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Zygotically transcribed genes
Signaling pathways involved in planar cell polarity
Jun mediates Frizzled-induced planar polarity determination in the Drosophila eye
Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle and strabismus are thought to antagonize Fz signaling. They act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. Stbm and Pk interact physically; Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus the data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling (Jenny, 2003).
pk function is required in the R4 precursor, as opposed to fz PCP signaling in R3, for control of polarity establishment. Stbm, a transmembrane protein also required in R4, interacts genetically and physically with Pk. This interaction is important for the recruitment of Pk to the plasma membrane. In Xenopus animal-cap explants, Stbm and Pk relocalize each other to subdomains of the membrane. A model is proposed of how Pk/Stbm might regulate Fz/Dsh signaling activity (Jenny, 2003).
The in vitro molecular interaction between Pk and Stbm and their mutual relocalization in Xenopus animal caps suggest that they form multiprotein complexes. Several pieces of evidence indicate that the physical interaction is physiologically important: (1) correct membrane localization of Pk depends on stbm function because in stbm mutant tissue Pk staining is diffuse and absent (or strongly reduced) at the membrane; (2) Pk and Stbm interact genetically by mutually enhancing each other's GOF and LOF phenotypes in the eye; (3) pk is necessary for PCP signaling in the R4 precursor, the same cell in which stbm is required; (4) expression of the interacting domains of Pk or Stbm interferes with polarity establishment. In particular, a subfragment of 131 amino acids of the C-terminus of Pk, required for the molecular interaction, is sufficient to affect polarity (Jenny, 2003).
Both Pk and Stbm act as if they antagonize Fz signaling. (1) In zebrafish, Stbm overexpression can prevent Wnt11 from rescuing a wnt11 mutation. (2) In the Drosophila wing, overexpression of Pk leads to wing hairs pointing towards the source of the overexpressed protein, behaving like a fz LOF clone (whereas overexpression of Fz leads to hairs pointing away from the Fz source). stbm LOF clones show the opposite behavior to fz LOF clones: wing hairs point away from the mutant patch, consistent with the mutant tissue having a higher Fz-activity (Jenny, 2003).
In the Drosophila eye, evidence that pk acts antagonistically to fz comes from the fact that the Notch-signaling-responsive R4-specific reporter mdelta0.5-lacZ is expressed for a prolonged period in both R3/R4 precursors in a pksple1 mutant. This is explained if Fz activity in the R4 precursor is increased, resulting in higher levels of Dl there. This in turn leads to N activation and concomitant mdelta0.5-lacZ reporter expression in both cells of the R3/R4 pair. Conversely, in fz and dsh mutant eye discs (where Fz signaling is absent or reduced and thus Dl should not be upregulated) N-signaling activity and mdelta-lacZ expression is initially reduced in both cells. Fz activity is also antagonized by stbm in the eye. Mosaic analysis of stbm shows that it has the capability to instruct a cell to become R4 as long as the other cell of the R3/R4 pair is mutant for stbm. Therefore, in such an all-or-nothing situation, Stbm in the R3 precursor can override a positive signal of Fz, resulting in a cell fate switch to R4 fate (Jenny, 2003).
In a wild-type situation with all PCP components present in both cells, it is crucial that Stbm activity is higher in R4 than in R3 to ensure proper Fz-signaling regulation. Therefore it is an intriguing possibility that a Pk/Stbm complex in the R4 precursor ensures such higher Stbm activity, and the associated higher Fz repression there is important for a proper R3/R4 cell fate decision (Jenny, 2003).
How does the Stbm/Pk complex regulate Fz-signaling activity? During PCP establishment in the wing, Fz, Dsh, Dgo, Fmi and Pk are initially localized uniformly around the apical circumference of wing cells. During and after PCP signaling, these proteins relocalize and become differentially enriched: Pk concentrates on the proximal side of the cell, whereas Fz and Dsh become enriched distally. Fmi becomes enriched at both sides (Jenny, 2003).
In the eye, the situation is analogous. During PCP establishment, signaling components at the R3/R4 cell border are relocalized from a uniform to a more restricted pattern. Stbm-YFP is localized on the R4 but not on the R3 side, and Fz-GFP ends up on the R3 but not the R4 side. The analogy between the R4/R3 and proximal/distal cell borders is corroborated by the genetic requirements in R3 and R4: the distally localized factors Dsh and Fz are required in R3, while proximally localized Pk is required in R4. Fmi is localized on both poles of each wing cell and also required in both cells of the R3 and R4 pair. The function of fmi has been linked to both proposed complexes, the 'Fz/Dsh side' (fmi is required for apical localization of both Fz and Dsh) and the Stbm/Pk complex. In addition to the genetic interactions between fmi and stbm or pk, a reduced membrane staining of Pk in fmi- clones in wing cells has been shown (Jenny, 2003).
How do these changes in localization occur? Localization studies in Drosophila and Xenopus suggest that Pk and Stbm influence each other's localization and form clusters in subdomains of the cell membrane. Interestingly, such Stbm/Pk complexes also affect Fz-dependent Dsh membrane localization. Thus it is an intriguing possibility that the patches observed in animal cap cells upon coinjection of Stbm with Pk represent the result of a similar, though unpolarized, symmetry-breaking step during PCP signaling (Jenny, 2003).
The PET/LIM domain of Pk can interact with the DEP-domain/C-terminus of Dsh. This interaction has been suggested to prevent Dsh membrane recruitment. Also, the C-terminus of Stbm can interact with Dsh as long as the PDZ domain is present. Since the data suggest that Pk regulates the activity and localization of Stbm, this regulation might promote or stabilize the interactions of Dsh with Stbm and/or Pk, thereby helping to pull Dsh away from a Fz-signaling complex. The Stbm/Pk complexes could then cause active release of Dsh from the membrane or target it for degradation, resulting in low levels of Dsh (and by inference Fz) at places where Pk and Stbm are enriched. Furthermore, in the R3 cell (or distally in the wing) an unknown factor might act to prevent either the formation of the Stbm/Pk complex or its effect on Dsh (Jenny, 2003).
In conclusion, Pk and Stbm form a functional complex during PCP signaling in Drosophila and during convergent extension in Xenopus. Interestingly, in zebrafish, in addition to its function in convergent extension, Stbm is also required for the caudal migration of hindbrain motor neurons. This function of Stbm is independent of Dsh and the PCP genes tested so far. It will be interesting to determine whether Stbm and Pk function together in this context as well (Jenny, 2003).
Jun acts as a signal-regulated transcription factor in many
cellular decisions, ranging from stress response to
proliferation control and cell fate induction. Genetic
interaction studies have suggested that Jun and JNK
signaling are involved in Frizzled (Fz)-mediated planar
polarity generation in the Drosophila eye. However, simple
loss-of-function analysis of JNK signaling components does
not show comparable planar polarity defects. To address
the role of Jun and JNK in Fz signaling, a
combination of loss- and gain-of-function studies has been used. Like Fz,
Jun affects the bias between the R3/R4 photoreceptor pair
that is critical for ommatidial polarity establishment.
Detailed analysis of jun- clones reveals defects in R3
induction and planar polarity determination, whereas gain
of Jun function induces the R3 fate and associated polarity
phenotypes. Affecting the levels of JNK
signaling by either reduction or overexpression leads to
planar polarity defects. Similarly, hypomorphic allelic
combinations and overexpression of the negative JNK
regulator Puckered causes planar polarity eye phenotypes,
establishing that JNK acts in planar polarity signaling. The
observation that Delta transcription in the early R3/R4
precursor cells is deregulated by Jun or Hep/JNKK
activation, reminiscent of the effects seen with Fz
overexpression, suggests that Jun is one of the transcription
factors that mediates the effects of fz in planar polarity
generation (Weber, 2000).
Jun, as a member of the AP-1 family, is activated by many
distinct extracellular stimuli and acts downstream of several
signaling pathways. Besides its
involvement in stress response, Jun has been implicated in the
control of proliferation, apoptosis, morphogenesis and cell fate
induction. In Drosophila, Jun is critical for the
process of dorsal closure in embryogenesis acting downstream
of the JNK module. It has also been implicated
in cell fate induction downstream of Ras/ERK signaling in the
eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity
signaling in the eye. It is the first transcription factor implicated
in Fz/planar polarity signaling. Fz signaling also requires a
JNK (or related kinase) module, and thus in the
eye imaginal disc Jun acts downstream of both ERK and JNK.
How does Jun achieve a specific response in this context?
The S/T residues that are phosphorylated in Jun are the same
for both ERK and JNK. Thus, although differences in phosphorylation level
and/or preference for any of the serine/threonine target residues
cannot be excluded in vivo, differential phosphorylation is
unlikely to create specificity. A potential mechanism for
specificity might be provided by other transcription factors that
cooperate with Jun in the different processes. This is supported
by the observation that the sev-JunAsp (expression of a constitutively active Jun) phenotype is a composite
of two events, photoreceptor recruitment and ommatidial
polarity generation. These two effects can, however, be
separated by the reduction of specific interacting partners. In
the process of Ras/ERK signaling in photoreceptor induction
Jun interacts and synergizes with the ETS domain transcription
factor Pointed (Pnt). Pnt has been
characterized as a target of the ERK/Rl kinase in Drosophila
in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process.
Removing one dose of pnt strongly suppresses the Ras/ERK-related
extra photoreceptor phenotype of sev-JunAsp, whereas
the polarity defects persist and thus are more prominent. This observation indicates that,
in the absence of normal Pnt levels, sev-JunAsp specifically
affects polarity, suggesting that the interaction with Pnt is
important for its role in the ERK-mediated induction. It is
likely that for its planar polarity function other specific
transcription factors provide the specificity cues (Weber, 2000).
Although all components of the JNK module tested genetically
interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function
mutants did not show defects in planar polarity
establishment, suggesting a redundant role. Even null alleles of
the Drosophila homolog of JNKK hep have no effects on
planar polarity (Weber, 2000).
However, expression of a dominant negative (kinase dead) isoform of
Bsk interferes with planar polarity, giving rise to
typical polarity phenotypes, implying that Bsk and
JNK signaling are important in this process. Consistently,
homozygous mutant clones of the deficiency Df(2R)flp170B
that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild
polarity phenotype in the eye, including the presence of
symmetrical ommatidia (Weber, 2000).
What are the redundant kinases in this process? Genetic
interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep
and bsk, deficiencies affecting other MKKs and the Drosophila
p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially)
overlapping phosphorylation targets] might be responsible for the redundancy in this process. The
analysis with the dominant negative (DN) Bsk isoform and the
respective deficiencies suggests that the p38 kinase(s) are
contributing to this redundancy, because they enhance the DN-Bsk
phenotype in a manner very similar to that of the bsk deficiency. The
identification of specific mutant alleles of p38a/b and double
mutant analysis with bsk will be necessary to further clarify
this issue (Weber, 2000).
The available results indicate that the level of JNK/p38
signaling in planar polarity establishment is important, but that
the removal of a single kinase does not significantly affect this
level. In support, the observation that an allelic combination of
hep and puc hypomorphic alleles can give rise to planar
polarity eye phenotypes suggests that the balance
between negative and positive regulators of JNK and related
kinases is critical. Similarly, overexpression of the negative
JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to
those of fz or dsh mutants. It is likely that this phosphatase
negatively regulates all JNK-related kinases and thus reduces
the overall signaling more than the lack of a single kinase (Weber, 2000).
In summary, these data indicate that the transcriptional
events downstream of Fz in R3 specification and chirality
establishment (e.g. regulation of Dl) are mediated by Jun. The
factors with which Jun is redundant in the imaginal discs are
not yet identified. It is possible that other members of the AP-1
family are also involved in planar polarity signaling, since they
are related to Jun and could dimerize with it via the leucine-zipper
motif. A potential candidate is Fos, because like Jun, Fos is
required downstream of JNK in the process of dorsal closure
in the embryo. Similarly, the ETS domain protein Yan acts as a
negative regulator in dorsal closure and is inactivated by JNK
in the process. However, these factors do not show informative
planar polarity phenotypes in clones and thus their
involvement in this process remains unclear. Although AP-1
and ETS family members are attractive candidates,
transcription factors belonging to other families cannot be
excluded in this context (Weber, 2000).
Jenny, A., Darken, R. S., Wilson, P. A. and Mlodzik, M. (2003). Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. EMBO J. 22: 4409-4420. 12941693
Weber, U., Paricio, N. and Mlodzik, M. (2000). Jun mediates Frizzled-induced R3/R4 cell fate distinction and planar polarity determination in the Drosophila eye. Development 127: 3619-3629. 10903185
genes expressed in tissue polarity
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