XB-ART-49690Dev Biol. November 15, 2014; 395 (2): 317-330.
Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.
The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.
PubMed ID: 25224223
PMC ID: PMC4267880
Article link: Dev Biol.
Grant support: EY018000 NEI NIH HHS , EY022954 NEI NIH HHS , R01 EY017400 NEI NIH HHS , R01 EY018000 NEI NIH HHS , R01 EY022954 NEI NIH HHS , R01EY017400 NEI NIH HHS
Genes referenced: arx fezf2 foxg1 foxn4 hesx1 lhx2 mab21l2 myb myc otx2 pax6 plin1 rax six3 six6 tbx2 vsx1
Morpholinos referenced: fezf2 MO1 hesx1 MO4
Article Images: [+] show captions
|Fig. 1. A nonsense mutation in the retinal anterior homeobox gene (rax) results in an eyeless phenotype. (A) The Rax protein structure includes an octapeptide domain (OP, blue), homeodomain (HD, green) and OAR domain (orange). Nuclear localization signals (NLSs, red) are found in the homeodomain. A nonsense mutation results in a truncation prior to the homeodomain and NLSs. (B and C) Tadpoles homozygous for the rax mutant allele fail to form eyes (B, right; C, top). A small amount of retinal pigmented epithelium (RPE) is sometimes seen in mutant animals (B and C, orange arrows) whereas some mutant embryos have no sign of RPE (B, blue arrow); this variation is observed within the same clutch of embryos. The eyeless phenotype can be rescued with the injection of 5 pg of rax mRNA (C, bottom, 10 out of 24 injected). Sections through the mutant, wildtype, and mRNA-rescued mutant tadpole eye regions are shown in the middle panel, illustrating the loss of eye tissue, except small residual RPE in some mutant embryos, and rescue of the retinal and lens structures in mRNA-injected mutant. Far right of (C), insets of Sanger-sequencing showing point mutation in exon 2, which results in a premature stop codon. (D) A Xenopus tropicalis BAC clone containing a rax–gfp fusion gene can rescue eye formation when transiently expressed in mutant embryos. Note that BAC injection tends to be effective dominantly (or only) in one side as described before ( Fish et al. 2012). E1–E3 indicate rax exons; the gfp3 open reading frame (ORF) has been fused to the 3′ end of the final rax exon (E3) to produce a rax–gfp3 fusion gene. Quality of rescue is highly dose-sensitive, with the best rescue observed with 5 pg mRNA (C, data not shown) and 10 pg of BAC DNA injected (D).|
|Fig. 2. Key eye field transcription factor (EFTF) expression is unaffected in neural plate stage embryos. (A) The expression patterns and levels of rax, pax6, otx2, six3, lhx2, and six6 assayed by in situ hybridization in neural plate stage (St. 15) embryos with at least one wildtype allele versus homozygous mutant embryos reveal that key EFTF expression levels are unaffected in the rax mutant. Presumptive retinal regions indicated (arrows). (B) By early tailbud stage (St. 24), down-regulation of EFTFs can be detected in regions where the retina normally forms (arrows). (C) RNA-Seq analysis comparing wildtype and mutant St. 15 anterior neural plates confirms that EFTFs are not significantly affected in early stage rax mutant embryos. Fold change calculated using normalized read counts averaged from triplicate pooled samples. q-Values calculated using analysis utility Cuffdiff, part of the Cufflinks software package ( Trapnell et al., 2010), with a False Discovery Rate (FDR) of 0.05. High q-values indicate that listed genes are not significantly affected in neural plate stage rax mutant embryos.|
|Fig. 3. Morphological changes in rax mutant embryos can be detected by St. 21, prior to differential expression of key EFTFs, but following up-regulation of arx. Up-regulation of telencephalic marker foxg1 is also observed by St. 21. (A) Histological sections through St. 20 and St. 21 embryos reveal no detectable change in optic vesicle morphology at St. 20, but a lack of thinning of the posterior optic vesicle wall in mutant embryos at St. 21 (red arrows). The plane of sectioning is indicated above, and is the same plane for all stages shown throughout (serial sections were carefully examined to correctly match the plane of sectioning). The anterior and posterior ends of sections are marked A and P, respectively. (B) Examination of key EFTFs (pax6 and lhx2 shown) by in situ hybridization at St. 18 and St. 21 reveals no detectable change in expression. (C) By St. 19, anterior expansion of the arx expression domain is observed in mutant embryos (blue arrows). This expansion becomes more pronounced at St. 21 (red arrows). (D) Examination of telencephalic marker foxg1 at stages 21 and 24 reveals lateral expansion in rax mutant embryos (red arrows). At stage 24, lateral bulging of the optic vesicles can be observed in whole embryos from an anterior view (white arrows); these bulges are not observed in rax mutant embryos, and foxg1 expression is expanded into a region of the territory the optic vesicle would occupy in wildtype embryos. (E) Histological sections at St. 26, St. 28 and St. 30 show the increasing divergence of wildtype and mutant morphologies. By St. 30, the optic cup and lens tissue are forming in wildtype embryos (black arrows and arrowhead, respectively), but typically fail to form in rax mutant embryos. In panels A and E, a minimum of three embryos for each stage and genotype were sectioned and consistently displayed the shown morphology. Scale bars in panels A and E measure 75 μm.|
|Fig. 4. Diencephalic and telencephalic tissue is expanded in the rax mutant. (A) Dorsal views of St. 32 embryo heads assayed for diencephalic marker fezf2 (left) or telencephalic marker foxg1 (right) by in situ hybridization. Both fezf2 (blue arrows) and foxg1 (red arrows) expression is expanded into regions where the retina (r) would normally form in wildtype. (B) Frontal sections through St. 35 wildtype and mutant embryos detecting rax, fezf2, or foxg1 expression by in situ hybridization. In rax mutant embryos (bottom row), green arrows indicate small remaining rudiment still expressing rax. In the absence of functional rax, expression of fezf2 (blue arrows) and foxg1 (red arrows) is expanded into the tissue where the retina (r) would normally form. (C) Visualization of the parasagittal plane of sectioning performed on embryos in D- F. (D) Lateral views of parasagittally-bisected embryos at indicated stages stained by in situ hybridization for early diencephalic marker arx. In panels D-F, red dashed outlines indicate developing telencephalic tissue, and yellow dashed outlines indicate developing posterior secondary prosencephalon. White arrows cover the expression range of each marker, and dashed white arrows indicate into which region expression expands in rax mutant embryos (bottom row). arx expression is expanded ventrally in mutant embryos, and expands both anteriorly into the telencephalon and posteriorly into the secondary prosencephalon, with significant expansion observed by St. 21. (E) Parasagittally-bisected embryos at St. 21 show ventral expansion of telencephalic marker foxg1, which expands beyond the wildtype telencephalic domain in rax mutants. (F) fezf2 expression in the wildtype marks the posterior secondary prosencephalon. In rax mutant embryos this expression expands into the telencephalon and optic vesicle. (Note: for clarification of dense staining, this embryo was thinly parasagittally-sectioned by vibratome, instead of simply bisected as in panels D and E. The plane of sectioning is the same in panels D–F). (G) A cartoon illustrating the 3-dimensional movements of arx and foxg1 expansion into the presumptive optic vesicle regions of rax mutant embryos. Observed expansion of arx is moving ventrally, both anteriorly and posteriorly. Expansion of foxg1 is primarily lateral. Movement of these marker boundaries (and others) likely results in the expansion of diencephalic and telencephalic markers observed in the later mutant embryos shown in A and B. In all panels, A marks the anterior and P the posterior.|
|Fig. 5. Presumptive retinal tissue is transformed into diencephalic and telencephalic tissue in the rax mutant. (A–C) Genetic labeling of retinal fated tissue in wildtype and mutant embryos. A transgenic line expressing gfp3 under control of the rax-promoter was crossed into the rax mutant background. (A) Brightfield image of frontal sections from stage 32 embryos. Wildtype retinal tissue is indicated with "r". (B) Gfp3 protein expression detected by immunostaining. (C) Transgene (gfp3) mRNA expression visualized by in situ hybridization. (D) Telencephalic marker foxg1 mRNA expression visualized by in situ hybridization. In all panels, A marks the anterior and P the posterior. Red arrows mark regions of telencephalic expansion, and blue arrows mark diencephalic expansion into eye region. Green arrows mark the small rudiment that is pigmented (A) in this specific section and continues to express Gfp3 (B and C). Although Gfp3 protein can still be readily discerned, the mRNA is mostly absent in the expanded telencephalic region (black arrows). Five embryos from each phenotype (wildtype or mutant eye morphology) and treatment were scored for these analyses; expression patterns were highly stereotyped within each phenotype. A minimum of two embryos for each phenotype and treatment were then chosen for serial sectioning and imaging.|
|Fig. 6. Presumptive retinal tissue is transformed into both diencephalic and telencephalic fates in rax mutant embryos. (A) Retinal rudiment was labeled in St. 15 embryos using DiI marking. Embryos were then raised to St. 32 and scored for the fate of labeled tissues. Frontal sections of St. 32 embryos show DiI marking the retina (r, white arrow) and small amount of diencephalon (d, yellow arrow) in wildtype embryos, but not the telencephalon (t, red arrow). In mutant embryos, labeled tissues are found extensively in both the diencephalon and expanded telencephalon. Results shown in the table. *Since eyes fail to form in rax mutants, DiI labeling in the retina is not applicable (N/A). (B) Presumptive retinal tissue from St. 15 rax mutant or wildtype donor embryos was transplanted to the posterior of host embryos, which were raised to St. 37 and fixed. Donor embryos were allowed to develop in order to determine donor phenotype. In situ hybridization to detect telencephalic marker foxg1 was performed on hosts with donor tissue to determine the extent of telencephalic character in donor transplants. Left panels show lateral views of whole embryos, white arrows indicate regions positive for foxg1 expression; right panels show higher magnification view of transplanted region (outlined with white dots). Representative examples of each category (large or small region of transplant that is positive for foxg1 expression domain relative to entire transplant size) are shown in right panels. Results from scoring are tallied in the bottom table.|
|Fig. 7. Genes with altered expression in rax mutants include those necessary for retina formation and forebrain patterning. (A) Table listing key up-regulated (red) and down-regulated (blue) genes from RNA-Seq analysis. (All significantly affected genes identified in this analysis can be found in Table S4) (B) In situ hybridization for up-regulated genes in St. 15 embryos. White arrows indicate regions where up-regulation is observed in mutants in the presumptive retinal field. (C) In situ hybridization for down-regulated genes in St. 15 embryos. White arrows indicate regions in the eye field where expression is not detected in the mutant. (D) vsx1 expression (arrows) at neural plate stage is reduced in the heterozygote compared to wildtype and not detected in rax homozygous mutant embryos. (E) A time course of fezf2 and hesx1 expression from late gastrula (St. 12) through neural tube stage (St. 21) in rax mutant and wildtype embryos. White arrows indicate regions where repression is seen in wildtype embryos and not observed in rax mutants.|
|Fig. 8. Knockdown of fezf2 and/or hesx1 function reduces expanded foxg1- and arx- expressing region(s) in rax mutant embryos. (A) Representative example of the dorsal view of St. 32 rax mutant embryos injected with fezf2 and/or hesx1 MO(s), and assayed for foxg1 expression by in situ hybridization (left panel). Note that reduced signal is seen in the ectopically expanded foxg1-expressing region (arrow, left panel) on the injected side of mutant embryos (yellow asterisk, left panel), as visualized by FLDx tracer (right panel). (A׳) Percentage of embryos with mutant phenotype (expanded foxg1 expression) on injected side after receiving MO(s) injection. Embryos were injected into one dorsal blastomere at the 4-cell stage with either control, fezf2 or hesx1 morpholinos, or a combination of two morpholinos, as indicated in the y-axis. (B) Representative example of the frontal view of a St. 25 rax mutant embryo injected with combined fezf2 and hesx1 MOs, and assayed for arx expression by in situ hybridization (left panel). Dashed yellow outlines mark the region of arx expression, which is reduced on the injected side (marked with an asterisk, and visualized by FLDx tracer in right panel). (B׳) Percentage of embryos with mutant phenotype (expanded arx expression) on injected side after receiving MO(s) injection. Embryos were injected into one dorsal blastomere at the 4-cell stage with either control or combined fezf2 or hesx1 morpholinos. (A′ and B′) Injected doses of morpholinos is indicated in brackets, and numbers of embryos scored in parentheses. Asterisks indicate p-values of <0.05 by chi-squared test.|
|Fig. 9. Models of rax function. (A) Analysis of the rax mutant indicates that rax function is not necessary for activation of key EFTFs pax6, otx2, lhx2, or six3. rax expression is necessary for the correct expression of other key eye genes mab21l2, foxn4, myc, myb, vsx1, in addition to other factors. Dotted orange lines indicate putative, early interactions within the eye field gene regulatory network not supported by these analyses, although later, indirect effects of rax inactivation does affect their expression. Solid gray lines indicate previously described interactions not addressed in this work, but consistent with our data, and solid black lines indicate interactions supported by these analyses. (B) A key forebrain patterning event necessary for correct retina formation is the activation of a repressor(s) by rax, whose role is to down-regulate fezf2 and hesx1 in the anterior neural plate, creating a hole within their expression domains starting by late gastrula stages (St. 13). As shown in (C), when this repression of fezf2 and hesx1 fails to occur we propose that they contribute to establishment of non-retinal diencephalic (blue arrows) and telencephalic (red arrows) properties within presumptive retinal tissue, resulting in the expansion of these forebrain identities into the retinal region. Fate map in (C) adapted with permission from ( Eagleson and Harris, 1990).|