XB-ART-55848Dev Biol January 1, 2019; 446 (1): 68-79.
The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.
PubMed ID: 30529252
PMC ID: PMC6349505
Article link: Dev Biol
Genes referenced: ctrl eya1 fgf8 foxd3 irx1 myc neurod1 pax2 pax3 six1 sox11 sox2 sox3 sox9 tubb4b zic1 zic2 znrd2
Morpholinos: six1 MO1 six1 MO2
Article Images: [+] show captions
|Fig. 1. Animal pole view of a 16-cell stage embryo with dorsal to the top. Nomenclature on the left side indicates blastomere names according to Hirose and Jacobson (1979).|
|Fig. 2. Six1 is required for Irx1 expression in the PPR and otic vesicle. (A) Irx1 expression in the PPR (black arrows) is distinct on the control side (ctrl) but not detected on the MO-mediated knock-down (KD) side (red arrows). np, neural plate. (B) Irx1 expression in two placodes is smaller and fainter on the KD side (red arrows) compared to the control side (black arrows). nt, neural tube. (C) Irx1 expression in the control otic vesicle (left image) forms two distinct patches (black arrows), whereas the anterior patch often is undetectable on the KD side (red arrows). e, eye. A, B, anterior views; C, side views, all with dorsal to the top. Percentages are the frequencies of the phenotypes; numbers in parentheses are the sample sizes.|
|Fig. 5. Irx1 is required for PPR and otic gene expression (A) Injecting a dominant-negative Irx1 construct (DN-Irx1) results in loss of Six1 PPR expression (red arrows) in every embryo. Black arrows denote normal expression on control (ctrl) side. (B) Injecting DN-Irx1 results in loss of Sox11 PPR expression (red arrows) in every embryo. Black arrows denote normal expression on control side. (C) Injecting DN-Irx1 results in loss of Six1 otic expression (red arrow) in every embryo. Black arrow denotes normal expression on control side. (D) Injecting DN-Irx1 results in loss of Pax2 otic expression (red arrow) in every embryo. Black arrow denotes normal expression on control side. A, B are anterior views; C, D are side views, all with dorsal to the top. np, neural plate; e, eye.|
|Fig. 7. Irx1 may affect PPR gene expression by downregulating Fgf. (A) When Irx1 mRNA (400 pg) was injected into animal blastomeres, an open blastopore phenotype (arrows) was observed at closed neural tube stages. Dorsal view, anterior (a) to the top, posterior (p) to the bottom. (B) The frequency of this phenotype depended upon the cell injected (see Fig. 1 for blastomere nomenclature). (C) Irx1 mRNA (400 pg) injection reduced the size of the Fgf8 expression domain in the PPR. Black arrows denote Fgf8 expression in the PPR on the control side; red arrows denote the reduced domain on the injected side. (D) In the majority of embryos co-injected with Irx1 plus cFgfr1 mRNAs, Six1 expression was restored (76.7%, n = 60). Black arrows denote control side; red arrows denote injected side. C, D are anterior views, dorsal to the top.|
|Fig. 10. Increased Irx1 reduces neural stem and neural differentiation genes in placodes. (A) Left: increased Irx1 reduced the Sox2 placode domain (red arrow) compared to control side (between black arrows) in the majority of cases. Right: expression was expanded in the lateral ectoderm along the cranial neural border in some cases. (B) Left: increased Irx1 reduced the Sox3 placode domain (red arrow) compared to control side (between black arrows) in the majority of cases. Right: expression was expanded in the lateral ectoderm along the cranial neural border in some cases. (C) Increased Irx1 reduced p27, NeuroD and Tubb2 in their placode domains (red arrows) compared to control side (black arrows). (D) Increased Irx1 also reduced these three genes in embryos in Six1 morphants. Black arrows denote control sides; red arrows denote injected sides. np, neural plate; nt, neural tube. All embryos are anterior views with dorsal to the top.|
|Fig. S1. (A) Sequences of the two antisense morpholino oligonucleotides (Six1-MO1, Six1-MO2) used to knock down Six1 translation, aligned with the sequences of the endogenous, wild type Six1 mRNA and the 5’ Myc-tagged (MT) rescue mRNA. MT-Six1-rescue mRNA contains 6 Myc-tags (proximal sequence in light blue font) 5’ to the two ATG start codons (bold in wildtype Six1). It also contains three mutated nucleotides in the Six1 ORF (red font) that do not change the amino acid code. Underlined are those nucleotides in the MT-Six1-rescue mRNA that are not complementary to either MO. (B) Validation of efficacy and specificity of Six1-MOs. Expression of the MT-Six1-rescue mRNA is high when injected alone into oocytes (-MO), and its expression is unaffected by previous injection with Six1-MO1+MO2 (+MO). Expression of an mRNA that contains the native Six1 5’UTR and a 3’Flag tag (5’UTR-Six1-3’Flag) is high when injected alone into oocytes (-MO), but translation is prevented in oocytes previously injected with Six1-MO1+MO2 (+MO). Expression of 5’Flag-Six1 and 3’Flag Six1mRNAs in injected oocytes also are shown. Uninjected lane = control oocyte lysates. (C) Uninjected Xenopus tropicalis embryo from same clutch as those shown in (D) showing normal Irx1 expression in the neural tube (nt) and otocyst (black arrow). e, eye (D) Six1 F0 mutants created by CRIPSR-Cas9 injections show normal Irx1 expression in the neural tube (nt), but loss of expression in the otic vesicle (red arrows). These Xenopus tropicalis embryos were created by microinjecting Cas9 protein plus sgRNAs, designed by the National Xenopus Resource and demonstrated by them to efficiently cause N-terminal Six1 mutations, into the fertilized egg during the 2017 Xenopus Genome Editing Workshop.|