Regulation of Endomesoderm Segregation

In this study, sea urchins embryos were used to study how the embryonic endomesoderm segregates into endoderm and mesoderm in deuterostomes.

In future mesoderm, Notch signaling inhibits expression of the key endoderm transcription factor: Hox11/13b. In future endoderm, Hox11/13b activates a regulatory circuit that maintains transcription of the canonical Wnt (cWnt) ligand: wnt1 , which reinforces the endoderm state. To further reinforce the segregation of endoderm and mesoderm, Notch signaling directs the export of the essential β-catenin transcriptional coactivator: TCF, out of the nuclei of mesodermal cells. This renders the mesoderm resistant to cWnt signals and an endoderm fate. (1)

The proposed mechanism of endomesoderm segregation relies heavily on the interplay of the Wnt and Delta-Notch signaling pathways. The two following videos are quick reviews each pathway.

Wnt Signaling Pathway (2)

Delta-Notch Signaling Pathway (3)

Introduction: Mechanism of Endomesoderm Segregation

Figure 1: In the veg2 cells (striped yellow, endomesoderm), early cWnt activates endoderm and mesoderm specification. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

Endomesoderm segregation in deuterostomes is not very well understood. It is known that Notch signaling regulates the process, but is unclear how it does so.

In sea urchins, and other deuterostomes, at the blastula stage, the embryo is enriched asymmetrically in cWnt signaling effector nuclear β-catenin, which initiates endomesoderm specification through 3 major ways.

(1) At the blastula stage nuclear β-catenin establishes an early endoderm regulatory state in a tier of vegetal blastomeres (the veg2 tier, striped yellow cells in Figure 1) at cleavage stages. All the cells of this lineage will differentiate into endoderm unless signaled otherwise.

(2) In micromere descendants located immediately adjacent to the veg2 tier (red cells in Figure 1), nuclear β-catenin induces expression of the ligand Delta. Delta activates mesoderm gene expression in adjacent veg2 blastomeres by signaling through the Notch receptor.

(3) cWnt makes the adjacent veg2 cells competent to receive the Delta signal. The Delta signal initiates mesoderm differentiation in the inner veg2 cells, while the endoderm regulatory state persists in the outer veg2 cells. (1)

Figure 2: The descendants of veg2 cells differentiate to form outer (yellow, endoderm) and inner (green, mesoderm) rings. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

By the hatched blastula stage, the progeny have formed outer (endoderm precursors) and inner (mesoderm precursors) rings of cells, and differentiation is reinforced through 3 major ways.

(1) The inner veg2 progeny (green cells in Figure 2), adjacent to the Delta-expressing micromere progeny (red cells in Figure 2) continue transducing Delta and continue expressing mesoderm markers.

(2) Meanwhile, the endoderm regulatory state persists in the outer veg2 progeny (yellow cells in Figure 2) These cells detect the transcripts encoded by the endoderm regulatory genes, and begin expressing endoderm markers.

(3) In inner veg2 progeny, Notch inhibits expression of the endoderm markers foxa, blimp1b, and dac. (1)

At the mesenchyme blastula stage (6-8 hours later), nuclear β-catenin (which was previously distributed uniformly through the endomesoderm and is required for the maintenance of the endoderm state) is down-regulated in the inner veg2 progeny through a process requiring Notch. (1)

The authors set out to answer 3 major questions that are important in understanding the above mechanism.

How does Notch restrict endoderm fate to a subset of the endomesoderm cells? (What is the target of Notch inhibition?)

Notch deficient embryos were used to systematically assess the expression of each gene in the early endoderm gene regulatory network (GRN). The authors hypothesized that the GRN is initially induced by cWnt signaling, and that Notch inhibits a downstream element of the GRN in the mesoderm precursors.

Notch was knocked down to determine the target of Notch inhibition. Without Notch signaling, the transcripts of the endoderm markers hox11/13b, brachyury, foxa, and blimp1 accumulated abnormally in inner veg2 progeny (mesoderm precursors). Since the transcripts identified represent only a part of the GRN, Notch must inhibit the GRN at a downstream position that allows upstream cWnt signaling to persist. Notch must inhibit an intermediate GRN transcription factor that has already been activated by cWnt signaling. (1)

Figure 3: In wild-type early mesenchyme blastulae (Control), endodermal transcripts brachyury (bra), foxa, and blimp1b are localized to endodermal precursors (C to E) (corresponding to the yellow cells of Figure 2 and visualized by the pink fluorescence), and are down-regulated in mesodermal precursors (indicated by the white arrow and corresponding to the green cells of Figure 2). When Hox11/13b is knocked down (Hox11/13b MASO), brachyury (bra), foxa, and blimp1b are down-regulated everywhere in the endomesoderm (G to I), suggesting that these genes are Hox11/13b target genes. When Notch is knocked down (Notch MASO), brachyury (bra), foxa, and blimp1b are not down-regulated in mesoderm precursors and thus are expressed everywhere in the endomesoderm (K to M), but wild-type expression is recovered when Hox11/13b is knocked out in addition to Notch (Notch + Hox11/13b MASOs) (O to Q). Hox11/13b mRNA is up-regulated in Hox11/13bmorphants (B, F and N) because Hox11/13b represses its own transcription. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

First brachyury, foxa, and blimp1 were determined to be Hox11/13b target genes. The knockdown of Hox11/13b resulted in the down-regulation of brachyury, foxa, and blimp1 transcripts. (Figure 3, C to E compared to G to I). Thus it was hypothesized that Notch initiates endoderm restriction by suppressing hox11/13b expression in the mesoderm precursors. This hypothesis was tested by two experiments.

(1) When Notch was knocked down, brachyury, foxa, and blimp1 transcripts accumulated ectopically in mesoderm precursors (Figure 3, K to M). However. the knockdown of Hox11/13b restored wild-type function in Notch deficient blastulae (Figure 3, O to Q), suggesting that the down-regulation of Hox11/13b is required for wild-type expression.

(2) In an experiment that knocked down both Notch and any one of Brachyury, FoxA, or Blimp1b, the other Notch-suppressed genes still accumulated ectopically in inner veg2 progeny. None of these genes had regulatory effects. This experiment confirmed that Hox11/13b is the source of regulation. (1)

Thus it was confirmed that Notch initially suppresses mesodermal accumulation of the endoderm transcription factor Hox11/13b, preventing ectopic activation of the Hox11/13b-dependent regulatory circuit, consisting of Brachyury, FoxA, and Blimp1b.

Note. All of the gene knockdowns were accomplished through MASO (Morpholino-substituted antisense oligonucleotide) sequences: treatment with morpholinos.

How does Notch inhibit endomesoderm-inducing cWnt in mesoderm precursors?

Figure 4:In wild-type blastulae (Control), wnt1 is localized to endoderm precursors and is down-regulated in mesoderm precursors. When Notch is knocked down (Notch MASO), wnt1 is not down-regulated in mesoderm precursors, and wnt1 is expressed throughout the endomesoderm. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

cWnt induces endomesoderm formation, and therefore must be cleared from mesoderm precursors to allow these cells to complete differentiation. Since Notch inhibits the early endoderm gene regulatory network downstream of cWnt signaling, cWnt must be cleared from mesoderm precursors in a different manner. It was hypothesized that Notch clears nuclear β-catenin (the key component of cWnt signaling) from mesoderm precursors by inhibiting expression of the ligand wnt1 (which is responsible for stimulating nuclear β-catenin activity ). wnt1 mRNA is initially detected throughout the veg2 endomesoderm (Figure 4, A to B), but is down-regulated by Notch in the mesoderm, as knockdown of Notch results in ectopic expression of wnt1 in the mesoderm (Figure 4, C to D). (1)

Are these two Notch-dependent events mechanistically linked?

Figure 5: In wild type blastula (Control, F), wnt1 is localized to the pre-endodermal cells, visualized by the darker purple coloration. Notch is necessary to restrict wnt1 to the endoderm precursors; when it's knocked down (Notch MASO, I), wnt1 is expressed throughout the endomesoderm. In addition, knockdown of Hox11/13b (Hox11/13b MASO, G) and Brachyury (Brachyury MASO, H) resulted in no wnt1 expression, suggesting Hox11/13b and Brachyury are also necessary for normal wnt1 mRNA expression. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

Because Notch restricts wnt1 expression to the endoderm 6 to 8 hours after Notch confines Hox11/13b expression to the endoderm, the linkage of these events were assessed. wnt1 expression requires Hox11/13b and its target Brachyury (Figure 5, F to K). Therefore, by inhibiting mesodermal hox11/13b expression at the hatching blastula stage, Notch indirectly restricts wnt1 accumulation to the endoderm 6-8 hours later, where it maintains cWnt activity, reinforcing the endoderm state. (1)

Stabilization of the endoderm and mesoderm states

Figure 6: Endodermal expression of hox11/13b, brachyury (bra), foxa, and blimp1b mRNA is reduced in Wnt1 morphants. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

Because the endodermal gene regulatory network (GRN) depends on upstream cWnt signaling, it was hypothesized that ligands that activate cWnt signaling (such as Wnt1) stabilize the endoderm regulatory state by promoting expression of GRN transcripts. In support of this, when Wnt1 is knocked down (Figure 6, L to S), the endodermal regulatory state transcripts hox11/13b, brachyury, foxa, and blimp1 are down regulated. (1)

Figure 7: In wild type blastula (Control), Notch down-regulates nuclear TCF in mesoderm, indicated by the white bracket (A to C). Without Notch (Notch MASO, D to F) or NLK (NLK MASO, G-I), nuclear TCF persists in the mesoderm. Note. Adapted from “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos,” by A. J. Sethi et al., 2012, Science, 335, p. 590-593. Copyright 2012 by AAAS.

Delta-Notch signaling also inhibits cWnt activity in mesoderm by activating Nemo-like kinase (NLK) activity in inner veg2 progeny. NLK is a MAP kinase that modulates cWnt signaling by phosphorylating nuclear β-catenin’s essential transcription coactivator, TCF, and promoting its export out of the nucleus. TCF protein was detected in all nuclei until the late mesenchyme blastula stage. Just before gastrulation, TCF clears specifically from mesoderm nuclei in a Notch and NLK dependent manner (Figure 7, A to I). When Notch or NLK are knocked down TCF does not clear from mesoderm nuclei. (1)

Review of Paper
The visualizations of signal and marker localization in the paper were especially effective. In the introduction, the authors showed us where the endoderm and mesoderm cells are located at the blastula stage, and in the experiments that followed, they showed where the different endoderm and mesoderm markers localized in reference to these locations. This was an effective method of portraying the signaling that differentiated the mesoderm from the endoderm. The authors also set out a proposed mechanism in the introduction, and then in the main body expanded on the steps of this mechanism and filled in the previously unknown signaling process responsible. The authors could have stopped after they had discovered that Notch inhibits the Hox11/13b-dependent regulatory circuit in the mesoderm, and then 6 to 8 hours later, restricts wnt1 to the endoderm. Instead they explored further by asking if these two events were mechanistically linked. By doing this, the authors effectively elucidated the important steps of endomesoderm segregation.

A weakness of this paper was the conclusion that knockdown of Hox11/13b restored wild-type function in Notch deficient blastulae. Through observation of Figure 3 (O to Q), it does not appear that knockdown of Hox11/13b restores wild-type function. Knockdown of Hox11/13b restores partial wild-type function at best, but the authors may have been attempting to emphasize their point. This discrepancy highlights another weakness of the paper: all the evidence is visual and subject to interpretation, and there is no real quantitative evidence. Also, the authors did not adequately explain the link between the Notch directed nuclear export of TCF and the rest of the signaling mechanism, so the section on TCF felt more like a loose end rather than an important component of the paper. To be fair, the strengths of the paper outweighed its weaknesses, and overall it was an informative and interesting read.

References
1. Sethi, Aditya J., Radhika M. Wikramanayake, Robert C. Angerer, Ryan C. Range, and Lynne M. Angerer, “Sequential Signaling Crosstalk Regulates Endomesoderm Segregation in Sea Urchin Embryos.” Science, 2012. 335: p. 590-593.
2. WNT Signaling Pathway [Video file]. Retrieved from http://www.youtube.com/watch?v=X4PAMOTDC6M.
3. Delta Notch [Video file]. Retrieved from http://www.youtube.com/watch?v=-PTDq-vmIT4&feature=related.

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