Sunday, August 6, 2017

An Egg Asks: Which Way is Up?

One of the prototypical morphogens, bicoid, tells fly eggs what's head and what's tail.

Translating the digital instructions of the genome into an actual body is the complicated work of development. It is obvious that the instructions are not explicit or specific, mapping out where every organ, cell, and molecule is supposed to go. Rather, the code provides ingredients and rough guides to channel development in favorable directions, relying heavily on implicit, default processes to fill in the details.

One way to amplify a digital code is to use a morphogen- a substance with an analog character, whose concentation varies with location and can give many of those locations different instructions. Morphogens are used extensively in body and brain development, and one of the first to be found genetically was bicoid, one of the proteins responsible for telling Drosophila eggs which end is anterior, and which posterior. The name comes from bicoid mutants, which end up with two tails (bi-caudal) and no head- naturally a fatal condition.

Effects of a complete bicoid deficiency.

Bicoid mRNA is deposited by the mother in eggs in a strongly asymmetrical fashion, at their anterior poles, so they are pre-oriented. When the egg is fertilized, this mRNA starts getting translated / expressed into bicoid protein, which is the actual morphogen. This is a DNA and RNA-binding protein, and regulates the expression of other genes. Bicoid expression is inhibited in the posterior by the protein nanos, which is concentrated there. Conversely, bicoid inhibits the expression of caudal, another protein that is concentrated in the posterior and directs posterior cell fate. Bicoid regulates at least 70 other promoters/genes to activate anterior cell fates, among them the next levels of location specificity genes, the gap (hunchback, giant) and pair-rule genes (krüppel, even-skipped). The interplay of numerous positive and negative regulators of this sort, others of which take over subsequently at ever finer levels of detail, creates a relatively robust system of cell specification which allows bodies to be made consistently under a variety of temperature, nutritional, genetic, and other conditions. For details, the fly community resource is valuable, as is a reference work.

Expression of some key genes in fly embryos, bicoid at anterior (left, blue) and caudal at right (red). Evx (yellow), is a relative of even-skipped, both pair-rule genes that specify finer segmental-scale divisions in the embryo than do bicoid or caudal.

A recent paper used new methods to study the activity of bicoid in detail, turning it on and off at will. They replaced the normal version of the gene with one carrying two extra protein sequences, one of which rendered the protein conveniently fluorescent (in red), and the second of which, on exposure to light, binds to other proteins in the cell, typically turning it to an inactive state. This engineered protein provided normal bicoid function when the flies were raised in the dark, but turn on the lights, and activity dropped immediately, allowing study of exactly when it is needed, and for what. Unfortunately, this turn-off only applied to one of the protein's activities, its transcriptional activation. Its second activity, of translational repression (of caudal, among other genes) was unaffected by the light-switch protein fusion.

It had not been understood just how long during embryogenesis bicoid is needed, whether only at the start, or ongoing through many stages. What these researchers found is that bicoid is needed into surprisingly late stages, to within 10-30 minutes of gastrulation, which is very roughly the midpoint of early embryogenesis that goes from egg to hatched larva. What is bicoid doing through all these later points in time, when its fundamental job was simply to instruct the next set of location-specific genes, the gap and pair-rule genes, where to turn on- a job that is over long before gastrulation?
Impact of brief light exposure (i.e. shut-down of bicoid) on development. Note that even the briefest interruption, for 10 minutes just prior to gastrulation, causes mal-development of the anterior-most (left) head section, comparing B' and C'. "n.c." refers to nuclear division cycles, which are a standard / convenient way to time the progress of these early embryos. Proteins stained are engrailed (En), deformed (Did), sex-combs reduced (Scr), abdominal-B (Abd-B), and (not stained), ultrabithorax (Ubx).

It turns out that many genes and cell types of the anterior have evolved in the presence of bicoid, and so have come to depend on its presence. Genes farther back in the fly have to make do with lower levels of bicoid protein, are more sensitive, and need its actiavtion more briefly (such as the mid-section gap gene krüppel). But in the anterior, the large and durable amount of bicoid has fostered a dependence well beyond the early roughing-out of the segmentation pattern. For example, knirps, a gap gene with head expression, is highly sensitive to bicoid activity and plays a key role in anterior fate specification, needs bicoid activation right up till gastrulation.

Length of time bicoid is needed, with respect to position in the embryo. The colored lines depict when bicoid is required in the corresponding segment(s).

"A minimal 20 min illumination at the end of n.c. 14 abolishes the expression of the Knirps anterior domain (Kni1) capping the tip of the embryo, as well as the first Giant stripe (Gt1). "

While not terribly pathbreaking in its conclusions, this work shows progress in the level of detail being studied, enabled by remarkable technological advancements. The authors even resort to computer modelling to make sense of the complex network of regulators, only a few of which were touched on above. Fruit flies have been the leading model system of animal development for over a hundred years, and are still going strong.

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