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The neural crest (NC) is a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates and migrate extensively before giving rise to diverse derivatives. A number of components of the neural crest gene regulatory network (NC-GRN) are used reiteratively to control multiple steps in the development of these cells. It is therefore important to understand the mechanisms that control the distinct function of reiteratively used factors in different cellular contexts, and an important strategy for doing so is to identify and characterize the regulatory factors they interact with. Here we report that the LIM adaptor protein, LMO4, is a Slug/Snail interacting protein that is essential for NC development. LMO4 is expressed in NC forming regions of the embryo, as well as in the central nervous system and the cranial placodes. LMO4 is necessary for normal NC development as morpholino-mediated knockdown of this factor leads to loss of NC precursor formation at the neural plate border. Misexpression of LMO4 leads to ectopic expression of some neural crest markers, but a reduction in the expression of others. LMO4 binds directly to Slug and Snail, but not to other components of the NC-GRN and can modulate Slug-mediated neural crest induction, suggesting a mechanistic link between these factors. Together these findings implicate LMO4 as a critical component of the NC-GRN and shed new light on the control of Snail family repressors.
Fig. 1. LMO4 is expressed in the neural crest. Schematic diagram showing LMO4 protein domains. (B) Whole mount in situ hybridization examining LMO4 expression in early Xenopus embryos. LMO4 expression in neural crest forming regions is apparent by stage 14, and is maintained in premigratory and migratory neural crest cells. LMO4 expression is also seen in the neural plate, placodal regions, and paraxial mesoderm at neurula stages (stages 149). At stage 24 LMO4 is expressed in the otic vesicle and tailbud in addition to migrating neural crest cells. At stage 28 LMO4 expression is seen in post-migratory neural crest cells in the branchial arches as well as in the tailbud, otic vesicle and somites.
Fig. 2. LMO4 is required for neural crest formation. (A) Whole mount in situ hybridization of embryos co-injected in one animal micromere at the eight-cell stage with LMO4 morpholino and β-gal as a lineage tracer. Embryos were examined at mid-neurula stage (St17) with neural crest markers Slug, Snail, Sox8, Twist, Sox10, FoxD3. * indicates side of injection which is also denoted by red gal staining. (B) Effects of LMO4 depletion can be rescued with a morpholino resistant form of LMO4 (LMO4 M1). Whole mount in situ hybridization injected probed for neural crest marker Slug. (C) Whole mount in situ of stage 13 LMO4 depleted embryos probed for neural plate marker Sox3, epidermal marker Epk, and neural plate border markers Opl, and Pax3. (D) In situs of LMO4 MO injected embryos probed for mesoderm marker MyoD. Normal expression of mesodermal markers indicates that effects of LMO4 depletion on the neural crest are not a consequence of mesodermal defects. (E) Animal cap assay demonstrating that mesoderm independent induction of neural crest by Wnt/Slug is blocked by LMO4 depletion. (F) Western blot analysis validating the knockdown of LMO4 immunoblotting with antibodies against the tagged protein. Actin is used as a loading control. (G) Western blot of in vitro translated (IVT) LMO4 proteins demonstrating that the mutant form is resistant to translation blocking morpholino.
Fig. 3. Excess LMO4 interferes with neural crest formation. (A) Whole mount in situ hybridization of embryos injected in one cell at two-cell stage with LMO4 and β-gal lineage tracer. Embryos were examined at mid-neurula stage (St. 17) with neural crest markers Slug and Snail, which display significant ectopic expression. B) Animal cap assay demonstrating that, in contrast to Wnt/noggin, LMO4 cannot induce Slug expression in isolated ectoderm. (C) Whole mount in situ hybridization of embryos injected in one cell at two-cell stage with mRNA encoding LMO4 and β-gal. Embryos were examined at mid-neurula stage (St. 17) with neural crest markers FoxD3, Sox8, Sox9, Sox10 and Twist expression of which, in contrast to Slug and Snail, were all inhibited by LMO4 misexpression. (D) In situ hybridization of stage 13 embryos injected with LMO4 and β-gal probed for neural plate marker Sox3, placodal markers Opl and Six1, epidermal marker Epk, and neural plate border markers Msx1 (St. 13). The expression of Slug and Snail is massively expanded while expression of other markers is inhibited. (E) TUNEL staining of stage 15 embryos injected with LMO4 MO, mRNA encoding LMO4, or apoptosis inducing factor DNMT3B1 (as a positive control). No significant changes in cell death were noted following either LMO4 up or down regulation. (F) phospho Histone H3 staining of stage 15 embryos injected with LMO4 MO or LMO4 mRNA. No significant changes in cell proliferation were noted following either LMO4 up or down regulation. * indicates injected side of embryo which is also marked by red gal staining.
Fig. 4. LMO4 forms a complex with Slug and Snail. (A) Co-immunoprecipitation (IP) assay probing the ability of LMO4 to interact with neural crest regulatory factors. Embryos were injected with mRNA encoding flag-tagged LMO4 and indicated myc-tagged neural crest transcription factors. Whole embryo lysates were prepared at stage 10.5, immunoprecipitated with α-flag antibodies, resolved by SDS page and subjected to western analysis using α-myc antibody. LMO4 interacts strongly with Slug and Snail but not Sox10, FoxD3 or Twist. (B) GST pull-down assay demonstrating that in vitro translated LMO4 protein directly interacts with GST-Slug, but not with GST alone.
Fig. 5. The LIM domains of LMO4 and the N-terminus of Slug are necessary for interaction. (A) Schematic of showing deletion mutants of LMO4, Slug, or Snail used in this study. (B) Co-immunoprecipitation (IP) assay probing the LMO4 domains required for interaction with Slug and Snail. Embryos were injected with mRNA encoding flag-tagged LMO4 constructs and myc-tagged Slug or Snail. α-flag IP followed by α-myc western shows that both LIM domains are required for robust interaction with Slug and Snail. (C) Co-immunoprecipitation assay using flag-tagged LMO4 and myc-tagged forms of either Slug (left panel) or Snail (right panel). Deletion of the Snag domain has a greater effect on the ability of Snail to interact with LMO4. (D) Co-immunoprecipitation assays comparing the ability of the full length or N-terminus of Slug (left panel) or Snail (right panel) to interact with LMO4, HDAC or Ajuba. Both Slug and Snail interact comparably with all three co-regulatory factors, but the Snag domain containing N-terminus is not sufficient for this interaction. Lower molecular weight bands on Snail immunoblot are common Snail degradation products.
Fig. 6. Slug and Snail utilize different domains to recruit Ajuba and HDAC. (A) Co-immunoprecipitation (IP) assay comparing the protein domains of (myc-tagged) Slug and Snail required for interaction with flag-tagged HDAC1 (A) or Ajuba (B). Deletion of the SNAG domain of Snail but not Slug, leads to loss of interaction with both HDAC1 and Ajuba. Conversely, the zinc finger domain of Slug, but not Snail, is sufficient for both interactions. These findings reveal novel differences in how Slug and Snail interaction with transcriptional co-regulatory factors.
Fig. 7. Functional roles for LMO4 in neural crest induction. (A) Co-IP assay demonstrating interaction between two LIM domain containing adaptor proteins, LMO4 and Ajuba. (B) Co-IP assay demonstrating that HDAC interacts strongly with Ajuba but not LMO4, indicating that the functions of these two adaptor proteins can be distinguished. Co-expression of LMO4 abrogates the interaction between Ajuba and HDAC, most likely by competing for Ajuba binding. (C) LMO4 is necessary for Slug/Snail-mediated, but not Sox10-mediated, neural crest induction. Whole mount in situ hybridization of embryos co-injected in one cell at the eight-cell stage with LMO4 morpholino and β-gal lineage tracer alone or in the presence of mRNA encoding Slug, Snail or Sox10. Embryos were examined at mid-neurula stages for expression of neural crest marker Sox10. Whereas all three NC regulatory factors can induce ectopic Sox10 expression, only Sox10 can do so in LMO4 depleted cells. These findings point to a more direct the role for LMO4 in the function of Slug and Snail.
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