A brief overview of Development:
Above is an abbreviated portion of the Dictyostelium multicellular
developmental program and the spore germination program. Several key regulatory
phenomena are emphasized.
The amoeba grow and reproduce asexually by feeding on bacteria in the leaf
litter of deciduous forests throught the world. This vegetative state can
continue indefinately as long as a source of bacteria are present to support
growth and division. Upon depletion of the bacteria in a local area, the
resulting lack of amino acids initiates the multicellular developmental
program.
During aggregation and mound formation, cell differentiation occurs to give
rise to several cell types: prespore cells (red) and prestalk A and prestalk
O cells (blue) (defined by expression of different promoter regions--ecmA
region and ecmO region--of the ecmA gene). Subsequent cell
sorting and patterning occurs as tipped mounds form: prestalk A cells localize
to and constitute the anterior-most portion of an apical tip; a band of
prestalk O cells sits under the prestalk A cells to make up the lower portion
of the tip; and the remaining 70-80% of the cells posterior to the tip are
mostly prespore cells. The apical tip, composed of prestalk cells, is referred
to as an organizer, and via various signaling mechanisms it mediates several
events: the morphological changes and accompanying assortment of cells into
discrete prestalk and prespore regions during tip formation itself; the
maintenance of cell patterning in the migrating slug; the movement of cells
within the slug that results in its migration; and the response to the environment
that results in either slug migration or culmination of development.
The tipped mound elongates into the cylindrical first finger stage in which
the spatial arrangement of the cell types is retained. The first finger
may initiate culmination in which morphogenetic changes and differentiation
of pre-cell types to mature stalk cells and spores occurs to produce the
fruiting body with a sorus full of spores sitting on top of a cellulosidic
stalk. Alternatively, the finger may transition into a slug which migrates
until environmental conditions conducive for spore dispersal are sensed.
The ability to sense an appropriate environment, and hence "decide"
when to culminate, is mediated by cells within the anterior most tip of
the finger and slug. The term transitional period is used in this proposal
and is defined as the time of fingers transitioning to early culminants,
including the variable time spent, if any, as a migrating slug.
At the end of the transitional period, the initiation of culmination is
manifest by the formation of a small cone of prestalk AB cells within the
prestalk A region at the very anterior or tip of the finger or slug. The
prestalk AB cells derive from a subset of the prestalk A cells termed prestalk
A* cells; this event is visualized by the expression of a reporter driven
by the promoter of the ecmB gene. Prestalk AB cells produce a nascent
stalk tube into which surrounding prestalk A cells are recruited while the
tube elongates towards the substratum. The maturing stalk cells within the
tube secrete factors that signal to the prespore cells to begin differentiation
into mature spores, and the spore mass begins moving up the apically elongating
stalk.
Ammonia and regulation of development:
Ammonia is produced by growing amoebae and by cells throughout development
via oxidative degradation of exogenously obtained or endogenous proteins.
Protein degradation is the primary source of nitrogen, carbon, and energy
for growing and developing cells. During development, ammonia production
and its local concentration are a reflection of the producing cell type
and of the macro- and/or micro-environment of the cell. Developing Dictyostelium
cells have co-opted the endogenously produced ammonia as a signal for monitoring
the environment and for regulating a number of the morphological changes
and differentiation events that occur during the developmental program (click
here for a summary listing). For
example, within the slug ammonia concentrations are relatively high in the
anterior or prestalk region and in the very posterior (rearguard cells)
and relatively low in between (prespore region). The local ammonia concentration
in the immediate cellular environment and within a particular cell at this
or other times of development is due to the rate of cellular ammonia production,
to its assimilation into amino acids, to cellular transport via Amts, and
to its rate of loss via evaporation to the atmosphere or via diffusion into
the substratum. These processes in turn are influenced by a number of factors
such as the presence or absence of a slime sheath (and its thickness), stalk
tube, or spore coat, the position of a cell in relation to these, the light
level, the temperature, and of course the pH. Hence ammonia is well suited
as a signaling molecule as its levels can reflect and monitor many cellular
and environmental states. Such monitoring is essential for progression through
the developmental program, for coordinating morphogenesis and differentiation,
and for choosing the correct path when alternate states are best suited
for particular environments.
There are a number of specific developmental processes for which ammonia/ammonia
signaling appears to be involved. These include: modulating the rate of
movement of chemotaxing cells during aggregation; inhibiting stalk cell
differentiation within slugs and promoting differentiation of prespore cells
to spores during culmination; effecting cell proportioning in migrating
slugs by modulating the migration of ALCs (anterior like cells found within
the prespore region) to the anterior; stimulation of prespore gene expression
and depression of prestalk gene expression ; effects on spacing of culminating
structures; and others (click here
for a summary listing).
A major role of ammonia is regulating the alternative developmental pathways
of a migrating slug versus culmination of development. Low local ammonia
levels promote culmination while high concentrations result in slug migration
as high concentrations reflect a poor environment for spore dispersal. Ammonia
is thought to mediate the alternative outcomes of slug migration versus
culmination in part via modulation of cAMP dependent protein kinase A (PKA).
Our work has demonstrated that the histidine kinase DhkC controls PKA activity
to regulate the slug/culmination choice. This is accomplished via a phosphorelay
that modulates the activity of the cAMP phosphodiesterase RegA. Several
results suggest that the DhkC phosphorelay mediates ammonia signaling with
regard to the slug/culmination choice. We have proposed that high ammonia
levels activate the phosphorelay resulting in the activation of RegA and
thus inhibiting PKA activity and culmination. When local ammonia levels
are low, the phosphorelay would be inactive, allowing cAMP accumulation
to activate PKA and subsequently initiate culmination.
Our past and current work addresses how ammonia
transporters mediate ammonia signaling to carry out its many regulatory
roles. The current model of the relationship between AmtA and AmtC and the
DhkC phosphorelay can be found here.
Histidine Kinases and phosphorelays regulating development:
Two-component phosphorelays (click here for basic
models of these signaling systems), controlled by sensory histidine kinases,
regulate various important aspects of development by modulating the activity
of cAMP dependent protein kinase (PKA). We have investigated how the histidine
kinases DhkC and DhkB are involved in regulating development, and their
involvement in these regulatory processes are indicated in the diagram below.
The details of these pathways can be seen elsewhere (DhkC
or DhkB and DhkC).
As mentioned above, DhkC controls PKA activity to regulate the slug/culmination
choice via a phosphorelay that modulates the activity of the cAMP phosphodiesterase
RegA. During teminal differentiation and culmination, once again differentiation
and morphogenesis must be coordinated. The downstream components in the
DhkC phosphorelay are invovled in this coordination, but the input for regulating
their activities is probably not DHKC but an as yet unidentified regulator
functioning in response to secreted factors from prestalk cells.
During late spore maturation and once spores have fully developed, DhkB
negatively regulates the phosphorelay so that spore germination (yellow
emerging amoeba from red spores) is not initiated until spores are dispersed
and the environment will support amoebal growth. When these conditions are
met, DhkB become inactivated and DhkC activates the phosphorelay to result
in the intitiation of the spore germination pathway and re-establishment
of the growth and cell division programs.
