Download Questions, chapter 18

Chapter 18 Answers
1.
Asymmetric mRNA distribution, cell-to-cell contact, and secretion of signaling
molecules are all mechanisms that can be used to induce cells to adopt different cell fates.
Asymmetric mRNA distribution takes place in conjunction with cell division. In this case, a
regulatory mRNA is unevenly distributed within a cell along the same axis as that used for
division. In this way, the two daughter cells produced when the cell divides inherit different
amounts of the mRNA, and consequently adopt different fates. Cell-to-cell contact and
secretion of signaling molecules are similar in that in both cases one cell produces a molecule
that activates a signal transduction pathway in a second cell. The two mechanisms differ,
however, in the particular way that the molecule is transmitted from the first to the second
cell. In cell-to-cell contact, the signaling cell deposits the molecule directly on the plasma
membrane of the recipient cell. In secretion, in contrast, the signaling cell secretes the
molecule into the extracellular matrix, and it then diffuses away and can bind to receptors
present on the surface of cells that are not in direct contact with the signaling cell.
mRNA localization is especially useful in developmental contexts where it is
necessary that the two cells produced from a single division event adopt different fates. For
example, during budding of the yeast S. cerevisiae, only the daughter cell (and not the mother
cell) inherits ash1 mRNA. This difference in ash1 levels leads to differences in HO
endonuclease activity, and ultimately means that only the mother cell can undergo mating
type switching. Cell-to-cell signaling is often used to guide the precise development of cells
within a tissue, where it is critical, for example, that two neighboring cells take on distinct
fates. An example of this is seen in the development of the fly, where Notch-Delta signaling
helps ensure that one cell becomes a neuron, and its neighbor does not. Finally, secreted
signaling molecules are particularly useful for organizing large fields of cells, where the
development of complex structures depends upon the establishment of positional information
within a large number of cells. An example of this is provided by Shh signaling from the
floorplate within the neural tube, which helps organize the future spinal cord by positioning
the appearance of at least four different types of neurons.
mRNA localization cannot be used in a developmental context where there is no cell
division. Cell-to-cell contact is limited to situations where the cells involved in the signaling
are in immediate contact with each other; it is unable to act over large distances. Finally,
secretion of signaling molecules is less precise than the other types of signaling, because it
involves less control over which cells receive the signal.
2.
A morphogen is a secreted signaling molecule that acts to convey positional
information on cells. Morphogens are present in a gradient, and cells adopt particular fates
depending on how much of the morphogen they are exposed to.
Examples of morphogens discussed in the chapter include Sonic hedgehog, Dorsal,
Bicoid, and Hunchback.
Because morphogens work by causing cells to adopt different fates depending on how
much of the morphogen they are exposed to, a morphogen must be present as a gradient in
order to work (although, in some cases, the gradient can be something other than overall
concentration, such as the gradient of nuclear localization seen with Dorsal). Therefore, in
order to determine if a particular molecule is a morphogen, one important thing to do would
be to visualize its distribution and confirm that it indeed forms a gradient. In addition, you
could confirm that different concentrations of the molecule indeed cause cells to adopt
different fates. For example, if a putative morphogen directs cells to take on three potential
fates—with cells closest to the morphogen adopting cell fate A, cells at a medium distance
adopting cell fate B, and with the most distant cells developing into fate C—then you would
expect that overexpressing the morphogen throughout the tissue might cause all of the cells to
adopt cell fate A. Similarly, mutations that eliminate morphogen expression altogether might
make all of the cells adopt fate C. Finally, you could carry out experiments in which the
gradient is altered, for example by mildly overexpressing the morphogen from its normal
source. In this case, you would expect to shift the boundary between the A and the B cells
(and between the B and C cells) so that it is now located farther away from the source of the
morphogen.
3a.
The gradient of morphogen concentration can lead to different levels of target gene
expression because it causes a similar gradient of transcriptional regulators to be present
within the nuclei of recipient cells. This regulator gradient influences the expression of
individual target genes in accordance with the type of regulator binding sites that the target
genes have. For example, target gene promoters containing low affinity binding sites for a
particular activator will only be expressed in cells closest to the source of the gradient,
because only those cells will have sufficient levels of the activator to drive expression of the
gene. Alternatively, target gene promoters with high affinity binding sites will be expressed
in cells throughout the field, because even the low level of activator that is present in cells
farthest from the morphogen source will be sufficient to turn on the gene.
b.
The stripes of even gene expression occur because the level of transcription at a target
gene does not simply rise in a linear fashion with increasing activator concentration, but is
instead controlled by thresholds that determine whether a gene is simply on or off. Once the
threshold is reached, the gene is turned on, and the intensity of expression does not
necessarily rise with increasing amounts of activator.
c.
Dorsal does not activate rhomboid expression in cells containing the highest amounts
of Dorsal because the high levels of Dorsal in the cells cause the Snail repressor to be
produced, and Snail represses rhomboid expression.
4.
The forespore (but not the mother cell) contains an active form of the sigma factor F,
which promotes the forespore-specific expression of a gene encoding a secreted factor called
SpoIIR. The forespore secretes SpoIIR into the compartment located between it and the
mother cell, and once there, SpoIIR triggers the proteolytic cleavage of a membrane-bound
sigma factor precursor called pro-E in the mother cell. This cleavage activates E in the
mother cell, where it turns on the expression of a number of mother cell-specific genes.
This is most similar to the activation of Notch by Delta, which also involves the
proteolytic cleavage of a membrane bound factor (Notch, in the Drosophila case), allowing a
portion of the protein to enter the nucleus and activate the transcription of its target genes.
5.
A ligand can lead to alterations in gene expression in several distinct ways. First, it
can activate a signal transduction pathway that ultimately changes the properties of regulatory
proteins within the nucleus—for example, activating them through phosphorylation. Second,
it can activate a receptor and thereby stimulate the movement of DNA-binding proteins into
the nucleus (for example, if the receptor activation frees a DNA-binding protein that was
previously held at the plasma membrane). Finally, ligands can stimulate proteolytic cleavage
of the receptor, allowing its cytoplasmic domain to enter the nucleus where it can associate
with DNA-binding proteins and modify gene expression.
6.
Patterning genes have such large regulatory regions so that they can accommodate
different combinations of the large number of DNA-binding proteins that reflect positional or
other developmentally relevant information. These unique combinations of binding proteins
help produce the precise and complex gene expression patterns that are required for the
development of an animal.
eve is expressed in seven stripes in the embryo, whose positions are determined by
particular combinations of various regulatory molecules. Specifically, the eve promoter
contains five different enhancers that together produce the seven stripes of eve expression
(see Figures 18-23 and 18-24). The stripe 2 enhancer, for example, contains binding sites for
Bicoid, Hunchback, Giant, and Krüppel. Bicoid and Hunchback activate expression within
the stripe, and in fact are present at high enough levels to activate the stripe 2 enhancer
throughout the anterior half of the embryo. The domain of expression is restricted to the
stripe, however, because Giant and Krüppel act as repressors to define its anterior and
posterior boundaries, respectively. Krüppel, which is expressed in a broad stripe in the middle
of the embryo, represses eve transcription by binding to the enhancer at sites overlapping
Bicoid binding sites, and also by "quenching" the activity of any Bicoid that does bind. Giant,
which is present at high levels in the anterior part of the embryo, presumably blocks eve
expression to the anterior side of the stripe using a similar mechanism.
7.
Notch can probably play a productive role in such different developmental contexts
for several reasons. First, because of combinatorial control of gene expression, the presence
of Notch in a nucleus will not necessarily turn on the same genes in any two cells. For
example, a given class of Notch target genes may require the presence of a second activator
in order to be expressed, and the second activator may only be present in certain cell types.
Second, differences in chromatin packaging between cells may mean that many Notch target
genes are only accessible to Notch binding in particular cell types. Finally, if many of the
genes turned on by Notch are not specifically involved in a particular process (such as neuron
or wing development), but instead involve more generic functions (such as modulating
movement through the cell cycle, or promoting general cellular differentiation), then Notch
could play a productive but non-specific role in the development of multiple types of tissues.
8.
localized mRNAs
Bicoid
→
gap genes
orthodenticle
→
pair-rule genes
Krüppel
eve
Nanos
hunchback
Oskar
knirps
giant
bicoid (activates orthodenticle, hunchback, eve)
nanos (inhibits hunchback translation)
hunchback (represses Krüppel, knirps, giant; activates eve)
kruppel (represses eve)
giant (represses eve)
9.
The gene is named after its mutant phenotype. When dorsal is defective, no ventral
differentiation occurs, and all the cells adopt dorsal fates.
10.
The adjacent sites allow cooperative binding of Bicoid monomers to the DNA.
Cooperative binding increases the steepness of the curve that describes the binding of a
protein to its target site as a function of the protein's concentration. For this reason, even a
small difference in Bicoid concentration (for example, the difference between two adjacent
cells of a fly embryo along its anterior-posterior axis) can translate into Bicoid binding sites
being either empty or being occupied. This helps bring about the sharp boundaries in the
expression domains of Bicoid target genes.
11.
a.
low level of Hunchback in anterior part of embryo, none in posterior
b.
high levels of Hunchback in anterior part of the embryo, with highest levels at
anterior pole; lower level of protein in posterior half of embryo
c.
low, even level of Hunchback throughout embryo;
d.
high levels of Hunchback in anterior half of embryo, low level in posterior
half, decreasing towards the posterior pole;
12
e.
no Hunchback in embryo;
f.
no Hunchback in embryo;
g.
no Hunchback in embryo;
h.
high levels of Hunchback throughout embryo.
a.
The switch involves a Notch-mediated transformation in the activity of the
Su(H) DNA-binding protein, causing it to go from being a repressor to being an activator.
Specifically, Delta stimulates the cleavage of Notch at the cell surface, releasing the
intracytoplasmic domain (NotchIC) of the protein. NotchIC then enters the nucleus, where it
binds to Su(H)—which had previously been actively repressing Notch target genes in
association with Hairless, CtBP, or Groucho—and activates its target genes.
b.
The inhibition of delta expression by Notch activation is part of a mechanism
by which two neighboring cells (such as the neuronal precursor cell and the skin cell
discussed in the text) can ensure that they adopt different cell fates. The two cells each have
the capability of following either of two developmental paths, one involving the expression of
Notch (but not Delta), and the other involving the expression of Delta (but not Notch). If the
two cells are to adopt different fates, then, it is imperative that one of them follows the
Notch-expressing pathway, and that the other follows the Delta-expressing pathway. This is
accomplished through a mutually-reinforcing mechanism in which the Delta producing cell
promotes the Notch-expressing pathway, and inhibits the Delta-expressing pathway, in the
opposite cell. The inhibition of delta expression by Notch activation is part of this
mechanism, as it acts to inhibit the Delta pathway in the Notch-expressing cell.
13.
Examples of the action of broadly expressed activators being spatially restricted by
repressors include: activation of HO within mother and daughter cells, with restriction by
ash1 in daughter cells; activation of rhomboid and sog by Dorsal, with restriction by Snail in
mesoderm; Bicoid/maternal expression of hunchback, with restriction by Nanos in posterior;
activation of Krüppel, knirps, and giant, with restriction by Hunchback to the anterior;
activation of eve by Bicoid and Hunchback, with restriction by Giant and Kruppel.
14.
Microtubules have a polarity that can be exploited by the cell to achieve the
asymmetric distribution of molecules such as mRNA. For example, in the Drosophila oocyte,
the microtubules are oriented in the anterior to posterior direction, with their + ends located at
the posterior pole. mRNA can be localized to the anterior or posterior pole by associating
with adaptor molecules that bind to the 3'UTR of the mRNA as well as with one or the other
end of the microtubules. In this way, for example, oskar mRNA is transported to the posterior
pole of the embryo, and bicoid mRNA is moved to the anterior pole.
Depolymerizing the microtubules should disrupt the polar localization of bicoid and
nanos mRNA, since both of their localizations depend upon the polar movement of mRNA
along microtubules. Also, if bicoid and nanos are delocalized, then hunchback (whose
localized transcription depends on localized transcriptional activation by Bicoid, and by
localized translational repression by Nanos) should be as well. The disruption of the
localization of any of these molecules would disrupt development in a major way, eliminating
critical anterior-posterior positional information and preventing the development of the
animal.