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doi: 10.1242/10.1242/dev.00097

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Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein

Ignacio Muñoz-Sanjuán*, Esther Bell*, Curtis R. Altmann, Alin Vonica and Ali H. Brivanlou{dagger}

The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA



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  		Fig. 1. Experimental approach. Embryos were injected in both cells at the two-cell stage in the animal pole with 1 ng Smad7 RNA. Ectodermal explants were isolated at stage 9 from both Smad7-injected and uninjected embryos and cultured until stage 15 (neurula). Transcriptional differences were analyzed on a 5000 clone gastrula (stage 10.5) cDNA microarray in duplicate as inverse-dye labeled competitive hybridization (Altmann et al., 2001Go).

 


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  		Fig. 2. Confirmation of selected clones by RT-PCR. Individual clones were analyzed by RT-PCR in animal caps injected with 1 ng of Smad7 RNA and harvested at either stage 10.5 (gastrula; A) or stage 15 (neurula; B). Primers were designed to include either 5' or 3' UTR sequences. Ornithine decarboxylase (ODC) is used as a loading control and NCAM and NRP1 to control for neural induction by Smad7.

 


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  		Fig. 3. Microarray analysis of RNAs isolated from ectodermal explants neuralized by Smad7. Pie chart of the classification of clones identified in the array based on EGAD categories (TIGR, Maryland).

 


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  		Fig. 4. Whole-mount in situ hybridization of selected clones. (A) Clone 46-E2/FUSE-binding protein is expressed throughout the animal pole of the gastrula embryo (left and center) and at tadpole stages in the branchial arches and the brain (right panel). (B) Clone 45-H5/prothymosin is expressed at high levels throughout the epithelial layer of the animal pole (left panel) and in anterior neural tissue both at neurula (middle panel) and tadpole stages (right panel). It is also seen in the tailbud (arrow in right panel). (C) Clone 47-B11/plexin, is expressed at gastrula stages throughout the animal pole (left panel). At neurula transcripts are detected throughout the spinal cord and neural tube. At tadpole stages, it is detected in the brain, spinal cord, the facial and trigeminal ganglia, and the pronephros (right). (D) Clone 47-F3/novel is expressed throughout the gastrula embryo (left and center panels, arrow in middle panel depicts the dorsal blastopore lip). At later stages, expression is very strong in the brain and caudal end of the embryo (white arrows in right panel). It is also detected in ventral tissue (black arrow, right). (E) Clone 47-G3/hypothetical protein is strongly expressed in the animal pole at gastrula stages and in the dorsal blastopore lip (left). High levels are also detected in the nervous system and it is excluded from the cement gland (middle). At tadpole stages (right), it is expressed exclusively in the anterior neural tube. (F) Clone 51-A10/hypothetical protein is expressed at neurula stages in ciliated epidermal cells (arrows, left) and the presumptive otic placodes (arrow in center). In the tadpole (right), notice expression in the nervous system, branchial arches and pronephros. (G) Clone 51-D6/RNA bp EWS is expressed at high levels throughout the gastrula embryo excluding the vegetal pole (left panel), expression is very high in the anterior neural plate at neurula (middle) and is then expressed throughout the brain at tadpole stage (right). (H) Clone 57-G10/TAB3 is expressed throughout both the embryo at both gastrula and neurula stages (left and middle). At tadpole stage, expression is seen in the brain and branchial arches (right). (I) Clone 47-G6/DG42 is expressed throughout the animal pole in the gastrula (left) and is excluded from the neural plate at neurula stages (center); by tadpole stage it is only expressed in ventral regions of the gut (right, see arrow). a, anterior; ba, branchial arches; cg, cement gland; fb, forebrain; hb, hindbrain; mb, midbrain; nc, notochord; np, neural plate; nt, neural tube; ov, otic vesicle; p, posterior; pn, pronephros; tb, tailbud; viith, facial ganglia; *, dorsal blastopore lip.

 


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  		Fig. 5. Functional analysis in animal cap explants of clones implicated in post-transcriptional regulation. mRNAs for 13-A10/hnRNP-A/B, 46-B2/snRNP-B, 51-B11/snRNP-D, 54-G3/RNA helicase and 51-D6/RNA-bp EWS were injected into both animal cap cells at the two-cell stage, either alone or together with 15 pg of noggin RNA. RT-PCR analysis was performed at stage 20 (tadpoles). In all instances, the neural tissue induced is anterior in character, and no posterior (spinal cord) neural markers are detected. Similarly, there is no expression of mesodermal derivatives, as judged muscle actin (muscle) and collagen-type II (notochord) expression. Markers used are: NCAM and NRP-1, pan-neural; Otx2 and Pax6, forebrain/midbrain; XAG, cement gland; En2, midbrain/hindbrain boundary; Hoxb9, spinal cord; muscle actin, muscle.

 


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  		Fig. 6. Functional analysis of clones 56-G6/HMG-X and 57-G10/TAB3 in animal cap explants. (A) Clones 56-G6/HMG-X and 57-G10/TAB3 were injected into animal caps and analyzed for fate changes at stage 20. Both clones induce neural or cement gland marker expression. Notice no induction of posterior neural markers (En-2, mid/hindbrain; Krox-20, hindbrain; twist, neural crest) and weak induction of the heart marker xNkx2.5. (B) 56-G6/HMG-X synergizes with low amounts of Smad7 (5 pg). Notice inhibition of cement gland fates induced by Smad7 in the presence of 56-G6/HMG-X (lane 5). (C-H) Gain-of-function experiments with 56-G6/HMG-X in vivo. Embryos were injected into 1/2 cells in the animal pole and embryos analyzed at tadpole stages. Notice enlargement of the anterior neural plate (C-E), and ectopic expression of the anterior neural marker Pax6 (F-H). Asterisks indicate enlarged anterior neural territory (D,E) and ectopic Pax6 expression (G,H).

 


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  		Fig. 7. Analysis of xTAB3 involvement in BMP signaling and neural induction. (A) Dendrogram of the identified TAB2 and TAB3 family. Murine and human TAB3 were found in NCBI searches. Two TAB-related genes found in the fugu database are included (JGI 18320 and JGI 564). (B) Schematic of xTAB3 and xTAB3C proteins. Yellow box depicts the CUE domain (ubiquitin ligase interacting domain). Green box depicts the Zn-finger motif. (C) Injection of xTAB3C RNA into animal caps results in upregulation of epidermal keratin expression in gastrula stage explants, a marker responsive to BMP signaling. There is no mesodermal gene expression in these caps (xBra). (D-G) Animal cap explants (stage 23) injected with 50 pg Smad7 form neural and cement gland tissue (E). Notice pigmented cement glands (arrowheads). Co-expression with xTAB3C largely inhibits cement gland (F). (H) Analysis of explants injected with 50 pg of Smad7 or noggin in the absence (lanes 3, 6) or presence (lanes 4, 8) of xTAB3C shows that xTAB3C can inhibit neural and cement gland fates induced by BMP inhibitors.

 


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  		Fig. 8. Model for signal integration mediated by Smad7 in Xenopus laevis ectoderm. A proposed model of signaling integration in the context of neuralization might be mediated by Smad7. In this model, BMP signaling leads to the activation of both the Smad and TAK1 pathways. BMP signaling can be inhibited by 51-B6, a cerberus/gremlin-related factor (E. B., I. M.-S., C. R. A. and A. H. B., unpublished). We postulate that the transcriptional activation of TAB3 modulates the activity of TAK1, and might activate the NFkB and JNK pathways, as has been demonstrated for TAB2 in other contexts (Mazars et al., 2001Go; Wang et al., 2001Go). In addition, this switch might derepress some MAPK pathways, as TAK1/TAB1 complexes signal through p38 MAPK. Additional mechanisms to activate neural gene expression involve the HMG-X transcription factor and a variety of RNA-binding proteins implicated in post-transcriptional control. Red lines depict inhibitory signaling interactions. Green lines depict stimulatory signaling, either transcriptionally or through biochemical activation. Pink indicates genes identified in the array.

 




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