Saturday, October 15, 2016

The Odd Stripes

In the early fly embryo, a gene called odd-paired (opa), is essential for the transition from 7 to 14 segments.

Scientific data is sometimes boring, but other times quite aesthetic, even stunning. Astronomy can be a matter of graphs and spectra, but it can also be revelatory images from distant galaxies, or from weirdly amazing planets and moons in our own solar system. Fly embryology and genetics is one of the more conceptually, technically, and also aesthetically advanced areas of biology, leading the way in the understanding as well as visualization of early animal development.

Segmentation is a body plan principle shared by all chordates, most insects, and some worms. It provides a convenient blend of modularity that allows for repetition of a basic plan to organize cells into an extended, worm-like body, while also allowing variation and specialization in each segment, to the point that organisms like us end up not seeming segmented at all as adults. It may have evolved independently in those three lineages, though also shares some strategies and molecular details, so the full evolutionary story is not quite settled.
General plan and progression of fly embryogenesis, from rough location determination to finer and more precise specification. During this time what starts as a large mass of egg cytoplasm turns into a synctium of positioned, divided nuclei, which then lead to cell formation around the inner surface, and the beginning of tissue formation.

In each phylum, there is a progressive front-to-back and gross-to-fine scale process that is a little like an image coming into focus. A cascade of transcriptional regulation and related molecular events first decide which end of the fertilized egg is front. Then, as cell proliferation proceeds, this cascade subdivides the embryo into about seven zones, (in the fruit fly), and lastly divides those zones into fourteen parasegments, which eventually lead to formation of the ultimate physical segments, with their particular cells and organs, whether common or specialized.

A recent paper focused on one gene that participates in this process: the odd-paired gene of fruit flies. When this gene is missing, embryos don't form the 14 parasegments, but only the seven larger bands. Development is arrested, fatally. Yet odd-paired is expressed all over the body. It does not have the kind of localized segmental or parasegmenal expression that was expected, and is found for the other "pair-rule" genes, at least until quite a bit later on. How does that work?

Incidentally, the odd-paired gene is the homolog (or rough equivalent by sequence, and presumably, function) of a significant gene of humans, GLI1, which is found mutated in cancers, especially glioblastoma. It is typical for master regulators of early development, when cell proliferation is very high and differentiation is low, to be involved in cancer if they are activated through some kind of error at a later time, helping to re-create that embryonic condition in an uncontrolled form.
"Through gene regulation, the GLI1 family of proteins regulates a number of important cellular processes, such as, neural development, cell proliferation, oncogenesis, survival, epithelial-mesenchymal transition (EMT), migration, invasion and metastasis."

Detection of gene expression (mRNA) of selected pair rule genes in early embryos. Red is the odd-skipped gene, green is even-skipped, purple is runt, and turquoise is the paired gene. The time period in this panel (t4) is a slightly earlier stage of development vs the panel below (t6). The patten of seven stripes for each gene, offset slightly to its particular sub-location in the nascent double-segment, is very clear.
Under the influence of odd-paired, (wild-type case), the stripes from above have doubled after about 1.3 hours vs the panel above. 

The cascade of segmentation refinement is mostly a matter of gene expression. Transcription regulators are expressed in various places along the body axis, and their combination, overlapping in some places, absent in others, dictates (through their binding to enhancers at DNA of target genes, thus activating them) a refined pattern for the next step, which consists mostly of another set of transcription regulators. The "gap" genes each regulate portions of the ~seven broad bands of expression, and their combinations activate the next set of "pair-rule" genes that are typically expressed in the same seven bands, which over time (involving mutual back and forth regulation) resolve to and help form fourteen parasegments, each of which comprise the front half of one future physical segment, and the back half of another. Lastly, a set of "segment-polarity" genes are activated in one-cell wide stripes to specify one or the other side of each of these fourteen parasegments, preparatory to the more complex work of specifying the details of cell types, and tissues that are to reside there.

Expression of odd-paired (green) is all over the trunk, while another gene, odd-skipped, is nicely restricted to nascent double-segment zones. The phases refer to time points of embryogenesis, indicating that odd-paired comes on rather suddenly during this process.

The locations of gene expression can be visualized by constucting DNA molecules complementary to the expected mRNA from the respective pair rule or other genes. That DNA is labelled with a tag that can later be reacted to produce the intense fluorescent signals seen in the figures. This DNA (probe) is physically diffused into the chemically stabilized/fixed embryos under warm conditions so that it selectively hybridizes to the target mRNA, thus showing where it is located.

It is the endogenous refining transition of the "pair-rule" genes from seven to fourteen zones of expression that interested these researchers. They reason that perhaps the appearance of the odd-paired mutant, which misses features of each odd-numbered segment, indicating that it participates in specifying the parasegments, arises not from its own location of expression, but from its critically timed regulation of the other pair rule genes. It is known to come on right when this focusing event takes place, and binds to and regulates several of the pair-rule genes, as well as cooperates with them in their mutual regulation.

Embryos mutant for odd-paired (opa) compared to the normal wild-type (wt) case. Opa5 is a partial mutant, opa8 is more severe. The neat stripes in the wild-type are progressively deranged, and lack doubling during this later embryonic phase, gastrulation.

Unfortunately, the data of this paper consists entirely of anatomical expression patterns, rather than the enhancer structure and activation data they would need to support the final model, which was essentially replicated by another lab around the same time (and amplifies existing work). The model is that odd-paired cooperates selectively, and in a strictly timed fashion with the other pair-rule genes to regulate pair-rule as well as genes in the next level down, the segment polarity genes, to accomplish the positional refinement and particularly the band doubling that is absent when odd-paired is absent. The phenomenology, as seen in the expression visualizations, is very striking, and reflects circuitry at the level of gene regulation that, while painstaking to analyze, would give definitive answers about exactly what each of the actors in this process does.
"In the even parasegment, our data support a combinatorial model wherein Ftz [fushi terazu] activates en [engrailed] expression and odd [odd-skipped] restricts this activation to the anterior-most Ftz-expressing cells. An essential facet of this model is that Opa [odd-paired] must repress odd transcription in a specific cell, the anterior-most Ftz-expressing cell, to allow induction of en by Ftz. The Run [runt] pair-rule protein is a candidate for a cofactor with which Opa may cooperate to specifically repress odd." (from Benedyk, et al.)

While the data relies on molecular biology for the visualization and modeling, it carries on a grand tradition of anatomy and genetics as well, in its beautiful micrographs and focus on the physical structure of the later organism, which at the points pictured here is quite invisible, only incipient in the molecular patterning that is going on incognito, as it were.

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