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Dev Biol
2005 Dec 01;2881:294-307. doi: 10.1016/j.ydbio.2005.09.015.
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Subdividing the embryo: a role for Notch signaling during germ layer patterning in Xenopus laevis.
Contakos SP
,
Gaydos CM
,
Pfeil EC
,
McLaughlin KA
.
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The development of all vertebrate embryos requires the establishment of a three-dimensional coordinate system in order to pattern embryonic structures and create the complex shape of the adult organism. During the process of gastrulation, the three primary germ layers are created under the guidance of numerous signaling pathways, allowing cells to communicate during development. Cell-cell communication, mediated by receptors of the Notch family, has been shown to be involved in mediating diverse cellular behaviors during development and has been implicated in the regulation of cell fate decisions in both vertebrate and invertebrate organisms. In order to investigate a role for Notch signaling during boundary formation between the mesoderm and endoderm during gastrulation, we manipulated Notch signaling in gastrula stage embryos and examined gene expression in resultant tissues and organs. Our findings demonstrate a much broader role for Notch signaling during germ layer determination than previously reported in a vertebrate organism. Activation of the Notch pathway, specifically in gastrula stage embryos, results in a dramatic decrease in the expression of genes necessary to create many different types of mesodermal tissues while causing a dramatic expansion of endodermal tissue markers. Conversely, temporally controlled suppression of this pathway results in a loss of endodermal cell types and an expansion of molecular markers of mesoderm. Thus, our data are consistent with and significantly extend the implications of prior observations suggesting roles for Notch signaling during germ layer formation and establish an evolutionarily conserved role for Notch signaling in mediating mesoderm-endoderm boundaries during early vertebrate development.
Fig. 1. Activation of Notch signaling using GRSu(H)VP16 increases expression of markers of endodermal cell types and decreases expression of markers of mesodermal cell types. (A–F) Increased expression of endodermal tissue markers (fibrinogen, endodermin, and vito) was observed in embryos injected with mRNAs encoding GRSu(H)VP16 as described in Materials and methods. Activation of the Notch pathway in gastrula stage embryos resulted in an increase of gene expression of the endodermal tissue marker, fibrinogen, on the injected side of embryos (A) when compared to the uninjected control side of embryos (B). (C–D) Similar effects were observed in GRSu(H)VP16-injected embryos examined for another endodermal tissue marker endodermin. Expansion of the normal region of expression of the early ventralmidgut endodermal marker, vito, was observed in GRSu(H)VP16-injected (DEX) embryos (E) when compared to the sibling-injected (no DEX) control stage-matched embryos (F). (G–N) In sharp contrast, expression of mesodermal tissue markers tbx5 (G–H), nkx2.5 (I–J), lim-1 (K–L), and 12/101 (M–N) decreased or was completely ablated on the injected side of embryos when compared to the uninjected control sides of embryos (or no DEX control embryos, data not shown). In all experiments, mRNAs encoding β-galactosidase were co-injected as a lineage tracer (pink). Embryos were induced with dexamethasone at stages 10–12 and cultured until fixation at stages 22–33. Arrows indicate the injected side of embryos. Most embryos are oriented laterally except panels I–J that include a ventral image shown in inset and ventral views of embryos shown in panels E and F. The white line denotes the midline of the embryo.
Fig. 2. Activation of Notch signaling using GRNotchICD increases expression of markers of endodermal cell types and decreases expression of markers of mesodermal cell types. The Notch pathway was activated by co-injection of mRNAs encoding a temporally inducible GRNotchICD and the lineage tracer β-galactosidase (pink). Injected constructs were induced with dexamethasone at stages 10–12 and cultured until fixation at stages 22–33. Similar to the results seen in GRSu(H)VP16-injected embryos, activation of the Notch pathway via GRNotchICD resulted in an increase in gene expression of the endodermal markers fibrinogen and endodermin (A, C) when compared to the control side of the embryos (B, D). Comparable to patterns observed in GRSu(H)VP16-injected embryos, expression of the mesodermal tissue markers nkx2.5, tbx5, and lim-1 (E, G, I) decreased or were completely eliminated on the GRNotchICD-injected side of embryos when compared to the uninjected control sides (F, H, J). Immunohistochemistry revealed a decrease in expression of a molecular marker of the somites 12/101 (K–L). Arrows indicate the injected side of embryos. All embryos are oriented laterally except panels E–F that contain an inset ventral image. The white line denotes the midline of the embryo.
Fig. 3. Suppression of Notch signaling using GRSu(H)DBM decreases expression of markers of endodermal cell types and increases expression of markers of mesodermal cell types. Unlike what was observed in embryos in which Notch signaling was activated, suppression of Notch signaling via an inducible dominant negative Suppressor of Hairless [GRSu(H)DBM] resulted in decreased expression of endodermal tissue markers and an increase in expression of markers of mesodermal cell types. (A–D) Embryos injected with mRNAs encoding GRSu(H)DBM and induced with dexamethasone at stage 10 displayed a decrease of expression of two endodermal markers, fibrinogen and endodermin (A, C), on the injected side of the embryos when compared to the uninjected control side (B, D). In addition, a dramatic decrease of expression of the early ventralmidgut endodermal marker, vito, was observed in GRSu(H)DBM-injected (DEX) embryos (E) when compared to the sibling-injected (no DEX) stage-matched control embryos (F). In contrast, mesodermal tissue markers tbx5, nkx2.5, lim-1, and 12/101 (G, I, K, M) showed an increase in expression on the injected side of the embryo when compared to the uninjected control side (H, J, L, N). In all experiments, mRNAs encoding β-galactosidase were co-injected as a lineage tracer (pink). Black arrows designate the injected side of embryos, and white lines mark the midline of the embryo. Most embryos are oriented laterally except panels I–J that include a ventral image shown in inset and ventral views of embryos shown in panels E and F.
Fig. 5. Prior to morphogenesis, perturbation of Notch signaling during gastrulation modulates endodermal and mesodermal cell types (A–D). All embryos are oriented dorsally. Embryos were injected on one side as described in Materials and methods with mRNA encoding either GRSu(H)VP16 or GRSu(H)DBM, left untreated until stage 10, and then induced with dexamethasone. In all experiments, mRNAs encoding β-galactosidase were co-injected as a lineage tracer (pink). Embryos were induced with dexamethasone at stage 10 and cultured until fixation at stages 16–17 (for mesoderm marker lim-1) or stages 19–20 (for endoderm marker endodermin [edd]). Arrows indicate the injected side of embryos. (A) Ablation of expression of lim-1 was observed in embryos injected with mRNAs encoding GRSu(H)VP16. (B) In sharp contrast, increased expression of lim-1 was observed in GRSu(H)DBM-injected embryos. (C) Activation of the Notch pathway in gastrula stage embryos resulted in an increase of gene expression of the endodermal tissue marker endodermin (edd). (D) The opposite effect was observed in GRSu(H)DBM-injected embryos where a decrease of edd expression was detected.
Fig. 6. Activation and suppression of Notch signaling result in morphological anomalies of the gut and perturbation of a molecular marker of pronephric duct cells. (A–C) Ventral views of embryos injected with inducible mRNAs encoding dominant negative Su(H) [GRSu(H)DBM] and induced at stage 10 to suppress Notch signaling. Injected tadpoles displayed miscoiling of the gut (B, C) in comparison to the normal coiling pattern observed in injected embryos cultured in the absence of DEX (no DEX) (A). (D–E) Ventral views with lateral inset images of embryos injected with mRNA encoding Su(H) [GRSu(H)VP16] and induced at stage 10 to activate Notch signaling. Panel E illustrates gut malformation observed in embryos exposed to DEX compared to normal phenotypes observed in injected no DEX sibling control embryos (D). (F–G) Lateral image of embryo injected with mRNA encoding Su(H) [GRSu(H)VP16] and induced at stage 10 to activate Notch signaling. Activation of Notch signaling substantially decreased expression of a late marker for pronephric duct cells 4A6 (F). Left bottom panels show schematic tracing of the observed 4A6 expression pattern. Right bottom panel shows a magnification of boxed areas.
