October 15, 1999;
The early expression control of Xepsin by nonaxial and planar posteriorizing signals in Xenopus epidermis.
The control mechanism of the anteroposterior axis specification in Xenopus epidermis
was studied by comparing the expression of a novel anterior
, with that of a panepidermal marker, type I keratin. Xepsin
mRNA, which encodes a novel Xenopus serine protease, is transcribed zygotically with the expression peak in neurula
stages. In normal development, its expression is limited to the anterior
-dorsal portions within epidermis
stages, respectively. In UV-irradiated ventralized embryos (dorsoanterior index, DAI 0 and 1), an expression boundary for Xepsin
is apparently formed within the epidermis
. In contrast, Xepsin
expression was observed throughout the epidermis
in LiCl-treated dorsalized embryos (DAI 10), as seen from an expression pattern indistinguishable from that of type I keratin. These data suggest that posteriorizing signals which suppress the transcription of Xepsin
are present in nonaxial regions and absent in the anterior
. That posteriorizing signals were present in nonaxial regions was also supported by a conjugation experiment in which Xepsin
expression was suppressed in ectodermal explants conjugated with lateral
marginal zone. Moreover, the partly suppressed expression of Xepsin
in the epidermal region of exogastrulae indicates that the signals may travel horizontally within the plane of the epidermis
. We also present data showing that both treatment with retinoic acid and the overexpression of a constitutively active form of a retinoic acid receptor caused the suppression of Xepsin
mRNA transcription, suggesting that anterior
patterning in the central nervous system and in the epidermis
may share common endogenous factors, i.e. , retinoids, in the Xenopus embryo
[+] show captions
FIG. 2. Temporal expression of Xepsin gene during early Xenopus development analyzed using reverse transcriptase-PCR (RT-PCR). Total RNAs (0.4 mg) were used as templates to generate first-strand cDNA. Radiolabeled Xepsin and ODC sequences were amplified from first-strand cDNA and separated on polyacrylamide gels. The numbers refer to embryonic stages. [Stage 2, two-cell; stage 8, morula; stage 9, late blastula; stage 10, early gastrula; stage 11, midgastrula; stage 12.5, late gastrula; stage 14, early neurula; stage 17, midneurula; stage 21, late neurula; stage 28, late tailbud; stage 36, swimming tadpole.] ODC was used as an internal standard control.
FIG. 3. Localization of Xepsin transcript in normal embryos, analyzed by whole-mount in situ hybridization. Stages are 15 (A, B), 18 (G), 20 (C, D), 22 (H, I), and 27 (E, F). (A, C, E) Lateral view. (B) Anterior view. (D, F) Dorsal view. (G) Sections of embryo after whole-mount in situ hybridization. (H) Transverse section. (I) Parasagittal section. The signal was not detected in the neural plate (np), neural tube (nt), notochord (n), somite (s), or cement gland (cg), and Xepsin expression was also absent from the midline of the most dorsal region of the epidermis (black arrowhead) (D, F, H).
FIG. 4. Expression of Xepsin (A, C, E, G, I, K) and epidermal keratin (B, D, F, H, J, K) in axis-perturbed embryos analyzed by whole-mount in situ hybridization. All embryos were allowed to develop until control embryos reached stage 23. (A, B) Normal embryos. (C, D) UV-irradiated embryos (DAI 0). (E, F) UV-irradiated embryos (DAI 1). (G, H) LiCl-treated embryos (DAI 10). (A) Blastopore is to the right. (I, J, K) Exogastrula embryos. Ectodermal half is on the top right. (K) Double in situ hybridization showing Xepsin expression (purple) in the more anteriorly confined domain than keratin (pink).
FIG. 5. Analysis of the Xepsin expression in conjugates. (A) Experimental scheme. Animal caps (AN) and other explants (DMZ, dorsal marginal zone; LMZ, lateral marginal zone; VMZ, ventral marginal zone; and EN, endoderm) were isolated from stage 10.25 embryos (lateral view, left; vegetal view, right). Each explants was incubated either alone or as conjugates with animal cap and harvested at stage 15 for RT-PCR. (B) The expression of Xepsin and other marker genes in conjugates were analyzed by RT-PCR. Total RNAs from one explant equivalent, one conjugate equivalent, or one embryo equivalent were used as templates to generate first-strand cDNA. Epidermal keratin (Epid. Ker.) is specific for epidermis. NCAM is a neural-specific marker. Xnot is a dorsal mesoderm markers. Xvent-1 is expressed in the ventrolateral mesoderm. Xsox17b is a endodermal marker. EF1-a was used as RT-PCR control for RNA recovery. 2RT, sibling control embryo without reverse transcriptase. (C) Radioactivity level of each Xepsin band in B was measured with a laser image analyzer. The Xepsin expression, normalized by the epidermal keratin, is presented as a histogram. One-hundred percent expression of Xepsin was defined as the level seen in animal cap alone.
FIG. 6. Xepsin mRNA expression is suppressed by exogenous RA. (A) Analysis of Xepsin mRNA expression in RA-treated whole embryos. Embryos were treated with 1026 M all-trans-RA for 1.5 h at the early gastrula stage, cultured until the tailbud stage, and processed for whole-mount in situ hybridization with the Xepsin antisense probe. (Top) Untreated sibling control embryo. (Bottom) RA-treated embryo. Anterior is left. (B) RT-PCR analysis of Xepsin expression in whole embryos or explants after RA treatment. Animal caps were isolated from early gastrula embryos and cultured with different concentrations of RA until siblings reached stage 20. (Lane 1) Control caps; (lane 2) 1025M RA-treated caps; (lane 3) 1026M RA-treated caps; (lane 4) 1027M RA-treated caps. Embryo was treated either with (lane 6) or without (lane 5) 1025 MRA until control embryos reached stage 20. Note that NCAM and muscle actin are not expressed in these animal caps. (Lane 7) cDNA preparation except for reverse transcriptase. Total RNAs from one animal cap equivalent or one-fifth equivalent of one embryo were used as template to generate first-strand cDNA. (C) Radioactivity levels of each Xepsin band in B was measured with a laser image analyzer. The Xepsin expression, normalized by the EF1-a, is presented as a histogram. One-hundred percent expression of Xepsin was defined as the level seen in control caps.
FIG. 7. Expression of Xepsin in embryos expressing unilaterally constitutively active xRARa1 (A), dominant negative xRARa1 (B), and in a control embryo (F). (C) Expression of Krox-20 in a dominant negative xRARa1-expressing embryo (C). All embryos were analyzed at stage 17 by whole-mount in situ hybridization. TRDX was used as a lineage marker, showing the injected side. (D) The TRDX luminescence image of A after in situ hybridization. (E) The TRDX luminescence image of B before in situ hybridization. All embryos are shown in dorsal views with the anterior to the left. (A) The injected side is on the bottom.
xepsin (epidermis specific serine protease) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.