally, by using diffusion-hindered versions of Fgf8a, and by trapping extracellular Fgf8a using Morpho-trap, we demonstrate that extracellular diffusion of the protein from its source is crucial for it to exert its morphogenic activity.

Were you surprised to find from your FCS measurements that a small proportion of Fgf8a-EGFP molecules are relatively slow moving?

RKH: No. A previous study in the lab involving exogenous Fgf8a, generated from artificial sources in the embryo by mRNA micro-injections, had also shown that a minor fraction of such molecules in the extracellular space is slow moving (Yu et al., 2009). In general, extracellular matrix constituents, particularly the HSPGs, by virtue of their heparan sulphate side chains, interact with extracellular signalling molecules, and this interaction plays an important role in shaping the distribution of such molecules (e.g. Belenkaya et al., 2004; Han et al., 2005). We also knew from another study in the lab that HSPGs are abundantly expressed in the zebrafish gastrula (Gupta and Brand, 2013). So it was more of a question of whether the endogenous Fgf8a would also exhibit such interactions with the extracellular matrix and whether we would be able to detect such minute quantities, given that the overall amount of endogenous Fgf8a in the extracellular space was itself very low (in the nanomolar range).

Visualizing endogenous Fgf8a in zebrafish embryos by CRISPR-Cas9 mediated fluorophore knock-in. Top and middle panels show Fgf8a-EGFP distribution along the animal-vegetal axis at early and mid-gastrula stages, respectively. Fgf8a diffuses extracellularly at these stages to form graded distributions. The bottom panel shows a more localized deposition of Fgf8a along the basal side of the midbrain-hindbrain boundary organizer epithelium.

Visualizing endogenous Fgf8a in zebrafish embryos by CRISPR-Cas9 mediated fluorophore knock-in. Top and middle panels show Fgf8a-EGFP distribution along the animal-vegetal axis at early and mid-gastrula stages, respectively. Fgf8a diffuses extracellularly at these stages to form graded distributions. The bottom panel shows a more localized deposition of Fgf8a along the basal side of the midbrain-hindbrain boundary organizer epithelium.

What implications do your findings have on the understanding of the modes of propagation of morphogens?

MB: Production from an endogenous source and extracellular diffusion of even a monomeric protein can generate a morphogen gradient, and several interactions of that protein collude to generate the ‘sink-part of the equation’ to shape the gradient. The implication is that, for instance, production and secretion rates at the source, interaction with HSPGs, and different shapes and mechanochemical properties of ECM are all important. These, along with formation of ternary receptor-ligand-HSPG complexes, removal from the target cell surface by receptor-mediated endocytosis, active or passive degradation of Fgf8a, and relative movement of cell sources to target cell fields are all ‘tuning variables’ that help shape a morphogen gradient. At a general level, simultaneous propagation of multiple morphogens through different cellular and extracellular compartments may permit parallel transmission of information – and we see this over and again, embryos are very ‘smart’ in exploiting the cell biological mechanisms that are available to them!

Rohit, was there any particular result or eureka moment that stuck with you when doing the research for this paper?

RKH: When I started working on the project, we had clear goals in mind. We knew that FCS could be utilized to ‘monitor’ the nanomolar levels of endogenous Fgf8a in the extracellular space, but we also wanted to try and ‘visualize’ the endogenous protein distribution. The problem here was that over the time frame that we were interested in, i.e. early gastrula, when Fgf8a was suggested to act as a morphogen in patterning the gastrula, the levels of the protein were too low to be properly visualized by conventional confocal microscopy. So it was indeed a eureka moment for me when, together with Hella Hartmann from our imaging facility (who is also a co-author on the paper), I could establish a pipeline to visualize the protein gradient. When I saw for the first time on screen an image of endogenous Fgf8a-EGFP in the early gastrula, devoid of autofluorescent background, all those long hours that I was spending in the confocal room suddenly became meaningful (and motivated me to spend more).

How about the flipside, any moments of frustration or despair?

RKH: Of course there were! In fact, I can associate most of my moments of frustration with FCS. For instance, during the initial stages of my PhD, I was trying to take FCS measurements from the embryo without using a marker to label the extracellular space. The problem with this approach was that the extracellular space at early gastrula is highly dynamic, and also very narrow, particularly closer to the embryonic margin where the cells are tightly packed. This often led to some really frustrating moments with me ending up focusing my confocal laser beam on top or very close to the cells, which causes the FCS signal to degrade. After several such sessions however, I realised that this couldn't go on and I decided to utilize a marker to visualize the space properly. Injecting fluorescently labelled dextran into the animal pole of blastula stage embryos, where there is an abundance of extracellular space, proved to be a good way out, since dextran, owing to its hydrophilic nature, does not cross the cell membrane barrier but instead distributes throughout the extracellular space in the embryo by fluid flow.

Why did you choose to submit your paper to Development?

MB: The reason was our belief that the readership of Development would be the most appropriate one to appreciate our work. I have published many of my papers in Development; it is my favourite journal! I have always been impressed by the high standards of the review process and the professional editorial process at The Company of Biologists and Development, as a leading journal in our field.

Michael, where will this story take your lab next?

MB: Direct and single molecule visualization of the endogenous Fgf8a morphogen and other morphogens, and the ability to manipulate properties by knock-in and knockout technologies, of course opens a treasure trove that will help us understand the degrees of freedom that the embryo uses to generate information about position and differentiation processes. In combination with some modelling approaches that we are doing, this has the potential to develop a deep understanding of morphogen signalling mechanisms in vivo. The tighter localization of Fgf8a to the basal lamina around the anterior midbrain-hindbrain boundary epithelium is also quite interesting, suggesting progressively more selective interaction as ECM is built up around cells and epithelia. We have described something similar already, with the previously unreported secreted molecule calymmin being localized selectively to only one layer of the ECM around the developing notochord sheath (Cerdà et al., 2002). Such selective interactions with ECM could further enrich the repertoire of compartmentalized morphogen signalling avenues.

Finally, let's move outside the lab, what do you like to do in your spare time?

MB: I enjoy being with my family, to go on bike tours, and I love to read – fictional and non-fictional literature, and to learn and discuss about all sorts of science. As an institute director, I have strongly supported the development of the Dresden research landscape, in particular in the developmental biology-related areas. And I am generally interested in historical and political events, and enjoy good music.

RKH: And as for me, I am a huge movie buff. I try to watch most new releases at the cinemas, and sometimes (okay, maybe often) even tweet my opinions about them. I also love to travel – in fact, I even have a YouTube channel where I vlog about the places I travel to!

TU Dresden, Center for Regenerative Therapies, Fetscherstr. 105, Dresden 01307, Germany.

E-mail: [email protected]

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