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Dev Growth Differ
2024 Apr 01;663:256-265. doi: 10.1111/dgd.12919.
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Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.
Sakagami K
,
Igawa T
,
Saikawa K
,
Sakaguchi Y
,
Hossain N
,
Kato C
,
Kinemori K
,
Suzuki N
,
Suzuki M
,
Kawaguchi A
,
Ochi H
,
Tajika Y
,
Ogino H
.
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Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deeptissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.
19K06689 Japan Society for the Promotion of Science (JSPS), 22K06240 Japan Society for the Promotion of Science (JSPS), 22H02796 Japan Society for the Promotion of Science (JSPS), Takeda Science Foundation
FIGURE 1
Identification of optimum heat treatment conditions to inactivate endogenous alkaline phosphatase in Xenopus. (a, b) Fixed wild-type embryos (stages 25 and/or 33) were treated at 65°C for 60 or 120 min and then subjected to a chromogenic alkaline phosphatase (AP) reaction at 37°C for 3 h (Conditions 1 and 2, respectively). (c) Fixed wild-type embryos (Stage 33) were treated at 65°C for 120 min and then subjected to a chromogenic AP reaction at 4°C overnight, followed by the AP reaction at 37°C for 9 h (Condition 3). (d, e) Fixed wild-type embryos (Stage 33) were treated at 75°C for 120 min and then subjected to a chromogenic AP reaction at 4°C overnight, followed by the AP reaction at 37°C for 9 or 24 h (Conditions 4 and 5, respectively). Representative embryos are shown for Conditions 1 to 5. Magenta triangles indicate AP staining in the eye and tissues surrounding the anus. The purple triangle indicates the timing for providing AP substrates to the heat-treated embryos.
FIGURE 2
Comparison of detection sensitivities between green fluorescent protein (GFP) fluorescence and placental alkaline phosphatase (PLAP) staining using six3 cis-regulatory elements. (a) Schematic representation of an alignment of a genomic sequence corresponding to the Xenopus tropicalis six3 gene and its upstream region with the orthologous genomic sequences of Nanorana parkeri, Anolis carolinensis, Gallus gallus, Mus musculus, Homo sapiens, and Takifugu rubripes. In the reference X.tropicalis sequence, the coding regions, intron, and 3' untranslated region (UTR) of six3 are shaded in purple, yellow, and cyan, respectively. Previously identified conserved enhancers (CNS3 and CNS5) and a conserved proximal promoter region including the 5'UTR of six3 are shaded in pink and orange, respectively. Sequences with more than 75% identity to the orthologous X.tropicalis sequence are shown in red, and sequences with 50% to 75% identity are shown in green in the six aligned species sequences. (b) GFP and PLAP expression driven by the enhancers CNS3 and CNS5 and the proximal promoter of six3. Upper panels show the injected GFP reporter construct, representative embryos exhibiting GFP fluorescence in the eyeprimordia at the neurula and tailbud stages (pink triangles, stages 19 and 28), and a representative tadpole (stage 45) exhibiting GFP fluorescence in the eye (pink triangle), telencephalon (pink arrow), diencephalon (white triangle), nasal placodes (white arrow), and somite (blue arrow). In the eye, the pigment epithelium mostly covers the neural retina, which obscures fluorescence signals from the neural retina. A long-pass filter was used in GFP epifluorescence microscopy to visualize the whole embryo shape with the yellowish autofluorescence of the yolk (stages 19 and 28). The pink asterisk indicates autofluorescence in the intestine (stage 45). Lower panels show the injected PLAP reporter construct, representative embryos exhibiting PLAP staining signals in the eyeprimordia (pink triangles) and in the telencephalon and diencephalonprimordia (pink arrows) at the neurula and tailbud stages (stages 19 and 28), and a representative tadpole (stage 44) exhibiting PLAP staining signals in the eye (pink triangle), telencephalon (pink arrow), diencephalon (white triangle), nasal placodes (white arrow), and somite (blue arrow). (c) Bar graphs summarizing the transgenic reporter experiments. For each construct, reproducible expression patterns consistent with the representative examples shown in (b) were scored as eye- (and telencephalon/diencephalon-) specific expression at the indicated stages. Expression patterns that were reproducible in <5% of the analyzed embryos were scored collectively as ectopic expression.
FIGURE 3
Comparison of green fluorescent protein (GFP), placental alkaline phosphatase (PLAP), lacZ, and lagoZ expression driven by an eef1a1 or tubb2b promoter. (a–d) Representative expression patterns of GFP, PLAP (ALPP), lacZ, and lagoZ driven by an eef1a1 promoter in tailbud embryos. The expression of GFP and PLAP was visible as green fluorescence and AP staining, respectively. Pink triangles indicate part of the X-gal staining signals. (e, f) Representative expression patterns of GFP and PLAP (ALPP) driven by a tubb2b promoter in tadpoles. White, gray, black, blue, yellow, and green triangles indicate GFP fluorescence (e) or AP staining signals (f) in the olfactory epithelium, telencephalon, diencephalon, midbrain, hindbrain, and spinal cord, respectively. Pink arrows indicate GFP fluorescence (e) or AP staining signals (f) in the cranial nerves. (f) The purple AP staining in the eye (brown arrow) appears to merge with the brownish color of the residual retinal pigment that remained after the bleaching reaction. Given the absence of AP staining in the lens (f, blue arrow), the GFP fluorescence observed through the lens likely originated from the neural retina covered by the pigment epithelium (e, blue arrow).
FIGURE 4
3D imaging analysis by PLAP-CoMBI. (a) Schematic representation of the experimental procedure. (b) Lateral view of a representative embryo injected with Tg(Xtr.tubb2b:ALPP) and analyzed by the CoMBI method. White dotted lines c, d, e, f, and g indicate approximate sectioning planes of block-face images that are shown in (c–g), respectively. Pink arrows indicate alkaline phosphatase (AP) staining ventral to the forebrain (c) and as two ventrolateral patches each in the hindbrain (d, e) and in the spinal cord (f, g). (h–k) Lateral, left frontal, right frontal, and leftposterior views of the 3D image reconstructed from block-face images of the embryo shown in (b). White triangles and white arrows in (b) and (h–k) indicate AP staining in the spinal cord and in the bridging structure located ventral to the forebrain, respectively. (l) Lateral view of a representative embryo injected with Tg(Xla.actc1:ALPP) and analyzed by the CoMBI method. (m–p) Lateral, transverse, left dorsal, and true dorsal views of the 3D image reconstructed from block-face images of the embryo shown in (l). Yellow triangles and arrows indicate AP staining in the somites and heartprimordia, respectively.
