Arrested Development{41}

It’s spring of 2014, I’m teaching a course that I have not taught since my SCALE-UP epiphany, and thus, this blog will morph yet again. But what could be more appropriate for a morphing blog than a course in Developmental Biology? It’s been a while, but I have taught this class before, and as I’ve already told my students, it is my absolute favorite class to teach. What? More fun than Cell and Molecular Biology? Even better than Cancer Biology? Yes, it’s that good.

I’m not entirely certain why but throughout my graduate education and career as a cell, molecular and developmental biologist, the title of developmental biologist was always the most comfortable fit for me. Developmental biology is about change, upon which I thrive, but even more than that, it’s about progress, about moving forward toward ambitious yet achievable milestones – gastrulation, organogenesis, hatching (or birth).

In my own research, I studied the development of frogs. I found frogs to be a particularly compelling model organism because, just when this creature seems to have mastered something (e.g. swimming from predators), BAM!, it’s time to switch gears completely, active new developmental programs, and start all over again. Let’s lose that tail and grown some legs!

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fertilized Xenopus eggs

My research program focused on a particular developmental milestone called   the midblastula transition, or MBT, in the South African clawed frog, Xenopus laevis. Prior to the MBT, Xenopus embryos undergo rapid cell divisions, such that within five hours of fertilization, one relatively large cell, the egg, has become 4096 microscopic cells. With mitosis occurring every 30 minutes in near synchrony across the embryo, there is little time for the embryo to do anything else. No time for growth, no time for gene expression (the embryo relies entirely upon maternally stored mRNA and proteins), and apparently no time to worry about mistakes. This last characteristic is where I made my contributions to the field of frog development. I was interested in cell cycle checkpoints, those mechanisms present in eukaryotic cells that put the breaks on the cell cycle when something is seriously wrong such as damaged or unreplicated DNA. But early Xenopus embryos didn’t seem to engage these checkpoints.  We blasted embryos with gamma rays and X-rays to damage the DNA and the embryonic cells kept dividing in synchrony with their untreated siblings, every 30 minutes. We treated the embryos with a drug called aphidicolin to completely block DNA replication and they kept on dividing. With no DNA….

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blastula stage (pre-MBT) embryos

….until the MBT, when Xenopus embryos with damaged or unreplicated DNA engaged in what I came to refer to as the mother of all checkpoints. At the stage when embryos should have initiated transcription of their own genomes for the very first time, those with damaged or unreplicated DNA committed apoptosis, or programmed cell death (Sible et al, 1997). And it wasn’t subtle. Dying cells would come spewing out of the blastocoel, the hollow cavity of the embryo, like a tiny little volcanic eruption. I spent hours upon hours peering through the microscope, in morbid fascination of these embryonic suicides.

embryos undergoing apoptosis

embryos undergoing apoptosis

Why in the world would this happen? I lost a lot of sleep over this question. My favorite speculation was that the “apoptotic checkpoint” as we called it, provided distinct advantages for the organism. Frog embryos do not have mouths to eat nor tails to swim away. Floating around in the pond, they are about as vulnerable as a human infant. It is in the best interest of the embryo to develop quickly into a tadpole. The first order of business is cell division, which as I mentioned, happens quickly. Up until the MBT, the embryo is little more than a ball of undifferentiated cells, and it is only after gene expression begins at the MBT that one cell is significantly distinctive from another. Perhaps, rather than wasting time engaging checkpoints at every cell cycle, Xenopus embryos have evolved to divide unchecked until the MBT, the first time when damaged or unreplicated DNA would have a consequence, and at that stage, use the apoptotic checkpoint to clean house, ridding the embryo of any genetically damaged cells. In our experiments, we used insanely high doses of ionizing radiation or chemicals, but in the real world, these damaging events would likely be few and far between, with just a few of those 4096 cells in need of removal. Recent experiments by undergraduates working in my lab have supported this  hypothesis. They were able to ablate a certain percentage of cells before the MBT and the embryo would develop into a more or less normal tadpole (Mulholland et al, unpublished).

This story might have had a scientifically satisfying ending if it weren’t for the spring peepers. I love listening to them through my bedroom window at night, which got me interested in local species of frogs such as those studied by my friend and colleague Dr. Lisa Belden. Virginia is home to not only spring peepers, but also wood frogs, bull frogs, American toads, green frogs, gray tree frogs, and more. Under Lisa’s tutelage, I began mucking through puddles and swamps.  My neighbors would catch me sneaking into their cultivated fish ponds at night, flashlight in hand, looking for frog eggs. I became obsessed with collecting embryos from as may amphibian species  as possible. My children do not have fond memories of this period. I dropped one of them, then a toddler, into an ice-crusted pond in February while reaching for wood frog eggs. She marks that as the day that she decided to become a lawyer, not a scientist.

My goal was simple – establish the apoptotic checkpoint in as many species as possible, then publish the findings in a good journal. Once I established some credibility with the ecology crowd, I could pursue the ‘why’ questions about the apoptotic checkpoint though field studies.

The experiments were straightforward. We collected pre-MBT embryos from native species, blasted them with X-rays, then waited for them to die at the MBT. We waited, and waited, and…..waited. Nothing happened. The embryos did not undergo apoptosis, but they did not move past the MBT into gastrulation either. They just sat there with arrested development. We waited for hours and hours. We waited days. We waited weeks. Nothing happened. Eventually the embryos died, but not in any developmentally meaningful way. The dishes in which we kept them would become contaminated and moldy. Or someone would forget to add more medium and they would dry up. So much for easy answers, or publications for that matter. I didn’t know what to make of these data, and honestly I still don’t. Xenopus laevis is one of a handful of established model organisms for studying vertebrate development. We extrapolate our findings to fish and mice and people. But these data suggested that the discovery of the apoptotic checkpoint, although it had later been demonstrated in zebrafish (Ikegami et al, 1999), did not apply to all frogs, certainly not to Virginia frogs. Even more puzzling, our data suggested that embryonic development could be arrested, for a long time, maybe even perpetually. This was not what I signed up for when I became a developmental biologist. I wanted embryos with ambition, with a future, that knew where they were going and got there fast. That’s how I rolled.

At least, that’s how I used to roll, until about a month ago when I was rolling at a good clip down a hill on my bike. Then, before one could say ” midblastula transition”, I was on the ground surrounded by a patch of black ice with a fractured inferior pubis ramus. “What can I do to accelerate the healing?”, I asked the orthopedic. “Rest”, he replied. “How about cycling on the trainer? Physical therapy? Hot yoga?”, I persisted. “Nothing”, he growled. “The only thing you can do to get better is do nothing!”

I was sentenced to at least 8-12 weeks of arrested development, something extremely difficult for a distance athlete to come to terms with. But I complied and did nothing. A whole lot of nothing. I held out as long as I could stand it, until I started to feel kind of moldy and dessicated. Then I thought, “Hmm, development always moves forward, and that’s not an option, but what if it could move backwards? What if, under the right circumstances, a frog could undergo reverse metamorphosis? What if global warming and melting of polar caps will eventually cause the de-evolution of land animals back into creatures with fins and gills?”

So I took to the water, and am learning to swim despite the nagging of my inner frog, “wrong direction, lady”.  It’s the one time I can move without asking anything of His Excellency Pubis Ramus. Perhaps becoming  a swimmer is my MBT, or my likely my MLC (mid-life crisis). At any rate, it feels good to be moving again.

tadpoles!

tadpoles!