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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02603

Development 133, 4341-4353 (2006)
Published by The Company of Biologists 2006
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Nemo-like kinase (NLK) acts downstream of Notch/Delta signalling to downregulate TCF during mesoderm induction in the sea urchin embryo

Eric Röttinger*, Jenifer Croce*, Guy Lhomond, Lydia Besnardeau, Christian Gache and Thierry Lepage{dagger}

UMR 7009 CNRS, Université de Pierre et Marie Curie (Paris 6), Observatoire Oceanologique, 06230 Villefranche-sur-Mer, France.


Figure 1
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  		Fig. 1. Temporal and spatial expression of nlk during sea urchin development. (A) Northern blot (e) Egg, (16) 16-cell stage, (60) 60-cell stage, (B1) very early blastula, (B3) early blastula, (B5) hatching blastula, (sB) swimming blastula, (mB) mesenchyme blastula, (eG) early gastrula, (G) gastrula, (Pr) prism, (Pl) pluteus. (B-Q) Spatial distribution of nlk and (R-Y) Delta transcripts. Embryos at the indicated stage were hybridized with sense (not shown) and antisense probes for nlk or Delta. (B) Egg, (C) 60 cell stage, (D) early blastula, (E) hatching blastula, (F,G,R,S) swimming blastula, (H,I,T,U) mesenchyme blastula, (J,K,V,W) early gastrula, (L,M,X) late gastrula, (N,O) prism, (P,Y) early pluteus, (Q) pluteus. All pictures are lateral views with the animal pole at the top and the oral side on the left, except G,I,K,M,O,S,U,W, which are (vv) vegetal pole views. Note the similarities in the salt and pepper expression pattern of nlk and Delta gastrula and early pluteus stages (arrows in panels N,P and X,Y).

 

Figure 2
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  		Fig. 2. nlk and Delta regulate each other. (A-L) Embryos were injected with mRNA encoding Delta (B,D), dnDelta (F,H,J) or nlk (L); (A,C,E,G,I,K) control uninjected embryos. In situ hybridization was performed with the indicated probes. (A-D) Mesenchyme blastula, (G,H,K,L) swimming blastula, (I,J) late gastrula.

 

Figure 3
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  		Fig. 3. Overexpression of nlk causes the same phenotypes as overexpression of Delta. (A) Morphology of embryos overexpressing nlk or Delta. (a,c,e) Control embryos. (b,d,f,g,h) Embryos injected with nlk mRNA. (i-l) Embryos injected with Delta mRNA. (a,b) Late gastrula, (c,d,i) prism stage, (e-h,j-l) pluteus stage. (vv) Vegetal pole view. (B) Effects of nlk misexpression on the expression of ectodermal, endodermal and mesodermal genes. In situ hybridization was carried out between blastula and gastrula stages. HE (a,g), bhmt (b,h), brachyury (c,i), goosecoid (d,j) and bpnt (e,f,k,l). (b-f) Control embryos. (g-l) Injected embryos.

 

Figure 4
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  		Fig. 4. Strong synergy between NLK and Delta in mesoderm formation. Embryos were injected with nlk mRNA together with Delta. (A,B) Control embryos. (C-F) Co-injected embryos. (D) Mesenchyme blastula, (A,E) late gastrula, (B,C,F) early pluteus.

 

Figure 5
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  		Fig. 5. Effects of nlk/Delta misexpression on mesoderm specification. (A-X) Following injections of the indicated mRNA, in situ hybridization was performed, with probes indicated on the left. vv, vegetal pole view.

 

Figure 6
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  		Fig. 6. Partial redundancy between the TAK1/NLK and the MEK/ERK signalling pathways. (A-R) Embryos were injected at the egg stage with the indicated reagents, treated or not with U0126 starting at the two-cell stage. In situ hybridization was performed for gcm (G-L) and papss (M-R). (A,G,M) Control embryos (B,D,F,H,J,L), U0126-treated embryos (C,D,I,J,O,P). Embryos injected with a morpholino oligonucleotide against nlk (Mo-nlk) or against tak1 (Mo-TAK1) (E,F,K,L,Q,R). (A-F) Early pluteus, (G-R) gastrula. vv, vegetal pole view.

 

Figure 7
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  		Fig. 7. NLK antagonizes the activity of TCF in TOP FLASH assays. (A) Partial sequence alignments between the sea urchin TCF (Pl.TCF) and vertebrate TCF proteins sequences, showing the conservation of the MAPK phosphorylation consensus site. (B) Ectopic expression of NLK downregulates TCF. Embryos were injected at the egg stage and treated with LiCl (30 mmol/l) from the two-cell stage on. Embryos injected with the TOP FLASH construct alone (grey column), plus lithium treatment (dark blue column). Embryos co-injected with the TOP FLASH construct and nlk mRNA (light blue column), plus lithium treatment (green column).

 

Figure 8
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  		Fig. 8. Lithium treatment strongly potentiates the mesoderm-inducing activity of NLK. Embryos were injected at the egg stage and treated with LiCl at the two-cell stage and either observed for the phenotype (A-H) or fixed for in situ hybridization (I-X) performed with the probes indicated on the left. (M-P) Mesenchyme blastula (I-L), early gastrula (A-D,Q-T,U-X), late gastrula (E-H), pluteus. vv, vegetal pole view.

 

Figure 9
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  		Fig. 9. Gastrulation and formation of the endomesoderm requires downregulation of TCF. (A) Scheme of the experiment. (B-R) Embryos were injected at the egg stage and treated continuously with Dexamethasone (DXM) starting either at the egg cell stage (C,G,K,P),or at the hatching Blastula (D,H,L,Q) or the early mesenchyme Blastula (vemB) stage (E,I,N,R). The morphology of the embryos is documented in B-E. (F-R) In situ hybridization with the probes indicated on the left. (B,F,J,O) Control embryos. vv, vegetal pole view.

 

Figure 10
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  		Fig. 10. Model of NLK function during germ layer specification and position of NLK in the endomesoderm gene regulatory network. (A) Model of NLK function during segregation of the mesoderm. Treatment with lithium increases the size of the endodermal territory and shifts the ectoderm/endoderm boundary towards the animal pole. Overerexpression of NLK or Delta causes the mesodermal territory to expand and the endodermal/ectodermal boundary to be displaced towards the animal pole. Co-injection of NLK and Delta converts most cells of the embryo into mesoderm. (B) Role of NLK in the gene regulatory network regulating mesoderm formation. During cleavage stages, the maternal Wnt pathway acting through TCF induces the endomesoderm. At the blastula stage, Notch/Delta signalling upregulates the expression of nlk in mesodermal precursors. NLK promotes segregation of the mesoderm from the endomesoderm by downregulating TCF in the presumptive SMCs, allowing the establishment of a novel regulatory domain that expresses mesodermal genes such as gcm. NLK and Delta maintain the expression of each other. The NLK and ERK pathways converge to maintain specification of SMCs, while NLK, possibly acting upstream of the MAP kinase pathway, promotes epithelial mesenchymal transition by stimulating phosphorylation of ERK and expression of ets1.

 

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© The Company of Biologists Ltd 2006