Sunday, April 10, 2016

Who am I? Mechanics of Cell Identity




How do neurons in the fly know which segment they are in?

Organismal development is a biological mystery that is being gradually unravelled in labs all over the world in that heroic endeavor called "normal science". Which is the pedestrian counterpart to the Kuhnian revolutions termed paradigm shifts. That the endogenous materials and genetic code of the egg/embryo generate the later adult forms has been known ever since scientists gave up vitalistic and other religious ideas about our biology. But how that happens ... approaching that question has taken lots of modern technology and persistence.

Fruit flies are the leading model system for embryonic and organismal development, due to their marriage of complex body plans, simple experimental handling, and extraordinarily deep genetics. After almost a century of productive study, a revolution happened in the 1980s in fruit fly genetics, following new mutant screens that uncovered some of the most basic mechanisms in body plan development. The genes found and analyzed during this period established a basic paradigm that has extended to all metazoans that have segmented body plans. Do we have segments? Yes, our backbone is a testament to our segmented ancestors.

The fly is built out of segments, whose cells know where/what they are by virtue of special genes expressed in them- the homeotic genes. The major genes of the fly homeotic complexes are, in order, Labial, Proboscipedia, Deformed, Sex combs reduced, Antennapedia, Ultrabithorax, Abdominal-A, and Abdominal-B.
The theme of these studies was that a series of genes, typically regulators of the expression of other genes, are turned on in sequence during development to identify progressively finer regions of the developing body. So at first, the two ends of the egg cell or synctium are set as different, then some gross regions are defined, and later on, each segment (and each side of each segment) expresses a few key genes that identify its cells, so that another cell, say a nerve cell migrating through the area, can tell exactly where it is. Each protein is expressed in a gradient within its zone, allowing the next regulator in the process to detect which end of that gradient it lies in, and thus whether to turn on or not. Late in this genetic series are the Hox genes, which are notorious for the complexity of their own regulation, for their ability, when mutated, to transform the identity of some segments entirely into other ones, and for the linear relationship between their chromosomal position and the locations on the body where they are individually expressed.

Progressive genetic specification of the fly embryo body plan, dividing it up into segments.  Gradients of one gene product allow the next gene product to detect the sides of its compartment and thus refine its cellular and body identity to a finer level.

A recent paper took up this adventure in the area around the head and neck, asking how embryonic nerve cells (neuroblast stem cells) originating in segments 4 to 6 know who they are and where to go. While one might not think that an animal head has segments at all, in embryological and molecular terms, heads encompass about 7 segments, (in the fly), which go through very messy convolutions into the complex mature structure. In comparison, body segments are far more orderly. Indeed, the central thoracic segment appears to be the default state, needing no Hox gene expression to develop normally:
"While thoracic identities seem to represent a ground state (T2, no input of Hox genes), identities of consecutive posterior segments are established by adding the function of Bx-C Hox genes Ultrabithorax (Ubx), abdominal-A (abdA) and Abdominal-B (AbdB), an evolutionary highly conserved phenomenon described as posterior dominance or prevalence of Hox genes. The terminal abdominal neuromeres A8-A10 exhibit a progressively derived character regarding size and composition. In these segments, NB [neuroblast, or neuronal stem cell] patterns and segmental identities are controlled by combined action of the Hox gene AbdB and the ParaHox gene caudal."

Map of the Drosophila head region, stained to show the Engrailed gene product. This is a homeotic segment polarity gene, expressed on one side of each segment throughout the embryo at this stage. At bottom is a map, coding the different segments accounted for within the head: red- antenna segment; purple- ocular segment; orange- intercalary segment; brown- labral segment; black- mandibular segment; green- maxillary segment; blue- labial segment; gray- first thoracic segment. In ensuing figure, the embryo is squashed to lay out the segments better.

The head segments likewise require extensive input from the Hox genes to keep their identities distinct. The researchers use a series of mutants to figure out how the local (segments 4 to 6) neuronal stem cells respond to missing genetic homeotic inputs. To do this, they use a few morphological characteristics and gene markers (assays for a gene whose expression is restricted to a certain lineage or cell type, in this case antibodies specific to the respective proteins) to identify the neuroblasts or stem cells they are interested in.

Stem neurons in three segments are stained with a combination of gene expression probes: Eagle in green, Runt in red, and Engrailed in blue. Note how combined expression renders some key cells aqua (green + blue) or yellow (green + red). Other diagnostic genes used for cell identification, which are all known to have developmental roles, are Deadpan, Deformed, Repo, Even-skipped, Eyeless, Sex combs reduced, Proboscipedia, and Gooseberry. The segments, from front [top] to back, are mandibular (mad), maxillary (max) and labial (lab). In back of the labial segment is the first thoracic segment. This stage of development (12) is quite early, well before the first larva forms.

Many figures of embryos later, stained for the expression of various proteins, in flies mutated for various key homeotic genes, and analyzed for the presence of notable cells at various stages, the authors draw several conclusions about the genetic influences that determine the identity and existence of neurons in these head segments, some of which will go on to contribute to the adult fly's brain. First, the maxillary segment, including its neuronal stem cells, expresses Deformed and Sex combs reduced from the Hox genes, while the next labial segment expresses Labial, but not in its neuronal cells. These seem to be the principal determinants of segmental identity. Yet when Deformed is mutated, only about half the cells are transformed from maxillary identity to a labial or thoracic identity. Only when another homeotic gene is also mutated, either Antennapedia or Labial, is the transformation more complete.

The curious thing about this is that neither Antennapedia nor Labial are normally expressed in the maxillary head segment, so the effect of their mutation must not be what the resarchers term cell-autonomous. These other genes must be acting from some distance away, instead of directly via their own expression in the cells being affected. This gets these researchers quite excited, and they track down some of the mechanism behind this extra cell fate specification.
"We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity."

Summary of findings, where Deformed is the main, local homeotic specifier for the maxillary segment neurons. But additional help comes from the next-door labial segment which expresses the homeotic gene Sex combs reduced, which influences expression in turn of the diffusible protein Amalgam, which helps the nearby maxillary segment keep its identity, via repression of the gene cyclin E. Interestingly, the Amalgam gene is located in the homeotic cluster right next to Deformed.

Summary of findings, where Deformed is the main, local homeotic specifier for the maxillary segment neurons. But additional help comes from the next-door labial segment which expresses the homeotic gene Sex combs reduced, which influences expression in turn of the diffusible protein Amalgam, which helps the nearby maxillary segment keep its identity, via repression of the gene cyclin E. Interestingly, the Amalgam gene is located in the homeotic cluster right next to Deformed.

So what had originally been though of as a fully cell-autonomous system, whereby each homeotic gene or combination thereof dictates the identity of cells in each respective segment where it is itself expressed, turns out to be a bit more messy, with neighbor effects that refine the identity code. Obviously this is getting into the deep weeds of developmental biology, but at the same time is an outstanding example of where the field is today, filling in ever-finer details of how development happens, using sophisticated techniques and backbreaking amounts of work.



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