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Cells Dev
2024 Apr 03;:203918. doi: 10.1016/j.cdev.2024.203918.
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Brain enlargement with rostral bias in larvae from a spontaneously occurring female variant line of Xenopus; role of aberrant embryonic Wnt/β-catenin signaling.
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Increased brain size and its rostral bias are hallmarks of vertebrate evolution, but the underlying developmental and genetic basis remains poorly understood. To provide clues to understanding vertebrate brain evolution, we investigated the developmental mechanisms of brain enlargement observed in the offspring of a previously unrecognized, spontaneously occurring female variant line of Xenopus that appears to reflect a genetic variation. Brain enlargement in larvae from this line showed a pronounced rostral bias that could be traced back to the neural plate, the primordium of the brain. At the gastrula stage, the Spemann organizer, which is known to induce the neural plate from the adjacent dorsal ectoderm and give it the initial rostrocaudal patterning, was expanded from dorsal to ventral in a large proportion of the offspring of variant females. Consistently, siamois expression, which is required for Spemann organizer formation, was expanded laterally from dorsal to ventral at the blastula stage in variant offspring. This implies that the active region of the Wnt/β-catenin signaling pathway was similarly expanded in advance on the dorsal side, as siamois is a target gene of this pathway. Notably, the earliest detectable change in variant offspring was in fertilized eggs, in which maternal wnt11b mRNA, a candidate dorsalizing factor that activates Wnt/β-catenin signaling, had a wider distribution in the vegetal cortical cytoplasm. Since lateral spreading of wnt11b mRNA, and possibly that of other potential maternal dorsalizing factors in these eggs, is expected to facilitate lateral expansion of the active region of the Wnt/β-catenin pathway during subsequent embryonic stages, we concluded that aberrant Wnt/β-catenin signaling could cause rostral-biased brain enlargement via expansion of siamois expression and consequent expansion of the Spemann organizer in Xenopus. Our studies of spontaneously occurring variations in brain development in Xenopus would provide hints for uncovering genetic mutations that drive analogous morphogenetic variations during vertebrate brain evolution.
Fig. 1. Genealogy of spontaneously variant females of Xenopus laevis.
(A) Morphology of F0-1 larvae classified at stage 29/30. The table shows the proportions of each class in larvae from F0-1 to -3. n is the total number of larvae examined in a mating.
(B) The proportions of each class in larvae from F1-1 to -28 (colored bars). The solid circles on the bars show the levels of wnt11b mRNA in unfertilized eggs of the respective F1 females, quantified from the data in Fig. 8A as described in Materials and methods (4.4).
Fig. 2. Enlargement of brain structures in Class I larvae.
(A, B) Morphology of normal and Class I larvae at stage 29/30 used for WISH analysis in (A') and (B′). The top, middle, and bottom panels in (A) and (B) show lateral, dorsal, and ventral views, respectively. Scale bar: 1 mm.
(A') Expression of sox2 in normal and Class I larvae. The arrangement of the images is the same as in (A).
(B′) Expression of bf1 (red asterisk) and en2 (light blue asterisk) in normal and Class I larvae. Red boxed areas in (B) are magnified. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Serial cross-sections through the head of normal and Class I tailbud larvae at stage 29/30.
Serial cross-sections were taken from normal and Class I larvae shown in bright field at upper left with scale bar: 1 mm. The part of the head indicated by the bidirectional arrow in the lateral view was sectioned at 8 μm and the resulting serial sections were stained with DAPI. Every 6th section from section 7 (s7) to section 43 (s43) and a typical section at some distance (s78) are shown for normal (upper row) and Class I (lower row) larvae. The tissues of interest in the sections are indicated by the numbers given to each tissue at the top of Fig. 3. Specifically, forebrain (1), midbrain (2), and hindbrain (3) are outlined with yellow dotted lines. Scale bar: 0.25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Serial cross-sections through the head of normal and Class I tadpoles at stage 40.
Serial cross-sections were taken from normal and Class I larvae shown in bright field at upper left with scale bar: 1 mm. The part of the head indicated by the bidirectional arrow in the dorsal view was cross-sectioned at 15 μm and the resulting serial sections were stained with H/E. The tissues of interest in the sections are indicated by the numbers assigned to each tissue at the top of Fig. 4. Neural tissues are indicated by a red number. Specifically, forebrain (1), midbrain (2), and spinal cord (8) are outlined with red dotted lines. Scale bar: 0.25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Enlargement of the neural plate and its subdivisions in stage 13 neurulae of variant females.
(A, B, C) Examples of sox2 (A), rax and hes7.1 (B), and foxb1 (C) expression in stage 13 neurulae from control female (upper panels) and from F0-1 (lower panels).
(A') Histograms generated from sox2 expression data. The abscissa is the d/D value (% of embryo width). The mean ± SD for control and F0-1 neurulae are 62.6 ± 3.8 and 86.2 ± 10.2, respectively. The difference between the two groups is significant by t-test at p =1.6 x 10−14.
(B′, B″) Histograms generated from rax (B′) and hes7.1 (B″) expression data. Abscissa is d/D in (B′) and (d1 + d2)/D in (B″). Mean ± SD values (% of embryo width) for control and F0-1 neurulae are 54.4 ± 6.2 and 71.0 ± 12.3 in rax, and 61.0 ± 6.1 and 69.0 ± 10.9 in hes7.1 expression, respectively. Differences between the two groups are significant by t-test at p=3.0 x 10−11 for rax expression and p=8.5 x 10−7 for hes7.1 expression.
(C′) Histograms generated from foxb1 expression data. The abscissa is the d/D value (% of embryo width). The mean ± SD for control and F0-1 neurulae are 43.2 ± 6.0 and 46.8 ± 4.7, respectively. The difference between the two groups is significant by t-test at p=1.3 x 10−3.
Fig. 6. Expanded expression of chordin and cerberus in gastrulae of variant females.
(A, B) Examples of chordin (A) and cerberus (B) expression in stage 11 gastrulae from control female (top panels) and those from F0-1 (lower panels). Dorsal, lateral, and vegetal views are shown in the left, middle, and right panels, respectively. PFMH in the top middle panel of (B) indicates prospective forebrain, midbrain, and hindbrain regions, and white arrows in middle panels of (A) and (B) point to the the dorsal lip of the blastopore (see Discussion, 3.1).
(A') Histograms generated from chordin expression data. The abscissa is the value of the expression angle defined by ‘Angle’ in (A). The mean ± SD values (degrees) for control and F0-1 embryos are 86.1 ± 12.2 and 130.6 ± 47.3, respectively. The difference between the two groups is significant by t-test at p=3.0 x 10−25.
(B′) Histograms generated from cerberus expression data. The abscissa is d/D value (% of embryo width). The mean ± SD for control and F0-1 embryos are 43.7 ± 8.3 and 62.4 ± 11.9, respectively. The difference between the two groups is significant by t-test at p = 5.4 x 10−19.
Fig. 7. Expanded expression of siamois in blastulae of variant females.
(A, B, C) Examples of siamois (A) and chordin (B) expression at stage 9.5 and cerberus (C) expression at stage 10 in embryos from control female (upper panels) and F0-1 (lower panels). Dorsal views are shown.
(A', A") Histograms generated from siamois expression data. Blastulae from F0-1 (A') and F1-36 (A") were used. F1-36 is a progeny of F0-1 from a different cross than the one that produced the F1 progeny listed in Fig. 1B. Abscissas are d/D values (% of embryo width). Mean ± SD values for control and F0-1 embryos are 42.8 ± 11.6 and 64.4 ± 16.8, respectively (A'), and 48.2 ± 8.0 and 77.0 ± 13.2 for control and F1-36 embryos, respectively (A"). Differences between control and variant groups are significant by t-test at p=5.9 x 10−32 for (A') and p=1.8 x 10−31 for (A").
(B′, C′) Histograms generated from chordin (B′) and cerberus (C′) expression data. Abscissas in (B′) and (C′) are d/D values (% of embryo width) with each term defined in (B) and (C), respectively. Mean ± SD values for control and F0-1 embryos are 44.4 ± 5.3 and 70.3 ± 10.0 for chordin expression, respectively (B′), and 43.3 ± 9.1 and 54.4 ± 7.1 for cerberus expression, respectively (C′). The difference between control and F0-1 groups is significant by t-test at p=1.4 x 10−36 for chordin expression and p=8.8 x 10−13 for cerberus expression.
Fig. 8. Dispersion of maternal wnt11b mRNA in the vegetal cortical cytoplasm of variant female eggs.
(A) RT-PCR products of wnt11b mRNA in unfertilized eggs of females of the F1 generation. N in (F1-Nsingle bond) indicates the assigned number of F1 females examined as shown in Fig. 1B.
(B) Protocol for WISH with the bisection procedure and method for displaying stained samples in (C).
(C, D) Examples of wnt11b mRNA expression at stage 2−. In (C), cut-surfaces of WISH-processed embryos from control female (upper panels) and F1-36 (lower panels) are shown in the arrangement illustrated in (B). In (D), embryos from control F1-14 (upper panels) and variant F1-1 (lower panels) were used. Vegetal views of the recombined embryos are shown.
(C′) Histograms generated from the rotational shift data of wnt11b mRNA. The abscissa is the mean of the angles defined by ‘Angle’ on the pair of cut surfaces in (C), lower right panel. The mean ± SD (degrees) for control and F1-36 embryos are 12.8 ± 7.7 and 22.0 ± 9.7, respectively. The difference between the two groups is significant by t-test at p = 1.3 × 10−7.
(C″, C‴, D′) Histograms generated from the dispersion data of wnt11b mRNA. Abscissas are (d1 + d1')/2D in (C″), (d2 + d2')/2D in (C‴), and (d3 + d3')/D in (D′), respectively, with each term defined in (C), upper right panel, or (D), upper panel. Mean ± SD values (% of embryo width) for control and variant F1 embryos are 56.3 ± 5.2 and 66.7 ± 5.8 for dorsoventral dispersion in (C″), 23.1 ± 4.7 and 29.1 ± 2.9 for inward dispersion in (C‴), and 34.7 ± 9.3 and 59.0 ± 9.9 for lateral dispersion in (D′), respectively. Differences between control and variant F1 groups are significant by t-test at p=2.7 x 10−10 in (C"), p=2.070−7 in (C‴), and p=1.2 x 10−17 in (D′).
Fig. S1. Supplementary data to Fig. 3. Consecutive series of sections are shown. The sections
used in Fig. 3 are outlined in red. The upper sections are from a normal tailbud and the lower
sections are from a Class I tailbud.
Fig. S1. Supplementary data to Fig. 3. Consecutive series of sections are shown. The sections
used in Fig. 3 are outlined in red. The upper sections are from a normal tailbud and the lower
sections are from a Class I tailbud.
Fig. S2. Supplementary data for Fig. 4. Consecutive series of sections are shown. The
sections used in Fig. 4 are outlined in red. The upper sections are from a normal tadpole and the
lower sections are from a Class I tadpole.
Fig. S2. Supplementary data for Fig. 4. Consecutive series of sections are shown. The
sections used in Fig. 4 are outlined in red. The upper sections are from a normal tadpole and the
lower sections are from a Class I tadpole.
Fig. S3. Expansion of the Speamann organiser and neural plate structures in D2O-treated
embryos. Fertilised eggs from normal crosses were treated with D2O as reported (Scharf et al.,
1989). (A) Expanded expression of chordin at stage 11 in D2O-treated embryos. Histograms
were generated as described for Fig. 6A’. Mean + SD values (degrees) for control and
D2O-treated embryos are 90.1 + 14.0 and 133.2 + 333, respectively. The difference between the
two groups is significant by t-test at p=3.6 x 10-23
. (B) Expanded expression of sox2 (left panels)
and bf1 (right panels) in D2O-treated embryos at stage 13. (C) Expanded expression of sox2 (left
panels), bf1 (middle panels) and emx1 + fez (right panels) in D2O-treated embryos at stage 15.
emx1 expression is light blue (BCIP) and fez expression is purple (BM purple). The double in
situ procedures were essentially the same as described previously (Hongo and Okamoto, 2022).
Fig. S4. Suppression of siamois expression in late blastula by depletion of FGF2. (A)
Injection protocol. Note that the injection site of MOs in both animal and vegetal blastomeres at
the 8-cell stage was near the central equatorial region on the dorsal side of the embryo, so that
the MOs would be distributed in the dorsal marginal zone of the embryo grown to the blastula
stage. (B) FGF2 MO or 5mis-FGF2 MO was injected into a dorsal animal blastomere at a dose
of 6.0 ng/blastomere and into a dorsal vegetal blastomere at a dose of 12.0 ng/blastomere, each
co-injected with tracer gfp RNA (20 pg/blastomere). Whole-mount in situ hybridization was
performed on blastula embryos at stage 9.5. The dorsal view is shown. The numbers on the
panels indicate the number of embryos typically seen in the photographs relative to the total
number of embryos analyzed. Scale bar = 0.20 mm. Microinjection and whole-mount double in
situ hybridization were performed as previously described (Hongo and Okamoto, 2022).
