XB-ART-44906Development March 1, 2012; 139 (6): 1213-24.
RIPPLY3 is a retinoic acid-inducible repressor required for setting the borders of the pre-placodal ectoderm.
Retinoic acid signaling is a major component of the neural posteriorizing process in vertebrate development. Here, we identify a new role for the retinoic acid receptor (RAR) in the anterior of the embryo, where RAR regulates Fgf8 expression and formation of the pre-placodal ectoderm (PPE). RARα2 signaling induces key pre-placodal genes and establishes the posterolateral borders of the PPE. RAR signaling upregulates two important genes, Tbx1 and Ripply3, during early PPE development. In the absence of RIPPLY3, TBX1 is required for the expression of Fgf8 and hence, PPE formation. In the presence of RIPPLY3, TBX1 acts as a transcriptional repressor, and functions to restrict the positional expression of Fgf8, a key regulator of PPE gene expression. These results establish a novel role for RAR as a regulator of spatial patterning of the PPE through Tbx1 and RIPPLY3. Moreover, we demonstrate that Ripply3, acting downstream of RAR signaling, is a key player in establishing boundaries in the PPE.
PubMed ID: 22354841
PMC ID: PMC3283127
Article link: Development
Genes referenced: eya1 fgf8 frzb2 h4c4 psmd6 rab40b ripply3 six1 sox9 tbx1
Morpholinos: rara MO2 ripply3 MO1 ripply3 MO2 tbx1 MO1 tbx1 MO2
Article Images: [+] show captions
|Fig. 1. Expression of Tbx1 and Ripply3 across developmental time. (A,B)Whole-mount in situ hybridization of Tbx1 (A) and Ripply3 (B) gene expression at Nieuwkoop and Faber developmental stages 13, 16, 18, 20, 22 and 26 (B). (C)QPCR showing Tbx1 gene expression over developmental time. (D)QPCR showing Ripply3 gene expression over developmental time. In C and D, the y-axis represents 2#61508;Ct values normalized to the housekeeping gene histone H4.|
|Fig. 4. RARalpha2 is required for expression of PPE genes. Embryos were injected unilaterally at the two- or four-cell stage. The injected side is on the right, as indicated by the magenta beta-galactosidase lineage tracer staining. (A,D,G,J) Control expression of Tbx1, Ripply3, Six1 and Eya1. (B,E,H,K) 10 ng RARalpha2 MO reduced expression of Tbx1 (14/15 embryos), Ripply3 (9/10), Six1 (6/11) and Eya1 (8/9). (C,F,I,L) 10 ng RARalpha2 MO + 0.5 ng RARalpha2 mRNA rescued expression of Tbx1 (5/11 embryos), Ripply3 (8/11), Six1 (11/14) and Eya1 (13/17).|
|Fig. 5. Double whole-mount in situ hybridization reveals spatial relationship of PPE genes at stage 18. (A-C)Ripply3 (stained with BM Purple) is lateral to Tbx1, Six1 and Eya1 (stained with BCIP/Tetrazolium Blue), overlapping with Tbx1 and the posterior placodal (pp) area of Eya1. (D)Ripply3 (stained with Fast Red) is lateral to but does not overlap with Fgf8 (stained with BM-Purple). (E-G)Tbx1 (stained with BM Purple) is lateral to Fgf8, Six1 and Eya1 (all stained with Fast Red) in stage 18 embryos, overlapping significantly with Fgf8, and in the posterior placodal (pp) area of Eya1.|
|Fig. 6. Lateral view of double wish staining and schematics. (A-G)Lateral views of double whole-mount in situ hybridization from Fig. 5, including schematics of the overlapping or non-overlapping PPE genes. pp, the posterior placodal area; pPrV, profundal placodal area. (H)Relationship of PPE genes at stage 18.|
|Fig. 7. FGF8 establishes the PPE, but TBX1 maintains Fgf8 expression. All embryos were injected unilaterally at the two- or four- cell stage. The injected side is on the right. (A,C) Control expression of Tbx1 and Ripply3. (B,D) Embryos injected with 30 ng Fgf8 splice MO; Tbx1 (8/16 embryos) and Ripply3 (19/23) expression were knocked down or knocked out. (E,G,I,K) Embryos injected with 10 ng Tbx1 MO showed knockdown of Fgf8 (12/16 embryos), Ripply3 (17/21), Eya1 (21/26) and Six1 (25/36) expression. (F,H,J,L) 10 ng Tbx1 MO + 0.5 ng Tbx1 mRNA rescued Fgf8 (5/8), Ripply3 (6/8), Eya1 (6/8) and Six1 (8/11) expression.|
|Fig. 9. Knockdown of RIPPLY3 causes posterolateral shift of PPE gene expression. Embryos were injected at the two- or four-cell stage and the injected side is on the right. (A-H)Anterior (A,C,E,G) and dorsal (B,D,F,H) views of control expression of Fgf8, Tbx1, Eya1 and Six1. (I-P)Anterior (I,K,M,O) and dorsal (J,L,N,P) views of embryos injected with 50 ng Ripply3 MO 1+2 showing a posterolateral shift of Fgf8 (21/29 embryos), Tbx1 (14/19), Eya1 (12/18) and Six1 (12/20).|
|Fig. 10. Ripply3 gain of function causes loss of Tbx1 expression. Embryos were injected at the two- or four-cell stage and the injected side is on the right. (A,B)Anterior (A) and dorsal (B) views of embryos injected with 0.25 ng Ripply3 mRNA showing a knockdown of Tbx1 (11/11). (C,D)Anterior (C) and dorsal (D) views of embryos injected with 0.25 ng Ripply3WRPWrAAAA mRNA showing no change in Tbx1 (15/16) expression. (E,F)Anterior (E) and dorsal (F) views of embryos injected with 0.25 ng Ripply3FPVQrAAAA mRNA showing no change in Tbx1 expression (10/11).|
|Fig. S1. RARα2 is required for proper expression of Fgf8. All embryos were injected unilaterally at the two or four cell stage. The injected side is on the right as indicated by the magenta β-galactosidase lineage tracer staining. (A) Control expression of Fgf8. (B,C) 10 ng RARα1 MO + 10 ng RARα2 MO shifts Fgf8 expression posterolaterally (10/14 embryos shifted). (B) Anterior view. (C) Dorsal view. (D) 10 ng RARα1 MO + 10 ng RARα2 MO + 0.5 ng RARα2 mRNA rescued Fgf8 expression (8/16 embryos).|
|Fig. S2. Double WISH reveals spatial relationship of Tbx1 and Ripply3 expression at tailbud stage. (Left) Ripply3 (stained with BM Purple) and Tbx1 (stained with Fast Red) show overlapping expression in the epibranchial placodes (EB). Ripply3 is also expressed in the pronephros (PN), and Tbx1 in the otic placode (Ot). (Right) Schematic of expression patterns observed.|
|Fig. S4. Double WISH reveals spatial relationship of Fgf8 and Eya1 or Six1 at stage 18. Fgf8 (stained with BM Purple) overlaps with Six1 except at the bottom of the crescent. Fgf8 is expressed largely between the posterior placodal (pp) area and the profundal placode (pPrV) of Eya1, although some overlap is observed.|
|Fig. S10. Knockdown of Ripply3 disrupts placode morphology in the tailbud stage. All embryos were unilaterally injected at the two- or four- cell stage with 50 ng Ripply3 MO 1+2. Lateral views of the same embryo are shown facing each other. (A,C,E) Uninjected side showing control expression of Eya1, Six1 and Sox9. (B,D,F) Injected side showing altered placode morphology in Eya1 and Six1 in the spacing of the epibranchial placodes (EB), blurring of the lateral line placodes (LL), otic placode (Ot) and trigeminal placode (TG). The spacing of the hyoid crest (HC) and branchial crest (BC) is also altered in Sox9 expression. MC, mandibular crest; L, lens.|
|Fig. S11. Ripply3 MO embryos still form an otocyst. All embryos were unilaterally injected at the two- or four-cell stage with 50 ng Ripply3 MO 1+2. Paraffin-embedded tailbud stage embryos stained for Eya1, Six1 and Sox9 showing apparently solid otocyst morphology were sectioned transversely, 10 thick. Photographs (from different levels of the same embryo) are spliced from different sections (indicated by dotted line), to allow a direct comparison of otocyst morphology from the same embryo.|
References [+] :
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