hunchback
There are two promoter regions. The posterior regulatory region is several kb upstream of the P1 (maternal & zygotic) transcript. The anterior regulatory region is upstream of the P2 (zygotic) transcript within the large maternal intron.
hunchback expression in the tail initiates from two promoters. A cap, covering the terminal 15% of the embryo, is composed only of mRNA from the distal
transcription initiation site (P1). A posterior stripe generated later is composed of mRNA from both
the distal and proximal (P2) transcription initiation sites, upstream of the P2 site of initiation.
The posterior regulation region, a 1.4 kb fragment of the hb upstream region is both necessary and sufficient for posterior expression. Sequences within this fragment
mediate activation by the terminal gap gene tailless and repression by terminal gap gene huckebein, which together direct the
formation of the posterior hb stripe. The TLL protein binds in vitro to specific sites
within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of
gene expression by TLL (Margolis, 1995).
In adult Drosophila females maternal transcripts of hunchback
are produced only by the distal (P1) promoter. This expression is largely restricted to the ovarian nurse cells. A deletion analysis of the hb promoter
using lacZ reporter constructs defines a 1.2-kb genomic DNA fragment surrounding the P1
promoter sufficient to reproduce the wild-type pattern of hb ovarian transcript accumulation (Margolis, 1994).
The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides
positional information for pattern formation. However, since bicoid seems to be a recently acquired gene
in flies, the question has been raised as to how embryonic patterning is achieved in organisms with more
ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein acts as a morphogen in abdominal patterning, it was asked which functions
of Bcd could be performed by Hb. By reestablishing a proposed ancient regulatory circuitry in which
maternal Hb controls zygotic hunchback expression, it has been shown that Hb is able to form thoracic segments in the absence of Bcd (Wimmer, 2000).
A functional hb transgene has been generated that is
missing all P2 promoter sequences and relies solely on the P1 promoter (hbP1only). hbP1only
constructs do not respond to bcd and do not
mediate gene expression in the anterior cap domain. Therefore, hbP1only uncouples the direct link between the
Bcd and Hb morphogen systems. Zygotic hb mutants derived
from heterozygous parents do not develop labial
or thoracic structures, and they also show a fusion of abdominal
segments A7 and A8. When one copy of the hbP1only transgene is provided
zygotically (by the father) to a hb mutant embryo, it
rescues the posterior phenotype, and A7 and A8 developed normally. The labial/thoracic phenotype is not rescued. However, when
hbP1only is provided as one copy by the mother to a
hb mutant embryo, the posterior and part of the anterior phenotype are rescued. These embryos exhibit normal labial and
prothoracic (T1) segments, and only lack meso- and meta-thoracic segments (T2 and T3). The anterior
rescue is due to the maternal contribution of hbP1only
because sibling embryos that do not inherit the hbP1only
construct zygotically also exhibit the partial anterior (but not the
posterior) rescue. This indicates that restoring high levels
of maternal hb expression (i.e., two copies: one wild type
plus one copy of hbP1only) is sufficient to rescue the
labial and prothoracic segments in the zygotic hb mutant
progeny. Therefore, the lack of zygotic
hb leads only to the loss of T2 and T3 and to the
fusion of A7 and A8, whereas the previously reported zygotic
hb phenotype represents a
combination of a haploinsufficient maternal plus a zygotic phenotype (Wimmer, 2000).
The loss of zygotic hb activity affects regions of the
embryo that correspond to the two late stripes of zygotic hb
expression: The A7-A8 fusion corresponds to the posterior stripe,
whereas the loss of T2 and T3 corresponds to the PS4 stripe, which starts as a fairly wide domain covering the anlagen of T2 and T3. This correlation between the zygotic
hb phenotype and the late stripe expression pattern led to a reconsideration of the importance of the early bcd-dependent anterior cap domain. Under some conditions, hbP1only
(maternal hb contribution plus stripe expression) might
suffice for normal segmentation of head and thorax, making superfluous
the bcd-dependent anterior cap domain. Hence, the hb PS4 stripe is activated without bcd-dependent
hb expression. This stripe is repressed by
the knirps abdominal gap-gene product and is
activated by high levels of Hb itself, either directly or indirectly
(through repression of kni). Embryos that lack the bcd-dependent
hb cap domain have been generated that contain an increased maternal
hb contribution (to four copies) and kni reduced
to one copy. These embryos display a range of
partially rescued hb phenotypes, including some embryos with a full set of head and thoracic segments. Thus,
bcd-dependent hb expression can in principle be
dispensable for embryonic segmentation, and the only critical anterior
domain of zygotic hb expression appears to be the PS4
stripe, with the bcd-dependent cap domain serving to
activate this stripe. This role is likely achieved by the maternal
hb contribution in species where zygotic hb is not under the control of bcd or where a bcd
homolog might not exist (Wimmer, 2000).
The rescue of T2 and T3 structures by bcd-independent
hb expression raises the question of whether these
structures could develop in a completely bcd-independent
manner. Embryos derived from bcd mutant mothers develop
ectopic tail structures that replace head and thorax and exhibit a
disruption of some abdominal segments. Although previous work
has shown that, in the absence of
bcd, high levels of maternal hb can rescue a
normal abdomen and some thoracic structures, no complete thoracic
segments can be induced. A bcd-independent source
for high levels of zygotic hb expression was introduced into a bcd mutant background. By establishing this artificial zygotic Hb
gradient, two notable results were obtained, with variable expressivity:
(1) about 20% of the embryos exhibit rescued T2 and T3 segments. The maintenance of high Hb levels that lead to the rescue of
thoracic segments is likely due to the activation of the hb
stripe element because the hbP1AB reporter is activated as a stripe where T2 and T3 form. (2) Most of the ectopic tail structures that are anteriorly duplicated in bcd mutants are suppressed, suggesting
further redundancy between Hb and Bcd. However, Hb and Bcd must act at different levels in suppressing these tail structures, which depend on
the activity of the caudal (cad) gene: Bcd acts by repressing cad mRNA translation, whereas Hb does not but might instead
interfere with Cad protein function. This bcd-independent
suppression of cad function might be important in organisms
where the Cad gradient only forms late and represents
another variation as to how cad activity is suppressed at
the anterior of the embryo (Wimmer, 2000).
Different species use various strategies for repression of Cad
function: In Drosophila, translational repression of
CAD mRNA involves the Bcd homeoprotein,
whereas in Caenorhabditis elegans repression involves the
KH-domain protein MEX-3. In vertebrates, a mutually
antagonistic relation between otd-like and
cad-like genes has been proposed to reflect an ancestral
system to pattern the anteroposterior axis of the embryo. In arthropods, ancestral head determinants are
probably encoded by otd-like genes as well. Thus, in the
beetle Tribolium, where no bcd homologs but
Bcd-like activities have been found, these activities
are probably also covered by Otd or KH-domain proteins. This is
consistent with the Otd-like DNA binding specificity of Bcd, which is
atypical for a factor encoded by a gene duplication in the Hox cluster.
This change in specificity was probably crucial for Bcd to acquire its
key role in anterior patterning, because it allowed Bcd to function both as
an RNA binding protein and as an Otd-like transcription
factor. In this respect, it is not surprising that the zinc-finger
protein Hb cannot completely replace Bcd in the head region. Even the
highest levels of Hb obtained in these experiments were not able to
induce head formation in the absence of Bcd. However, Hb is required
for the posterior head region (maxillary and labial segment) and supports anterior head development by synergizing
with Bcd. It will be interesting to see whether such a
synergism can also take place between Hb and other more ancestral head
determinants (Wimmer, 2000).
These results indicate that the two morphogenetic systems,
Bcd and Hb, do not need to be directly linked. Hence, the direct
regulation of hb by Bcd might represent a recent
evolutionary addition to the insect body plan. In
Drosophila, the abundance of bcd-dependent
hb expression eventually renders superfluous the maternal
hb contribution, which is widespread within
arthropods. Consistent with the idea that the
bcd-dependent hb expression represents a recent evolutionary acquisition, the P2 promoter contains only activator sites
that allow the direct response to a specific threshold level of a
morphogen. This might be a unique situation,
given that most other developmentally regulated promoters contain, in
addition to activator sites, repressor elements for setting the exact
borders of gene expression. By tinkering with the
rather plastic mechanisms of early development, the
ontogeny of Drosophila could be changed toward an inferred ancestral state
where maternal Hb controls zygotic hb. This change could be
brought about by altering patterns and levels of gene expression; this
presents the most likely variation on which evolutionary processes are
based (Wimmer, 2000).
The Drosophila mophogenetic protein Bicoid (Bcd) can activate transcription
in a concentration-dependent manner in embryos. It contains a self-inhibitory
domain that can interact with the co-repressor Sin3A. A
Bcd mutant, BcdA57-61, that has a strengthened self-inhibitory function and is unable to activate the hb-CAT reporter in Drosophila cells, has been used to analyze the role of co-factors in regulating Bcd function. Increased
concentrations of the co-activator dCBP in cells can switch this protein from
its inactive state to an active state on the hb-CAT reporter. The C-terminal
portion of BcdA57-61 is required to mediate such activity-rescuing function of dCBP. BcdA57-61 has a normal ability to bind to a single TAATCC site when analyzed in vitro. Although capable of binding to DNA in vitro, BcdA57-61 is unable to access the hb enhancer element in cells, suggesting that its DNA binding defect is only manifested in a cellular context. Increased concentrations of dCBP restore not only the ability of BcdA57-61 to access the hb enhancer element in
cells but also the occupancy of the general transcription factors TBP and TFIIB
at the reporter promoter. These and other results suggest that an activator can
undergo switches between its active and inactive states through sensing the
opposing actions of positive and negative co-factors (Fu, 2005).
As a molecular morphogen, Bcd can undergo switches, in a
concentration-dependent manner, between its active and inactive states in
activating transcription of its target genes. The experiments described in this
report suggest another mechanism that can facilitate on-off switches of Bcd
activity in a Bcd concentration-independent manner. In particular, the mutant
BcdA57-61 is incapable of activating the hb-CAT reporter gene in
S2 cells at all concentrations tested. The
inability of this mutant Bcd to activate the hb-CAT reporter reflects a
distinct functional state of this protein rather than its defects in protein
stability. In fact, this same mutant protein is only modestly weaker than the wt
protein on another reporter gene, kni-CAT, which contains the
Bcd-responsive kni enhancer element. These and
other results suggested that the A57-61 mutation may cause its functionally
inactive state on hb-CAT by more efficiently interacting with a
co-repressor protein(s), such as Sin3A and its associated complex(es).
The experiments described in this
report show that increased concentrations of dCBP can restore activity to
BcdA57-61 on the hb-CAT reporter in cells. These results suggest
that the opposing actions of positive and negative co-factors can facilitate Bcd
to switch between its active and inactive states in a manner that is Bcd
concentration-independent (Fu, 2005).
Although BcdA57-61 can bind to both a single site and natural enhancer
elements in vitro, it is unable to access the hb enhancer element
in cells. These
results suggest that the DNA binding defect of this mutant protein is only
manifested in a cellular context. This notion is consistent with the finding
that the PAH domains of Sin3A do not exhibit any increased ability to reduce DNA
binding by BcdA57-61 in vitro when compared with wt Bcd. It is proposed
that other co-repressors or those
that are associated with Sin3A, such as the HDACs, can reduce the ability of Bcd
to access a natural enhancer in cells. It is possible that the enzymatic HDAC
activity that is more stably associated with BcdA57-61 makes it unable to
negotiate with histones for accessing DNA. It is also possible that a more
stable Bcd-co-repressor complex may sterically hinder the interaction between
BcdA57-61 molecules and prevent cooperative binding to the enhancer
element in cells (Fu, 2005).
The most striking finding of this report is that high levels of dCBP can switch
BcdA57-61 from its inactive state to an active one on the hb-CAT
reporter in cells. ChIP data further show that dCBP increases both the
ability of BcdA57-61 to access the hb enhancer element in cells
and the occupancy of GTFs at the reporter promoter. How does dCBP switch the activity states of BcdA57-61 on
hb-CAT in cells? Since Bcd and dCBP can physically interact with each
other through multiple domains, it is possible that dCBP may increase the DNA binding ability of Bcd in
cells by stabilizing the interaction between Bcd molecules and thus enhancing
its cooperativity. It is also possible that dCBP may physically compete with
co-repressor complexes in interacting with Bcd. Co-IP results suggest that
dCBP may negatively affect the interaction between Bcd and Sin3A in cells.
dCBP could also play a role in facilitating the
interaction between Bcd and the transcription machinery. For all these actions,
dCBP may play a structural (rather than enzymatic) role.
Finally, the fact that the HAT-defective mutant of dCBP does have a
reduced ability to restore activity to BcdA57-61 indicates that its enzymatic activity has a positive role, possibly
through modifications of histones. It is likely that dCBP can affect the
BcdA57-61 activity through multiple mechanisms that may be weak
individually but, when
combined, can lead to a dramatic switch from its inactive state to an active one
on the hb-CAT reporter in cells (Fu, 2005).
Currently, it is poorly understood how precisely Bcd activates transcription.
Previous studies suggest that much of its activation function is conferred by
the C-terminal portion of Bcd.
This portion of the protein contains several domains, including the acidic,
glutamine-rich and alanine-rich domains, that are characteristic of activation
domains capable of interacting with components of the transcription machinery.
Interestingly, the alanine-rich domain previously thought to play an activation
role was shown recently to exhibit an inhibitory function instead.
The C-terminal domain of Bcd can also interact with dCBP, and the results show that this domain is responsible
for mediating the activity-switching function of dCBP.
Although much of the activation function of Bcd is provided by its
C-terminal domain, the N-terminal portion of the protein also contains some
activation function. Studies have shown that Bcd(1-246), a derivative
lacking the entire C-terminal portion of Bcd, can rescue the
bcd- phenotype when expressed at high levels.
These results suggest that Bcd can achieve its activation
function through multiple domains presumably by interacting with different
proteins, including co-activators and components of the transcription machinery.
The results described in this report further support the importance of dCBP in
facilitating activation by Bcd (Fu, 2005).
Bcd is a morphogenetic protein whose behavior can be regulated not only by its
own concentration but also by the enhancer architecture.
On the kni and hb enhancer
elements, the N-terminal domain of Bcd is preferentially used for either
cooperative DNA binding or self-inhibition, respectively. It is proposed
that the interaction between Bcd molecules bound
to the kni enhancer element, through its N-terminal domain, can interfere
with its interaction with co-repressors, such as Sin3A.
Co-activators such as dCBP and co-repressors such as Sin3A can also
functionally antagonize each other, possibly by competing for Bcd interaction as
part of the mechanisms. Bcd is more sensitive to
the self-inhibitory function on the hb enhancer element than on the
kni enhancer element: consistent with dCBP's
antagonistic role, dCBP increases the activity of Bcd more robustly on the
hb enhancer element than on the kni enhancer element.
However, the interplay between positive and negative
activities that regulate Bcd functions is probably far more complex than the
simple physical competition: as already discussed above, dCBP can affect Bcd
activity through multiple mechanisms in both HAT-dependent and independent
manners. Moreover, in the
presence of exogenous dCBP, high levels of BcdA57-61 cause a reduction in
its activity on the hb-CAT reporter in cells, a reduction that is not observed with wt Bcd,
suggesting that the optimal concentration ratio between Bcd and dCBP may vary
depending on the strengths of the self-inhibitory function and interaction with
co-repressors. In addition, high concentrations of dCBP can rescue the inactive
derivative BcdA57-61, but not another inactive derivative lacking the
C-terminal portion, Bcd(1-246; A57-61), suggesting that the
Bcd-dCBP interaction strength can also influence the balance between
positive and negative activities that regulate Bcd function (Fu, 2005).
The experiments described in this report suggest that an activator's function is
subject to intricate controls by both positive and negative activities in cells.
A fine balance between these activities is critical for normal cellular and
developmental processes. Transgenic experiments show that both
BcdA57-61, which has a strengthened self-inhibitory function, and
BcdA52-56, which has a weakened self-inhibitory function, cause embryonic
defects. In addition, embryos
with reduced dCBP activity exhibit defects in early expression patterns of a Bcd
target gene, even-skipped. Finally, mutations affecting
SAP18, a component of the Sin3A-HDAC complex, can alter Bcd function and
anterior patterning in embryos. In addition to the
co-factors discussed in this study (Sin3A, dCBP and SAP18), Bcd likely has the ability to
interact with many other proteins, including not only regulatory proteins but
also components of the transcription machinery. Precisely how all these different proteins harmoniously
regulate and facilitate the execution of Bcd functions during development
remains to be determined. Recent studies have shown that the Bcd gradient in
embryos possesses a strikingly sophisticated ability to activate its target
genes in a precise manner.
These findings further underscore the need of intricate control mechanisms that
facilitate Bcd to switch between its active and inactive states in target gene
activation. These studies suggest that on-off switches of Bcd activity can be
achieved not only in a Bcd concentration-dependent manner but also in a Bcd
concentration-independent manner. It remains to be investigated whether and how
Bcd interacting proteins, including those yet to be identified, participate in
the precision control of target gene activation during development (Fu, 2005).
Bicoid activates hunchback's anterior to posterior zygotic gradient (Tautz, 1988 and Struhl, 1989).
Bcd contains three putative activation domains: a glutamine-rich region, which interacts in vitro with TAFII110; an alanine-rich domain, which targets TAFII60, and a C-terminal acidic region, which has an
unknown role. Transcriptional activation of
a bcd target, the hb promoter, is synergistically enhanced in vitro by Bcd and Hb. However, this effect is observed only when both TAFII60 and TAFII110 are present. It has been suggested that the synergy observed in vivo is because of the corecruitment of TAFII110 and TAFII60 by Bcd and Hb, respectively. Flies were generated carrying bcd transgenes lacking one or several of these domains to test their function in vivo. Surprisingly, a bcd transgene that lacks all three putative activation domains is able to rescue the bcdE1 null phenotype to viability. Moreover, the development of these embryos is not affected by the presence of dominant negative mutations in TAFII110 or TAFII60. This means that the interactions observed in vitro between Bcd and TAFII60 or TAFII110 aid transcriptional activation but are dispensable for normal development (Schaeffer, 1999).
Bicoid (Bcd), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions
require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of Caudal (CAD)
mRNA. The Bcd HD has been shown to be a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the
HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties
of the HD. Corresponding BCD mutants fail to repress CAD mRNA translation, whereas the transcriptional target genes are still activated (Niessing, 2000).
In order to characterize portions and individual amino acid residues of the Bcd HD that are specifically required for one or both Bcd regulatory functions, transgenes expressing wild-type or mutant bcd cDNAs were placed into the genome of homozygous bcd mutant females and their ability to rescue wild-type zygotic hb activation and cad mRNA translation in their embryos was assayed. Such embryos, referred to as 'bcd embryos,' fail to exert Bcd-dependent transcriptional activation of the zygotic target gene hb in their anterior half. Instead, the embryos show a duplication of the posterior Bcd-independent stripe of hb expression in the anterior region (Niessing, 2000).
Expressed Bcd mutant proteins that lack the helices I and II of the HD (BcdDeltaH1-2) or the amino acid interval between positions 42 and 51 in helix III (BcdT42-N51) fail to restore Bcd-dependent hb transcriptional activation and translational repression of CAD mRNA in the anterior region of bcd embryos. This indicates that the integrity of the Bcd HD is necessary for the control of transcription and translation. Transgene-dependent expression of BcdhIIIAntp, in which the C-terminal half of the Bcd HD is exchanged for the corresponding sequence of the Antennapedia (Antp) HD, rescues Bcd-dependent hb expression in the anterior region of bcd embryos, but no Cad gradient is formed. Bcd mutations in which two adjacent arginines at positions 53-54 and 54-55 of the HD, respectively, were replaced, fail to control Bcd-dependent transcription and translation. Thus, helix III of the Bcd HD is necessary for both transcriptional activation and translational repression, and amino acids within helix III are essential for specifying not only DNA binding but also RNA recognition by the HD. This proposal is consistent with the observation that part of the helix III of the Bcd HD has characteristics of an arginine-rich motif (ARM) (Niessing, 2000).
To test whether the conserved amino acids of Bcd's ARM are indeed required for RNA target recognition and whether single amino acid replacements may allow the DNA and RNA binding properties to separate, alanine replacement mutants of the Bcd HD were generated and their in vitro binding properties assayed. The Bcd HD (HDwt) binds both DNA and RNA, whereas HDK50A, HDN51A, HDR53A, and HDR55A failed to bind to both targets. Bcd HDR54A, which contains alanine in place of arginine in position 54 of the HD, bound DNA properly, but its RNA binding was reduced by more than one order of magnitude. The binding properties of HDK57A were indistinguishable from HDwt. In summary, arginine at position 54 of the HD is critical for specifying RNA versus DNA binding, and its replacement shifts the binding property of the HD to prefer DNA over RNA recognition (Niessing, 2000).
In order to test the in vivo relevance of these binding studies, the corresponding Bcd HD mutants were examined by transgene-dependent expression in bcd embryos. The Bcd mutants were generated in the context of an 8.7 kb genomic DNA fragment spanning the entire bcd locus, which fully rescues bcd embryos after P element-mediated transformation. The transgene-expressed BcdK57A protein, which contains an HD with normal DNA and RNA binding properties, causes Bcd-dependent hb expression and Cad gradient formation, and the embryos developed into normal-looking larvae and fertile adults. BcdN51A, BcdR53A, and BcdR55A, which contain HD mutations that cause the loss of DNA and RNA binding properties in vitro, fail to activate Bcd-dependent hb transcription and to repress translation of CAD mRNA; such embryos develop a bcd mutant phenotype. The BcdR54A mutant, which contains an HD with DNA, but no RNA, binding properties, was able to activate the transcription of hb but not to repress the translation of CAD mRNA. This observation is consistent with the result obtained using the transgene bearing the BcdR54S mutation, which contains a serine residue in place of arginine at position 54. Thus, both Bcd mutants that contain a replacement of arginine at position 54 of the HD fail to control CAD mRNA translation but do activate transcription of hb (Niessing, 2000).
Nanos functions as a localized determinant of posterior pattern. Nanos
RNA is localized to the posterior pole of the maturing egg cell. It encodes a protein that emanates
from this localized source. Nanos acts as a translational inactivator of hunchback and thereby establishes the early anterior to posterior gradient of Hunchback (Curtis, 1995).
Terminal gap genes tailless and huckebein direct the
formation of the posterior Hunchback stripe. The TLL protein binds in vitro to specific sites
within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of
gene expression by TLL. The anterior border of the posterior HB stripe
is determined by TLL concentration in a manner analogous to the activation of anterior hb expression
by Bicoid. In the posterior expression pattern of hb, the transcription factor serves as a (so-called) secondary gap gene regulated by "primary" gap genes tailless and huckebein, regulated in turn by torso (Margolis, 1995).
The Krüppel binds to the
sequence AAGGGGTTAA. Binding sites are present for KR upstream of
the two hb promoters. These could mediate the repression of hb by KR and perhaps
allow hb to influence its own expression. A 10 Kb genomic DNA fragment contains the hb coding sequence and both promoters. The proximal promoter directs early zygotic expression of hb in
the anterior part of the embryo The distal hb promoter is transcribed maternally and also directs later zygotic expression . This latter fragment contains the KR binding sites. 300 bp upstream of the transcription
start of the 2.9 kb transcript are sufficient for normal regulation of the
expression of this transcript. The two KR binding sites are located at -676 and -359 bp from
the proximal hb promoter (Treisman, 1989).
The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is
critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test
whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer
of the pair-rule gene even-skipped was used to express kni in an ectopic position.
Manipulating the stripe 2-kni expression constructs and examining transgenic lines with
different insertion sites led to the establishment of a series of independent lines that
display consistently different levels and developmental profiles of expression. Individual
lines show specific disruptions in pair-rule patterning that are correlated with the level
and timing of ectopic expression. No effect on the expression patterns of giant or Krüppel could be observed at any level of kni misexpression. However, the ectopic kni did significantly alter the hunchback pattern. Stripe 2-kni, centered on PS3, completely prevents the expression of the PS4 hb stripe. This expression occurs even in embryos that contain the lowest levels of ectopic kni (Kosman, 1997).
High local concentrations of NOS
protein in the posterior of the embryo are necessary to inhibit translation of the transcription factor
Hunchback in this region, and thus permit expression of genes required for abdomen formation (Gavis, 1994).
Nanos prevents the repressor hunchback from acting in the posterior half of the embryo. This allows Caudal to activate the gap genes giant and knirps (Rivera-Pomar, 1995).
Mutations in several Polycomb group (PcG) genes cause maternal-effect or zygotic segmentation
defects, suggesting that PcG genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other PcG
genes show gap, pair rule, and segment polarity segmentation defects.
Posterior sex combs and polyhomeotic interact with Krüppel and
enhance embryonic phenotypes of hunchback and knirps (McKeon, 1994).
Genetic experiments and a targeted misexpression approach have been combined to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo. The results suggest that gt functions in the repression of three target genes, the gap genes Kruppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve). The anterior border of Kr, which lies 4-5 nucleus diameters posterior to
nuclei that express GT mRNA, is set by a threshold repression mechanism involving very low levels of Gt protein. The gap gene Kr is activated in a broad central region of
precellular embryos. Midway through cleavage cycle 14, this domain extends from 41-59% egg
length. The initial positioning of the anterior border of this
domain is thought to be controlled by repression involving a
combination of maternal and zygotic hunchback transcripts. To test whether gt
is also involved in setting or maintaining this border, the Kr expression pattern was analyzed in embryos containing the st2-gt transgene, a modified version of the 480 bp eve stripe 2
enhancer. These embryos show no changes in the
initial positioning of the Kr expression domain early in
cleavage cycle 14, but slightly later there is a dramatic
retraction of the anterior Kr border. The delay in the observed repressive effect on the Kr anterior border is probably due to the fact that the Kr domain is expressed earlier
than the st2-gt transgene. Higher levels of ectopic gt result in a more severe retraction, suggesting that Kr transcription is very sensitive to repression by gt. To test whether gt affects Kr expression during normal development, Kr expression was examined in embryos that carry
a strong hypomorphic gt allele. The initial Kr expression
pattern was correctly established in these gt hypomorphic embryos. However, slightly later, a significant anterior expansion (from 59% to 65% egg length) is observed,
suggesting that gt-mediated repression is essential for
maintaining the position of the anterior border of the Kr domain (Wu, 1998).
gt is required for repression of zygotic hb expression in more anterior
regions of the embryo. Zygotic expression of hb is initially activated by
the bcd and maternal hb gradients in a broad domain that spans the
anterior half of the embryo. This expression is then rapidly refined
during nuclear division cycle 14, leaving a secondary pattern that
includes a variable head domain, a stripe at the position of
parasegment 4 (PS4), and a posterior stripe. The PS4 stripe overlaps
the anterior border of the Kr domain. By examining
hb expression in gt mutants, significant changes in
this secondary pattern were detected. Initially, hb expression at the
position of PS4 is greatly reduced, possibly because of the
anterior expansion of the Kr domain in gt mutants. High levels of hb expression persist in more anterior regions of gt mutant embryos. The persistent hb
expression domain appears very similar in shape to the
normal gt domain, suggesting that gt may
act as a repressor to clear hb expression from this part of the
embryo during wild-type development. To test whether
endogenous gt levels were required for this repression, hb expression was examined in gt mutants that also contained the st2-gt transgene. hb expression is repressed normally by a single copy of the st2-gt5 transgene, suggesting that relatively low levels of ectopic gt can replace this function of the endogenous gene. Since gt seems to be involved in repression of hb in anterior regions, it is possible that this repression is important for
setting the anterior border of the hb PS4 stripe during wild-type
development. To test this, hb expression was examined in
embryos containing the st2-gt transgene. The position of the anterior border of the hb PS4 stripe appears unchanged in these embryos, suggesting that the levels of
ectopic gt tested here are not sufficient to repress hb PS4
expression. However, a slight posterior expansion of this stripe
could be detected in embryos with high levels of misexpression, which is probably caused by the retraction of the Kr domain. This supports the hypothesis that Kr activity is
important for setting the posterior PS4 stripe border, and further demonstrates the importance of gt-mediated restriction of Kr expression to central regions of the embryo (Wu, 1998).
Anterior terminal development is controlled by several
zygotic genes that are positively regulated at the anterior
pole of Drosophila blastoderm embryos by the anterior
(bicoid) and the terminal (torso) maternal determinants.
Most Bicoid target genes, however, are first expressed at
syncitial blastoderm as anterior caps, which retract from
the anterior pole upon activation of Torso. To better
understand the interaction between Bicoid and Torso, a
derivative of the Gal4/UAS system was used to selectively
express the best characterized Bicoid target gene,
hunchback, at the anterior pole when its expression should
be repressed by Torso. Persistence of hunchback at the pole
mimics most of the torso phenotype and leads to repression
at early stages of a labral (cap'n'collar) and two foregut
(wingless and hedgehog) determinants that are positively
controlled by bicoid and torso. These results uncovered an
antagonism between hunchback and bicoid at the anterior
pole, whereas the two genes are known to act in concert for
most anterior segmented development. They suggest that
the repression of hunchback by torso is required to prevent
this antagonism and to promote anterior terminal
development, depending mostly on bicoid activity (Janody, 2000).
The results indicate that early anterior expression of a labral
determinant, cnc, and of two foregut determinants, wg and hh,
is repressed when zygotic expression of hb is allowed to persist
at the anterior pole of the Drosophila blastoderm embryo.
Expression of cnc, wg and hh is under the positive regulation
of bcd and torso but no zygotic gene has yet been implicated
in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and
that torso-induced anterior repression of hb is necessary for
their positive control by torso. To determine whether the
positive control of cnc, wg and hh by torso could be the result
of a double negative control involving hb, expression of these
genes was analysed in hb zygotic mutant embryos derived from
torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb
at the pole, expression of these genes should be recovered in
hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is
not recovered in hb minus embryos derived from torso females
whereas it is normal in hb minus embryos. This indicates
that, although necessary, the anterior repression of hb is not
sufficient to mediate Torso positive control on cnc, wg and hh
early anterior expression (Janody, 2000).
During embryonic development, orderly patterns of gene expression eventually
assign each cell in the embryo its particular fate. For the anteroposterior axis
of the Drosophila embryo, the first step in this process depends on a spatial
gradient of the maternal morphogen Bicoid (Bcd). Positional information of this
gradient is transmitted to downstream gap genes, each occupying a well defined
spatial domain. The precision of the initial process has been determined by comparing expression domains in different embryos. The Bcd gradient
displays a high embryo-to-embryo variability, but this noise in the
positional information is strongly decreased ('filtered') at the level of
hunchback (hb) gene expression. In contrast to the Bcd gradient, the hb expression pattern already includes the information about the scale of the embryo. Genes known to interact directly with Hb are not
responsible for its spatial precision, but the maternal gene staufen may be
crucial (Houchmandzadeh, 2002).
Among all the mutations studied, the only ones that
affect Hb boundary precision are certain alleles of staufen. In embryos from mothers homozygous for either stauHL or staur9, the Hb boundary position shows a variability of
6%, comparable to the observed Bcd variability.
Surprisingly, this variability is largely reduced (to 2%) in another
strong allele of stau, D3. Mutations in stau disrupt bcd and osk mRNAs and decrease Bcd protein level about twofold. Whether the effect of stau on Hb is simply an indirect effect of its variable effect on bicoid was tested. From the pool of embryos in stauHL background that were double stained for Bcd and Hb,
two populations were selected: one that displayed an anterior Hb boundary shift,
and one that displayed a posterior shift. The corresponding
average Bcd profiles for these two populations are very similar, both
in the Bcd level and in its spatial distribution. Thus, the
observed variability in the Hb boundary position may reflect an
activity of staufen independent of bcd. The disruption of Hb
precision in stauHL
is transmitted to downstream genes, and is
not corrected before gastrulation. For instance, double staining for
Hb and Kr shows that the variability of the Kr
boundary in the stauHL background is similar to that of the Hb boundary. Moreover, the positions of these two boundaries remain tightly correlated, as in the wild type (Houchmandzadeh, 2002).
By quantitatively analyzing the protein profiles of maternal
morphogens and zygotic gap genes in numerous wild-type and
mutant embryos, two phenomena that take
place in the early Drosophila development have been demonstrated: (1) at a very early stage, noise associated with the maternal gradient of Bcd is filtered out,
and (2) at the same time, the genetic network, which includes the Hb
gap gene, establishes spatial proportions (scaling) in the embryo.
It is potentially significant that staufen, the one gene affecting the
process, makes a product that localizes to both poles of the
egg. More work is needed to establish the mechanisms that
control the spatial scaling and precision. It would then be
interesting to investigate whether similar phenomena are present in other developmental processes in Drosophila and other organisms (Houchmandzadeh, 2002).
Cooperative interactions by DNA-binding proteins have been implicated in
cell-fate decisions in a variety of organisms. To date, however, there are few
examples in which the importance of such interactions has been explicitly tested
in vivo. This study tests the importance of cooperative DNA binding by the Bicoid
protein in establishing a pattern along the anterior-posterior axis of the early
Drosophila embryo. bicoid mutants specifically defective in
cooperative DNA binding fail to direct proper development of the head and
thorax, leading to embryonic lethality. The mutants do not faithfully stimulate
transcription of downstream target genes such as hunchback (hb), giant, and
Krüppel. Quantitative analysis of gene expression in vivo indicates that bcd cooperativity mutants are unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis; they display a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo (Lebrecht, 2005).
In Drosophila, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, a potent transcriptional activator, Bicoid (Bcd), was ectopically expressed in pole cells. Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. Evidence is presented indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells (Deshpande, 2004).
A number of maternally derived gene products are likely to contribute to transcriptional quiescence in the pole cells of Drosophila. One of these is Germ cell less (Gcl), a component of the germ plasm that is necessary for the formation of pole cells. gcl appears to be involved in the establishment of transcriptional quiescence and in embryos lacking gcl activity, newly formed pole buds are unable to silence the transcription of genes such as sisterless-a and scute.
Conversely, when Gcl protein is ectopically expressed in the anterior of the embryo it can downregulate the transcription of terminal group genes such as tailless (tll) and huckebein
(Leatherman, 2002). A second maternally derived gene product involved in transcriptional quiescence
is Nanos. In the soma, Nanos, together with Pumilio, plays a key role in
posterior determination by blocking the translation of maternally derived
hunchback (hb) mRNA. Nanos (Nos) also plays a role in down-regulating transcription in pole cells, and in embryos produced by nos mutant mothers: genes that are normally active only in somatic nuclei are inappropriately transcribed in pole cells. These
include the pair-rule genes fushi tarazu and even
skipped, and the somatic sex determination gene Sex-lethal (Deshpande, 2004 and references therein).
Ectopic expression of Bcd in pole cells can induce the
transcription of the bcd target gene hb. In addition to
activating hb transcription, Bcd protein perturbs the migration of
the pole cells to the primitive somatic gonad and causes abnormalities in cell cycle control. These effects on germ cell development resemble those observed in embryos from nos mutant females. Moreover, as in the case of nos- pole cells, the Sxl promoter Sxl-Pe is also turned on in pole cells by Bcd in a sex-nonspecific manner.
Surprisingly, transcriptional activation in pole cells by Bcd requires the
activity of the terminal signaling system. This observation is unexpected, since previous studies have established that the transcription of a downstream target gene of the terminal pathway, tailless (tll) is shut down completely in pole cells. Moreover, the doubly phosphorylated active isoform of MAP kinase ERK, which serves as a sensitive readout of the terminal pathway,
is nearly absent in pole cells. Taken together, these findings argue that the activity of terminal signaling pathway in pole cells of wild-type embryos must be substantially attenuated, but not shut off completely. What mechanisms are responsible for downregulating terminal signaling in the presumptive germline? Evidence indicates that polar granule component (pgc) functions to attenuate the terminal pathway in newly formed pole cells. pgc encodes a non-translated RNA that is localized in specialized germ cell-specific structures called polar granules (Nakamura, 1996). Loss of pgc function in newly formed pole cells results in the ectopic phosphorylation of ERK and the activation of the ERK dependent target gene tll. pgc is required to block the establishment of an active chromatin architecture in pole cells (Deshpande, 2004).
Thus Bcd protein expressed from a
bcd-nos3'UTR transgene (the 3' UTR of nos serves to localize the bcd message to pole cells) can activate the transcription of its target gene hb in pole cells, overcoming whatever mechanisms are responsible for transcriptional quiescence. In addition to activating transcription of hb, Bcd has other phenotypic effects. It prevents the pole cells from properly arresting their cell cycle and disrupts their
migration to the somatic gonad. Because similar defects in pole cell
development can be induced by the inappropriate expression of Sxl protein in these cells, one plausible hypothesis is that Bcd not only activates the hb promoter, but also turns on the Sxl establishment
promoter, Sxl-Pe. Consistent with this idea, the Sxl-Pe:lacZ
reporter is turned on in the pole cells of male and female bcd-nos
3' UTR embryos and Sxl protein accumulates in these cells. Although
previous studies indicate that Sxl-Pe is responsive to Bcd, it is somewhat surprising that Sxl-Pe is not only inappropriately turned on in pole cells by Bcd, but that it is activated in both sexes. This suggests that Bcd activation of Sxl-Pe in pole cells must proceed by a mechanism that bypasses the X/A chromosome counting system which controls Sxl-Pe activity in the soma. It is interesting to note that the activation of Sxl-Pe in pole cells in the absence of nos function also seems to depend upon a mechanism(s) that circumvents the X/A chromosome counting system (Deshpande, 2004).
That Bcd protein depends upon other ancillary factors to turn on
transcription in pole cells is demonstrated by the requirement for
tsl function in the activation of both the hb and
Sxl-Pe promoters. tsl is a component of the maternal
terminal signaling pathway that activates the zygotic genes, tll and huckebein (hkb), at the poles of the embryo. In addition, the terminal pathway has opposing effects on the expression of
bcd-dependent gap genes. At the anterior pole, where terminal signaling activity is highest, Bcd targets such
as hb and orthodenticle (otd) are repressed. At a distance from the anterior pole, where both the
concentration of Bcd protein and the strength of the terminal signaling
cascade is much lower, the terminal pathway has an opposite, positive effect on hb and otd expression. Two mechanisms are thought to account for the positive effects of the terminal pathway on bcd target genes: (1) Bcd is a direct target for phosphorylation by the terminal signaling cascade; (2) regulatory regions of bcd target genes have sites for other transcription factors whose activity can be directly modulated by the terminal system (Deshpande, 2004).
Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).
To analyze the effects of dsRNA on mRNA stability in
Drosophila oocytes, dsRNAs corresponding to the
maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and
localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi,
fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that
dsRNA-coated gold particles randomly introduced into fertilized eggs by
a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of
a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with
bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).
Whether and when transcripts become sensitive to dsRNAs during
oogenesis was determined. dsRNA was injected into staged oocytes and their
consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage
14 oocytes could be examined for RNAi activity.
Levels of bicoid and hunchback mRNAs were unchanged
in stage 14 oocytes after injection of dsRNA, indicating that oocytes
at this stage are unable to carry out RNAi (Kennerdell, 2002).
Oocytes of most animals arrest at species-specific stages of meiosis
while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded
with RNAs and proteins. Some of these
molecules are differentially localized within the oocyte, imparting
positional information to be used for embryonic axis formation. When
Drosophila oocytes reach stage 14, they undergo meiotic arrest
once more, this time at metaphase I. These arrested oocytes remain
translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process
of maturation or activation that precedes fertilization. In the case of
Drosophila, it appears that ovulation triggers activation of
the oocyte to resume meiosis. When oocytes are
activated, meiosis is completed and translation of maternal RNAs is
dramatically elevated. Shortly thereafter,
the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).
RNAi-like effects are not detected in arrested stage 14 oocytes
injected with dsRNA. Was this a general feature of the female germ
line? To explore this issue, dsRNA was injected into mature activated
oocytes. Injection of dsRNA causes reduction in bicoid and
hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This
maturation procedure reactivates meiosis, mRNA translation, and
vitelline membrane cross-linking. After
maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a
decrease in bicoid mRNA. Thus, immature
Drosophila oocytes that are coordinately blocked for meiosis
and translation are resistant to RNAi, and the block to these processes
can be released by maturation or activation of oocytes (Kennerdell, 2002).
There are several possible ways in which RNAi might be blocked in
arrested oocytes. One possibility is that an essential component of the
RNAi machinery might be missing at this stage. Oocyte maturation would
then involve synthesis of the component. To address if synthesis of a
missing component is responsible, oocytes were activated in the presence
of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in
vitro in the presence of cycloheximide. This treatment inhibits
>95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid
dsRNA and, strikingly, they showed a decrease in bicoid mRNA
levels that was comparable to that of normal mature oocytes.
RNAi is established during oocyte maturation even when protein
synthesis is blocked. Thus, RNAi establishment during oocyte
maturation does not likely occur by synthesis of an essential
protein component of the RNAi machinery (Kennerdell, 2002).
The stage 14 oocyte is coordinately blocked in both translation and
RNAi. The two processes are released near simultaneously from this
block, suggesting perhaps that a shared mechanism links their
regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after
oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C
mRNA occurs, even though transcripts at this stage are associated with
ribosomes and are competent to drive translation in vitro. The stable pool of
alphaTubulin67C mRNA is comparable to levels of
bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).
Several Drosophila genes have been identified that affect
translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).
The aub gene is a member of a family of genes implicated in
RNAi and PTGS. Indeed, aub has been implicated in PTGS
regulation of the Stellate repeats and Su(Ste) genes
on X and Y chromosomes. Another member of the
family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this
gene family in Drosophila have been implicated in RNAi and
PTGS at various steps (Kennerdell, 2002).
It was of interest to determine if other translational regulatory genes
are involved in RNAi. To test this possibility, two genes
that possibly act through interactions with RNA were examined. vasa and
spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or
hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).
Arrested Drosophila oocytes are unable to generate RNAi
silencing of endogenous maternal mRNAs, but selectively establish this
capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene
expression, may conceivably be one way in which mRNA is blocked from
RNAi attack. Alternatively, targeting of mRNA might require transcripts
be assembled onto active polysomes. This may be the case, because
siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no
evidence to indicate that dsRNA-targeting requires ribosome
translocation on transcripts, because it is found that cycloheximide
inhibition of ribosome translocation does not block RNAi activity in
activated mature oocytes (Kennerdell, 2002).
Coupling RNAi to translated mRNA might facilitate base-pairing
interactions between siRNAs and an unfolded mRNA target, or it might
simply be a means to mark RNAs to be scanned for destruction. The key
evidence suggesting that transcript translation is linked to transcript
degradation by RNAi comes from experiments in which dsRNA
against the alphaTubulin67C message was tested. dsRNA is
ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry
out RNAi against translated bicoid and hunchback
transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).
Thus Aub and Spn-E are required for RNAi in Drosophila oocytes. Aub and
Spn-E might play a specific role in gene silencing mechanisms,
including RNAi, that nevertheless have a widespread impact on many
features of development. Alternatively, Aub and Spn-E could be required
for RNAi because they activate translation of germ-line transcripts
including those for bicoid and hunchback. Although
there is no evidence for translational control of bicoid mRNA
in aub mutants, these mutants may perturb
steps in the translation of transcripts that are essential for
triggering RNAi. Future experiments should define the specific roles
for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).
The reproducibility and precision of biological patterning is limited by the accuracy with which concentration profiles of morphogen molecules can be established and read out by their targets. This study considered four measures of precision for the Bicoid morphogen in the Drosophila embryo: the concentration differences that distinguish neighboring cells, the limits set by the random arrival of Bicoid molecules at their targets (which depends on absolute concentration), the noise in readout of Bicoid by the activation of Hunchback, and the reproducibility of Bicoid concentration at corresponding positions in multiple embryos. Through a combination of different experiments, it was shown that all of these quantities are 10%. This agreement among different measures of accuracy indicates that the embryo is not faced with noisy input signals and readout mechanisms; rather, the system exerts precise control over absolute concentrations and responds reliably to small concentration differences, approaching the limits set by basic physical principles (Gregor, 2007).
The development of multicellular organisms such as Drosophila is both precise and reproducible. Understanding the origin of precise and reproducible behavior, in development and in other biological processes, is fundamentally a quantitative question. Two broad classes of ideas can be distinguished. In one view, each step in the process is noisy and variable, and this biological variability is suppressed only through averaging over many elements or through some collective property of the whole network of elements. In the other view, each step has been tuned to enhance its reliability, perhaps down to some fundamental physical limits. These very different views lead to different questions and to different languages for discussing the results of experiments (Gregor, 2007).
The goal of this study was to locate the initial stages of Drosophila development on the continuum between the 'precisionist' view and the 'noisy input, robust output' view. To this end the absolute concentration of Bcd proteins was measured and these measurements were used to estimate the physical limits to precision that arise from random arrival of these molecules at their targets. The input/output relation between Bcd and Hb was measured, and it was found that Hb expression provides a readout of the Bcd concentration with better than 10% accuracy, very close to the physical limit. The mean input/output relation is reproducible from embryo to embryo, and direct measurements of the Bcd concentration profiles demonstrate that these too are reproducible from embryo to embryo at the ~10% level. Thus, the primary morphogen gradient is established with high precision, and it is transduced with high precision (Gregor, 2007).
Analysis of the Bcd/Hb input/output relations is similar in spirit to measurements of noise in gene expression that have been done in unicellular organisms. The morphogen gradients in early embryos provide a naturally occurring range of transcription factor concentrations to which cells respond, and the embryo itself provides an experimental 'chamber' in which many factors that would be considered extrinsic to the regulatory process in unicellular organisms are controlled. Perhaps analogous to the distinction between intrinsic and extrinsic noise in single cells, this study has distinguished between noise in the responses of individual nuclei to morphogens within a single embryo and the reproducibility of these input signals across embryos. Although there are many reasons why antibody staining might not provide a quantitative indicator of protein concentration, the results show that coupling classical antibody staining methods with quantitative image analysis allows a quantitative characterization of noise in the potentially more complex metazoan context. This approach should be more widely applicable (Gregor, 2007).
A central result of this work is the matching of the different measures of precision and reproducibility. Near its point of half-maximal activation, the expression level of hb provides a readout of Bcd concentration with better than 10% accuracy. At the same time, the reproducibility of the Bcd profile from embryo to embryo and from one cycle of nuclear division to the next within one embryo, is also at the ~10% level. Importantly, these different measures of precision and reproducibility must be determined by very different mechanisms. For the readout, there is a clear physical limit which may set the scale for all steps. This limiting noise level is sufficient to provide reliable discrimination between neighboring nuclei, thus providing sufficient positional information for the system to specify each 'pixel' of the final pattern (Gregor, 2007).
Previous work has shown that the Bcd profile scales to compensate for the large changes in embryo length across related species of flies, but evidence for scaling across individuals within a species has been elusive, perhaps because the relevant differences are small. This study found that the Bcd profile is sufficiently reproducible that it can specify position along the anterior-posterior axis within 1%-2% when position is expressed in units relative to the length of the embryo. But embryos have a standard deviation of lengths. Even if the Bcd profile were perfectly reproducible as concentration versus position in microns, this would mean that knowledge of relative position would be uncertain by 4%, which is more than what was see. This suggests that the Bcd profile exhibits some degree of scaling to compensate for length differences. New experiments will be required to test this more directly (Gregor, 2007).
The results suggest that communication among nearby nuclei, perhaps through a diffusable messenger, plays a role in the suppression of noise. The messenger could be Hb itself since in the blastoderm stages the protein is free to diffuse between nuclei, and hence the Hb protein concentration in one nucleus could reflect the Bcd-dependent mRNA translation levels of many neighboring nuclei. This model predicts that precision will depend on the local density of nuclei and hence will be degraded in earlier nuclear cycles unless there are compensating changes in integration time. Such averaging mechanisms might be expected to smooth the spatial patterns of gene expression, which seems opposite to the goal of morphogenesis; the fact that Hb can activate its own expression may provide a compensating sharpening of the output profile. There is a theoretically interesting tradeoff between suppressing noise and blurring of the pattern, with self-activation shifting the balance. Note that the idea of spatial averaging, although employed in this study in a syncitial embryo, can be extended to nonsyncitial systems (e.g., via autocrine signaling or via small molecules that can freely pass through cell membranes or gap junctions) (Gregor, 2007).
The reproducibility of absolute Bcd concentration profiles from embryo to embryo literally means that the number of copies of the protein is reproducible at the ~10% level. Understanding how the embryo achieves reproducibility in Bcd copy number is a significant challenge. Feedback mechanisms, explored for other morphogens, could compensate for variations in mRNA levels, but the linear response of the Bcd profile to halving the dosage of the Bcd-eGFP transgene argues against such compensation. The simplest view consistent with all these data is that mRNA levels themselves are reproducible at the ~10% level, and this should be tested directly (Gregor, 2007).
At a conceptual level the results on Drosophila development have much in common with a stream of results on the precision of signaling and processing in other biological systems. There is a direct analogy between the approach to the physical limits in the Bcd/Hb readout and the sensitivity of bacterial chemotaxis or the ability of the visual system to count single photons. In each case the reliability of the whole process is such that the randomness of essential molecular events dominates the reliability of the macroscopic output. There are several examples in which the reliability of neural processing reaches such limits, and it is attractive to think that developmental decision making operates with a comparable degree of reliability. The approach to physical limits places important constraints on the dynamics of the decision making circuits. Finally, it is noted that the precision and reproducibility which observed in the embryo are disturbingly close to the resolution afforded by the measuring instruments (Gregor, 2007).
Drosophila neuronal stem cell neuroblasts (NB) constantly change character upon
division, to produce a different type of progeny at the next division.
Transcription factors Hunchback (Hb), Kruppel (Kr), Pdm, and Castor are
expressed sequentially in each NB and act as determinants of birth-order
identity. How any NB switches its expression profile from one transcription factor
to the next is poorly understood. The Hb-to-Kr switch is directed
by the nuclear receptor Seven-up (Svp). Svp expression is confined to a
temporally restricted subsection within the NB's lineage. Loss of Svp function
causes an increase in the number of Hb-positive cells within several NB
lineages, whereas misexpression of svp leads to the loss of these early-born
neurons. Lineage analysis provides evidence that svp is required to switch off HB at the proper time. Thus, svp modifies the self-renewal stem cell program to allow chronological change of cell fates, thereby generating neuronal diversity (Kanai, 2005).
The expression profile of Svp in the CNS is extremely dynamic. For example, at stage 11, Svp is expressed in NB2-4 but not in NB7-3 just after NB2-4
formation. After NB7-3 has divided, Svp is expressed in NB7-3
and in the GMC that it has generated but Svp is no longer detectable in NB2-4.
Thus, the expression of Svp is confined to temporally restricted
subsections of the NB lineage. While Svp is expressed in many NB and GMCs, only
a small number of neurons are Svp positive. This indicates
that, unlike Hb and Kr, the expression profile of Svp in the NBs is not
maintained in their neuronal progeny (Kanai, 2005).
In the svp mutant, NB7-3 does not switch
its expression pattern from the Hb, Kr double-positive state to the Kr
single-positive state until one division after the normal transition period.
This prolonged expression of Hb results in overproduction of Hb-positive
neurons exhibiting characteristics of early-born neurons. The timing of the
expression of Svp protein in NB7-3 coincides with the transition in the
expression of Hb to Kr, and precocious expression of Svp causes the loss of Hb
expression within the lineage. These results indicate that Svp has an
instructive role in determining the period of Hb expression in the NB and the
proper generation of neuronal diversity. While this work places Svp upstream of
Hb, how the expression of Svp itself is regulated is not well understood. In
hb mutant embryos, Svp is still expressed transiently at the time that
NB7-3 produces its first GMC. Thus,
it is unlikely that the temporal delay of Svp expression with respect to Hb is
due to a negative feedback loop in which Hb induces its own repressor (Kanai, 2005).
Svp is a well-conserved nuclear receptor whose human homolog, COUP, has been shown to
act as a transcriptional repressor. Because a reporter gene that
contains only an enhancer element of the hb gene also responded to Svp,
Svp can affect hb expression at the level of its transcription. It is
thus possible that Svp directly represses hb transcription by binding to
its cis-element. Interestingly, misexpression of Svp in postmitotic
neurons does not affect their Hb expression, consistent with the observation that
the regulatory mechanism of Hb expression differs between the NBs and their
progeny. The repressor activity of Svp on hb expression likely requires other factors that are present in precursor cells of neurons (Kanai, 2005).
In svp mutant embryo, augumented expression of Hb was seen in many NBs, resulting in overproduction of early-born neurons in at least three NB lineages. This suggests that Svp may have a common function in many NB lineages regulating hb expression. However, of 30 NBs within each hemisegment, four do not express svp. Indeed, in an
svp-negative NB1-1 lineage, the number of the early-born neurons aCC and
pCC in svp mutant embryo is normal. How do these NBs
generate birth-order-dependent progeny without svp expression? Since some
NBs are known to start their lineage without expressing Hb, they may not need
Svp to regulate Hb expression. Indeed, svp-negative NB6-1,
which expresses Cas at the time of formation never expresses Hb. It
is also possible that there are other factors or mechanisms to regulate
hb expression. In the nematode C. elegans, hb homolog
lin57/hbl-1 (which controls developmental timing as a
heterochronic gene) is regulated by a micro RNA that binds its 3'UTR. Since Drosophila hb 3' UTR contains putative micro RNA binding sites, transcription
factor switching in Drosophila NBs might also be regulated
posttranscriptionally by micro RNAs (Kanai, 2005).
While the overproduction of Hb-positive
neurons is consistent with the idea that prolonged expression of Hb in
svp mutant NBs causes production of supernumerary GMC-1s, examination of
postmitotic neurons reveals that the number of neurons with particular identity
does not always correspond to duplicated GMC-1s. In the NB7-3 lineage, GMC-1
divides to produce two neurons, EW1 and GW, whereas GMC-2 gives rise to EW2
neuron and its sibling which undergoes programmed cell death. In svp
mutant, two EW1 neurons are present consistent with duplicated GMC-1, but only one
GW-like neuron is observed. Likewise, when Hb is misexpressed in the
NB7-3 lineage, not all GMCs that were transformed toward GMC-1 produced GW
neurons. These
data suggest that the fate of postmitotic progeny from GMCs is dependent not
only on the birth-order identity of GMCs determined by transcription factors
such as Hb and Kr, but is also influenced by signals that come from outside of
the NB lineage. Since the decision for the sibling of the EW2 neuron to undergo
cell death depends on the activation of Notch signaling, it is possible that
signals for Notch activation originate outside the NB7-3 lineage, and are not
affected by genetic manipulations altering the birth-order identity of the GMCs (Kanai, 2005).