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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
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arx
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tropicalis
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NF stage 18
to
NF stage 21
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diencephalon
,
optic vesicle
,
neural plate
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pax6
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tropicalis
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NF stage 18
to
NF stage 21
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retina
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diencephalon
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pre-chordal neural plate
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eye
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neural tube
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lhx2
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tropicalis
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NF stage 18
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retina
,
eye
,
eye primordium
,
optic vesicle
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lhx2
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tropicalis
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NF stage 21
|
retina
,
diencephalon
,
pre-chordal neural plate
,
eye
,
optic vesicle
,
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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. |
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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. |
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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. |
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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. |
