cactus
Other IkappaB interactions The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel
transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor
is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response
domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to
identify regulators of the I kappaB alpha inhibitory activity, a yeast two-hybrid genetic
screen was undertaken, using the amino-terminal end of I kappaB alpha as bait: 12 independent
interacting clones were identified. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence
encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid
assay, Dlc-1 also interacts with full-length I kappaB alpha protein, but not with
N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacts in vitro with a
glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) does not displace this association,
demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in
HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitate with
Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy
indicates that Dlc-1 and I kappaB alpha colocalize with both nuclear and cytoplasmic distribution.
Dlc-1 and I kappaB alpha are found to associate with the microtubule organizing
center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalizes
with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between
the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins
and provide a potential link between cytoskeleton dynamics and gene regulation (Crepieux, 1997).
Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. Modified IkappaBalpha has been detected, conjugated to the small ubiquitin-like protein SUMO-1 (see Drosophila SUMO), which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugates SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation (Desterro, 1998).
The nuclear factor-kappaB (NF-kappaB) signalling pathway participates in a multitude of biological processes, which imply the requirement of a complex and precise regulation. IkappaB (for Inhibitor of kappaB) proteins, which bind and retain NF-kappaB dimers in the cytoplasm, are the main contributors to negative regulation of NF-kappaB under non-stimulation conditions. Nevertheless, increasing evidences indicate that IkappaB proteins exert specific nuclear roles that directly contribute to the control of gene transcription. In particular, hypophosphorylated IkappaBbeta can bind the promoter region of TNFalpha leading to persistent gene transcription in macrophages and contributing to the regulation of the inflammatory response. Recently, it was demonstrated that phosphorylated and SUMOylated IkappaBalpha resides in the nucleus of the cells where it binds to chromatin leading to specific transcriptional repression. Mechanistically, IkappaBalpha associates and regulates Polycomb Repressor Complex activity, a function that is evolutionary conserved from flies to mammals, as indicate the homeotic phenotype of Drosophila mutants. The implications of chromatin-bound IkappaBalpha function is discussed in the context of tumorigenesis (Espinosa, 2014).
Mutation of IkappaB To study the importance of IkappaBalpha in signal transduction,
IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from
IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta
similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that
cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of
wild-type cells with tumor necrosis factor alpha (TNF-alpha) results in rapid, transient nuclear localization
of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of
NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is
termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and
NF-kappaB's role in regulating inflammatory and immune responses, is the normal development of
IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10
days of age. An increased percentage of monocytes/macrophages are detected in spleen cells taken from 5-,
7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of
TNF-alpha mRNA in the skin (Klement, 1996).
Hematopoiesis occurs in the liver and the bone marrow (BM) during murine development. Newborn mice with a ubiquitous deletion of I kappa B alpha develop a severe hematological disorder characterized by an increase of granulocyte/erythroid/monocyte/macrophage colony-forming units (CFU-GEMM) and hypergranulopoiesis. This particular myeloproliferative disturbance is mediated by continuously deregulated perinatal expression of Jagged1 in I kappa B alpha-deficient hepatocytes. The result is a permanent activation of Notch1 in neutrophils. In contrast, in mice with a conditional deletion of I kappa B alpha only in the myeloid lineage and in fetal liver cell chimeras, a cell-autonomous induction of the myeloproliferative disease was not observed. Coculture of I kappa B alpha-deficient hepatocytes with wild-type BM cells induced a Jagged1-dependent increase in CFUs. In summary, cell-fate decisions leading to a premalignant hematopoietic disorder can be initiated by nonhematopoietic cells with inactive I kappa B alpha (Rupec, 2005).
Rel/NF-kappaB transcription factors and IkappaBalpha function in an autoregulatory network. Avian
IkappaBalpha transcription is increased in response to both c-Rel and v-Rel.
IkappaBalpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). A 386 bp region of
the IkappaBalpha promoter (containing two NF-kappaB and one AP-1 binding sites) is
necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1
proteins. In addition, an imperfect NF-kappaB binding site is found to overlap the AP-1 binding site.
Mutation of either of the NF-kappaB binding sites or the AP-1 binding site dramatically decreases the
response of the IkappaBalpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of
c-Rel results in the formation of DNA binding complexes associates with the imperfect NF-kappaB
binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappaB site stronger than
c-Rel, and overexpression of v-Rel also results in the formation of a v-Rel containing complex bound to a
consensus AP-1 site (Kralova, 1996).
Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit
synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but
the mechanism underlying this activity has been unclear. Glucocorticoids are potent
inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated
by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic
complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli,
the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids (Auphan, 1995).
To investigate the determinants of promoter-specific gene regulation by the
glucocorticoid receptor (GR), the composition and function of
regulatory complexes at two NFkappaB-responsive genes that are differentially
regulated by GR were compared.
Transcription of the IL-8 and IkappaBalpha genes is
stimulated by TNFalpha in A549
cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2
phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The
proximal kappaB elements at
these genes differ in sequence by a single base pair, and both recruited RelA
and p50. Surprisingly, GR is recruited to both of these elements, despite the
fact that GR fails to repress the IkappaBalpha promoter.
Rather, the regulatory complexes formed at IL-8 and IkappaBalpha
were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb.
Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small
interfering RNA selectively blocks TNFalpha stimulation
of IL-8 mRNA production. GR competes with P-TEFb
recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis is repressed
by GR at a post-initiation step, demonstrating that promoter proximal
regulatory sequences assemble complexes that impact early and late stages of
mRNA synthesis. Thus, GR accomplishes selective repression by targeting
promoter-specific components of NFkappaB regulatory complexes (Luecke, 2005).
This study investigated recruitment of coactivators (SRC-1, SRC-2, and SRC-3) and corepressors (HDAC1, HDAC2, HDAC3, SMRT, and NCoR) to the IkappaBα gene promoter after NF-kappaB activation by tumor necrosis factor-α. The data from chromatin immunoprecipitation assay suggest that coactivators and corepressors are simultaneously recruited to the promoter, and their binding to the promoter DNA is oscillated in HEK293 cells. SRC-1, SRC-2, and SRC-3 all enhanced IkappaBα transcription. However, the interaction of each coactivator with the promoter exhibited different patterns. After tumor necrosis factor-α treatment, SRC-1 signal was increased gradually, but SRC-2 signal was reduced immediately, suggesting replacement of SRC-2 by SRC-1. SRC-3 signal was increased at 30 min, reduced at 60 min, and then increased again at 120 min, suggesting an oscillation of SRC-3. The corepressors were recruited to the promoter together with the coactivators. The binding pattern suggests that the corepressor proteins formed two types of corepressor complexes, SMRT-HDAC1 and NCoR-HDAC3. The two complexes exhibited a switch at 30 and 60 min. The functions of cofactors were confirmed by gene overexpression and RNA interference-mediated gene knockdown. These data suggest that gene transactivation by the transcription factor NF-kappaB is subject to the regulation of a dynamic balance between the coactivators and corepressors. This model may represent a mechanism for integration of extracellular signals into a precise control of gene transcription (Gao, 2005).
TNF-induced NF-kappaB activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, two temporal amplification steps have been detected - one determined by the obligate negative feedback regulator IkappaBα - that define the duration of the first phase of NF-kappaB activity. The second phase is defined by A20 (a ubiquitin-editing protein that is involved in the negative feedback regulation of NF-kappaB signaling), whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. These results delineate the nonredundant functions implied by the knockout phenotypes of ikappabα and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses (Werner, 2008).
As the molecular connectivity within signaling networks has increasingly become a focus of biomedical research, a surprising number of inducible negative regulators have been identified; these are usually categorized as negative feedback regulators. However, remarkably few have been examined to determine what functional roles their inducible expression may play; indeed, little is known about whether inducible expression can even allow for distinct functional roles. This analysis demonstrates distinct functions for IkappaBα and A20, whose expression is driven by similarly inducible promoters. In the case of IkappaBα, negative feedback is required for function; in other words, no value of constitutive IkappaBα expression parameters will provide the degree of NF-kappaB activation and post-induction repression that NF-kappaB-responsive expression of IkappaBα allows for. In contrast, there is a range of constitutive A20 expression values that can functionally replace A20 negative feedback. Hence, a distinction can be made between an obligate (IkappaBα) and a nonobligate (A20) feedback regulator. Indeed, the A20 regulatory mechanism may not fit a narrower definition of a negative feedback regulator (Werner, 2008).
Instead, the inducibility of A20 expression functions to tune a rheostat that controls cellular signaling responsiveness. This is demonstrated most clearly by the fact that A20 mediates signaling cross-talk between inflammatory stimuli when they are administered sequentially. However, A20's rheostat function is not limited to cross-talk between IL-1 and TNF; its promoter is inducible by all NF-kappaB-inducing stimuli tested so far. It provides short-term cellular memory by transiently 'tolerizing' (i.e., reducing the sensitivity of) the TNF signaling pathway. Whereas the dynamics of IkappaBα inducibility, but not the actual protein concentration, critically define NF-kappaB activity, the A20 protein concentration determines its attenuation function, regardless of whether the protein level was the result of inducible or constitutive expression. This distinction between the negative regulators may explain how subtle misregulation of A20 protein levels have been implicated in a range of physiological and pathological processes, including atherosclerosis, T-cell responsiveness, the homeostasis of signaling by pathogen-sensing receptors and of commensual bacteria, and suppression of autoreactive immune responses (Werner, 2008).
What might be the molecular basis for the differential functionality? There are differences in network connectivity (model topology) and rate constants (parameter values) that may be relevant to consider. Although both IkappaBα and A20 are rapidly and highly inducible at the level of mRNA transcripts (which show a similarly short half-life), producing the larger A20 protein takes more time. More importantly, a significantly longer protein half-life allows for not only a gradual build-up of the A20 protein, but also a memory function that was revealed in cross-talk or priming experiments. Whereas the obligate negative feedback regulator IkappaBα functions as a stochiometric binder of the NF-kappaB activator, the nonobligate feedback attenuator A20 reaches back many more reactions into the pathway, making its effect more temporally diffuse at the NF-kappaB level. In addition, A20 possesses an enzymatic function, which further slows its total functional effect. These conclusions are insensitive to alterations of parameter values within the ranges set by experimental constraints. In fact, model topology aspects mirror prior theoretical considerations pertaining to metabolic networks. It is suggested that theoretical modeling work may prove useful in distinguishing between different categories of negative feedback regulators in signaling. In addition, a combined computational and experimental strategy may be applied to other signaling systems to characterize the functional diversity of negative feedback regulators (Werner, 2008).
TNF-induced NF-kappaB dynamics are encoded not only by IkappaBα and A20, but also by an IKK autorepression mechanism that provides powerful negative feedback on a faster scale than mechanisms involving de novo gene expression. Although the described model recapitulates the observed temporal IKK activity profile, it does not describe the actual regulatory mechanism(s) because further molecular characterization is required. Indeed, recent work suggests that the association of the essential IKK scaffold subunit NEMO with catalytic subunits IKK1 and IKK2 is regulated via phosphorylation. Similarly, the mechanism by which K63-linked ubiquitin chains activate IKK, the involvement of A20 in their removal, and whether and how TAK1/Tab2/3 is involved in IKK control remains to be characterized in more detail. New mechanistic insights should lead to a revision of the mathematical model, in turn enabling an investigation of their role in determining NF-kappaB dynamics. The iterative strategy of combined experimental and modeling work promises to result in amply validated and sufficiently detailed models that may function as standalone discovery tools (Werner, 2008).
Signaling pathways involving IkappaB Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are required for an
effective immune response to bacterial infection. These cytokines synergize in a variety of biological
responses, including the induction of cytokine, cell adhesion, and inducible nitrous oxide synthase gene
expression. Typically, the synergistic effect on gene expression is due to the independent activation of
nuclear factor kappaB (NF-kappaB) by TNF-alpha and of signal transducers and activators of
transcription or IFN-regulatory factor 1 by IFNs, allowing these transcription factors to bind their
unique promoter sites. However, since activation of NF-kappaB by TNF-alpha is often transient and
would not activate long-term kappaB-dependent transcription effectively, the effects of
IFN-gamma on TNF-alpha-induced NF-kappaB activity were explored. IFN-gamma, which typically does not activate NF-kappaB, synergistically enhances TNF-alpha-induced NF-kappaB nuclear translocation via a
mechanism that involves the induced degradation of I kappaBbeta and that apparently requires tyrosine
kinase activity in preneuronal cells but not in endothelial cells. Correspondingly, cotreatment of cells
with TNF-alpha and IFN-gamma leads to persistent activation of NF-kappaB and to potent activation
of kappaB-dependent gene expression, which may explain, at least in part, the synergy observed
between these cytokines, as well as their involvement in the generation of an effective immune
response (Cheshire, 1997).
The Rho family of small GTPases includes critical elements involved in the regulation of
signal transduction cascades from extracellular stimuli to the cell nucleus. Other family members are the
JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6
kinase. A novel signaling pathway is activated by the Rho proteins; this pathway
may be responsible for biological activities carried out by Rho proteins, including cytoskeleton organization,
transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1
proteins efficiently induce the transcriptional activity of nuclear factor KB (NF-KB) by
a mechanism that involves phosphorylation of IKappaBalpha and translocation of p50/p50
and p50/p65 dimers to the nucleus, independent of the involvement of Ras GTPase and the Raf-1
kinase. Activation of NF-KB by TNFalpha depends on CDC42 and
RhoA because this activity is drastically inhibited by CDC42 and RhoA dominant-negative mutants. In contrast, activation of NF-KB by UV light
is not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus,
members of the Rho family of GTPases are involved specifically in the regulation of
NF-KB-dependent transcription (Perona, 1997).
IkappaB and early development The Spatzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a
gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose
patterning functions are conserved between insects and vertebrates. Although there is no experimental
evidence that upstream events are conserved as well, the following question was posed: can a vertebrate embryo
respond to maternal components of the fly Dorsal pathway? A dorsalizing activity is demonstrated
for the heterologous Easter, Spatzle and Toll proteins in UV-ventralized Xenopus embryos; dorsalization is
inhibited by a co-injected dominant Cactus variant. Thus the epistatic relationships between upstream and downstream components of the Drosophila dorsoventral (d/v) pathway are maintained in the frog, as is evident from the inhibtion of Spz and Easter activity by the dominant Cactus mutation. It is concluded that the Dorsal signaling pathway is a
component of the conserved d/v patterning system in bilateria (Armstrong, 1998).
The rel/NF-kappaB transcription factor Dorsal controls dorsoventral (DV) axis formation in Drosophila. A stable nuclear gradient of Dorsal directly regulates ~50 target genes. In Tribolium castaneum (Tc), a beetle with an ancestral type of embryogenesis, the Dorsal nuclear gradient is not stable, but rapidly shrinks and disappears. Negative feedback accounts for this dynamic behavior: Tc-Dorsal and one of its target genes activate transcription of the IkB homolog Tc-cactus, terminating Dorsal function. Despite its transient role, Tc-Dorsal is strictly required to initiate DV polarity, as in Drosophila. However, unlike in Drosophila, embryos lacking Tc-Dorsal display a periodic pattern of DV cell fates along the AP axis, indicating that a self-organizing ectodermal patterning system operates independently of mesoderm or maternal DV polarity cues. The results also elucidate how extraembryonic tissues are organized in short-germ embryos, and how patterning information is transmitted from the early embryo to the growth zone (da Fonsaca, 2008).
Tc-Toll transcription appears to start evenly along the DV axis at the syncytial blastoderm stage but is rapidly enhanced at the ventral side, where higher levels of nuclear Tc-Dorsal accumulate. This positive feedback between Toll expression and nuclear import of Dorsal could explain an initiation of DV axis formation at ectopic positions of the embryonic blastoderm, a situation which has been observed upon experimental manipulations in beetles and various hemimetabolous insects. During normal development, however, ectopic axis formation has to be prevented, and this can be achieved by coupling positive feedback control to inhibitory processes. Linking self-enhancement to limiting mechanisms provides a general condition for pattern formation as has been shown by mathematical modeling. The Tc-Dorsal-dependent transcriptional activation of Tc-cact might provide the mechanism counterbalancing the positive feedback between Tc-Toll and Tc-Dorsal (da Fonsaca, 2008).
Within the limits of detection, Tc-cact expression appears to be restricted to the ventral side of the embryo. However, the knockdown of Tc-cact leads to nuclear import of Tc-Dorsal also at the dorsal side. To explain this long-range requirement of Tc-cact, one might speculate that detection of Tc-cact transcripts is not sensitive enough or that Tc-Cact protein is able to diffuse within the cytoplasm from ventral toward dorsal. Irrespective of the mechanism, a long-range action of Tc-Cact would meet an important prediction for pattern formation by reaction-diffusion systems, namely that the inhibitor should spread faster and thus act less locally than the activator (da Fonsaca, 2008).
Besides its potential role in pattern formation, Tc-cact activation seems also to be involved in the temporal control of the Dorsal gradient. During late blastoderm stages Tc-cact activation by Tc-dorsal is replaced through activation by Tc-twi. The Tc-twi knockdown phenotype shows that this shift is relevant to prevent Dorsal from accumulating in ventral nuclei during gastrulation. Thus, it seems that in Tribolium a Dorsal target gene is involved in terminating Dorsal function (da Fonsaca, 2008).
Collectively, these observations indicate that major evolutionary changes have occurred regarding Tc-Dorsal gradient formation and the network of downstream target genes. Nevertheless, traces of the feedback mechanisms uncovered in Tribolium have been preserved in the Drosophila lineage. Recently, zygotic enhancers of Dm-cactus and Dm-Toll were identified by ChIP-on-chip experiments and bioinformatics approaches. These enhancers contain Dm-Dorsal and Dm-twist binding sites and are active in the prospective mesoderm of Drosophila. However, the analysis of mutant phenotypes precludes an important function of these enhancers in DV patterning or cell type specification. A weak stabilizing function may explain why they were retained in evolution (da Fonsaca, 2008).
On an even larger evolutionary scale it is interesting to note that negative feedback control is a hallmark of NF-κB-mediated signaling. Like in Tribolium, the transcription of the Cactus homolog I-κB is activated by NF-κB in vertebrates both in the mesoderm and during innate immune response. The ensuing negative feedback loop can cause oscillatory signaling outputs or termination of signaling. Even an involvement of twist in negative feedback regulation of NF-κB has been demonstrated in vertebrate mesoderm cells. It has been proposed that the twi-NF-κB interactions represent an evolutionarily conserved regulatory module. The Dorsal/NF-κB- and twi-dependent activation of Tc-cact might be a relic of mesodermal and innate immune functions the pathway had in the common ancestor of vertebrates and arthropods. According to this scenario, the ancestral feedback mechanisms were adjusted to the needs of spatial patterning after the pathway was adopted for DV axis formation (da Fonsaca, 2008).
Classical fragmentation experiments have suggested two routes for pattern regulation along the DV axis: an early route which takes place before gastrulation and a later one which can be initiated after mesoderm internalization. Evidence has been provided for late autonomous patterning within the ectoderm that depends on the Dpp/Sog system and additional inhibitory processes. Tc-Toll knockdown embryos add additional support for this assumption. They show pattern duplications of ectodermal DV cell fates along the AP axis. This remarkable phenotype is not just restricted to the abdominal segments derived from the growth zone, but it occurs also within the anterior (thoracic) segments. Thus, it is unlikely to reflect a specific mechanism that operates only in the growth zone (da Fonsaca, 2008).
The modulation of Dpp activity underlying the periodic cell fate changes is likely to be due to periodic transcription of Tc-dpp and inhibition of Tc-Dpp diffusion or signaling along the AP axis. Since Tc-sog is not re-expressed in Tc-Toll1 RNAi embryos, the expression of other Dpp inhibitors was analyzed. Tc-bambi showed periodic expression in the same domains as Tc-dpp and thus might provide the inhibitory function. The fact that Tc-dpp is transcribed in regions of high pMAD activity suggests positive feedback control which is counterbalanced by Tc-bambi. Thus, the interaction might be similar to that described for Tc-Toll and Tc-cact (da Fonsaca, 2008).
The unusual orientation of the ectodermal patterning process might depend on the early AP asymmetry of Dpp signaling in Tc-Toll1 RNAi embryos. After Tc-Toll1 RNAi, Tc-dpp is expressed along the symmetric border between serosa and germ rudiment and in the posterior pit region (data not shown). These regions also have high levels of pMAD. Thus, the ectodermal patterning process is initiated with AP asymmetric boundary conditions after Tc-Toll RNAi. In WT embryos this process is oriented along the DV axis through the Toll-dependent activation of Tc-sog at the ventral side, which leads to a Dpp signaling gradient with peak levels along the dorsal midline (da Fonsaca, 2008).
Experiments clearly demonstrate that Tc-Dorsal is essential for establishing all aspects of normal DV polarity in Tribolium, including DV polarity of the growth zone from which the abdominal segments emerge. Thus, although DV patterning in the growth zone starts after gastrulation, when the Tc-Dorsal gradient has vanished, it is not independent of Tc-Dorsal. It is suggested that there are two ways by which early DV polarity is transmitted to the growth zone. First, distinct inner and outer cell layers are formed during gastrulation. The observation that the majority of the mesenchymal layer cells are absent in Tc-Toll RNAi embryos strongly suggests that the mesenchymal cells in the growth-zone are derived from cells internalized by ventral furrow formation in the early embryo. These cells cannot be resupplied by a growth zone-specific process of cell internalization. Thus, gastrulation-like mechanisms do not continue in the growth zone of Tribolium, as has been suggested for the tail-bud of vertebrate embryos. Second, DV patterning in the growth zone does not only depend on the generation of two separate cell layers. The ectoderm needs also to be patterned, a process which mainly depends on Dpp signaling. To a certain degree this process takes place in a Toll knockdown embryo. However, the orientation of the resulting pattern is incorrect. It is assumed that during WT development DV polarity is first established within the anterior (gnathal and thoracic) segments. Subsequently, this pattern is used as a template for the DV pattern of the abdominal segments emerging from the growth zone. This would require a process of forward-induction from differentiated to nondifferentiated tissues. Since there is no DV polarity in Tc-Toll RNAi early embryos, forward-induction cannot operate (da Fonsaca, 2008).
The loss of Toll signaling in Tribolium leads to phenotypes that are similar to those produced by the loss of the Dpp inhibitor sog. In both situations the ectoderm lacks normal polarity, the amnion and the CNS are largely deleted (the CNS is completely absent after Tc-sog RNAi and reduced to narrow periodic stripes after Tc-Toll RNAi), and the embryos form long tube-like structures. This situation is strikingly different in Drosophila. There, loss of Toll signaling leads to completely dorsalized embryos, while loss of sog causes only minor deletions in the CNS and subtle ectodermal patterning defects. These differences are due to the fact that Toll signaling in Drosophila provides functions which the Dpp/Sog system fulfils in Tribolium. For example, in Drosophila Dorsal represses dpp and activates brinker, an inhibitor of Dpp target genes, within the presumptive neuroectoderm and thereby specifies the CNS through mechanisms which act independently from and parallel to sog. These mechanisms do not exist in Tribolium. Apparently, the Dorsal gradient has a less direct role with regard to cell-type specification in Tribolium than in Drosophila, and DV patterning in Tribolium relies to higher degree on the Dpp/Sog system. Since the Dpp/Sog (BMP/Chordin) system is involved in DV axis formation in all bilaterian animals investigated so far, this is likely to represent the ancestral mode of DV axis formation. It is suggested that the trend observed by comparing Drosophila and Tribolium applies to other insect orders and that the functional shift between Dpp and Toll signaling with regard to DV axis formation will be even more prominent in basal hemimetabolous insects. Thus, the study of more basal insects groups might reveal the evolutionary path of how Toll signaling was co-opted for DV axis formation (da Fonsaca, 2008).
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