Dissecting the Segregation of Anterior Mesendoderm Lineages at the Single-Cell Level in Zebrafish

Core Concepts

Anterior endoderm cells originate from prechordal plate progenitors, and their segregation is regulated by a balance of Nodal signaling, chromatin remodeling, and the collaborative actions of transcriptional regulators Gsc and Ripply1.

Abstract

The study investigates the mechanisms underlying the segregation of anterior endoderm (Endo) and prechordal plate (PP) lineages from common mesendoderm progenitors in zebrafish.

Key findings:

1. Single-cell trajectory analyses and live imaging reveal that anterior Endo cells are derived from PP progenitors.
2. Perturbation of the Nodal-Lefty signaling system affects the balance between PP and Endo lineages, with higher Nodal activity promoting PP fate and lower Nodal favoring Endo.
3. Chromatin remodeling factors, such as the SWI/SNF complex component Srcap, play a role in regulating the differential chromatin accessibility states between PP and Endo, influencing the expression of key lineage regulators.
4. The transcriptional repressors Gsc and Ripply1 cooperatively suppress Endo specification by directly binding to the regulatory regions of endoderm genes like sox17 and sox32.

The study provides a comprehensive understanding of how Nodal signaling, chromatin remodeling, and lineage-specific transcriptional regulators orchestrate the segregation of closely related mesendoderm cell fates in zebrafish development.

Stats

"Nodal signaling activity was significantly enhanced in lft1 mutants, while decreased in ndr1 morphants compared to wild-type embryos."
"The proportions of both PP and Endo cells increased in lft1-mutant explants, while decreased in ndr1-morphant explants compared to wild-type explants."
"Genes related to 'chromatin organization' were significantly enriched in the differentially expressed genes of PP cells when comparing lft1 mutant or ndr1 knockdown samples to wild-type samples."

Quotes

"Nodal signaling levels play a conserved role in determining the cell fate specification, which influences the differentiation towards either Endo or PP lineages."
"Collectively, these regulators collaborate to finely regulate the segregation of mesendoderm cell fates."
"Our findings revealed the origin of anterior Endo from PP progenitors and unveiled a correlation between subtle differences of Nodal signaling levels and lineage commitment."

Key Insights Distilled From

Ripply1 and Gsc collectively suppress anterior endoderm differentiation from prechordal plate progenitors

by Cheng,T., Li... at www.biorxiv.org 05-29-2023

https://www.biorxiv.org/content/10.1101/2023.05.29.542713v2

Deeper Inquiries

How do the specific interactions between Fgf signaling and factors like Gsc, Ripply1, Sox32 regulate the fate diversification of PP and Endo lineages?

Fgf signaling plays a crucial role in the regulation of mesodermal and endodermal cell fate diversification, particularly in the context of the prechordal plate (PP) and anterior endoderm (Endo) lineages. The interaction between Fgf signaling and transcriptional regulators such as Gsc, Ripply1, and Sox32 is essential for determining the fate of these lineages.

Fgf Signaling and Cell Fate Specification: Fgf signaling has been shown to inhibit endoderm specification while promoting mesodermal characteristics. This is particularly relevant in the lateral margin of the zebrafish embryo, where Fgf signaling interacts with Nodal to regulate the balance between mesoderm and endoderm differentiation. The presence of Fgf signaling can lead to a decrease in the number of Endo cells derived from PP progenitors, thereby influencing the fate diversification.

Role of Gsc: Gsc acts as a transcriptional repressor that suppresses endodermal specification by binding to the promoter regions of key endodermal markers such as Sox17. By modulating the expression of Gsc in response to Fgf signaling, the balance between PP and Endo cell fates can be adjusted. High levels of Gsc expression, induced by Fgf signaling, can promote PP fate while inhibiting Endo specification.

Ripply1's Function: Ripply1 collaborates with Gsc to further repress endodermal fate. The interaction between Ripply1 and Gsc enhances the suppression of Sox32 and Sox17, which are critical for endoderm specification. This cooperative action ensures that cells remain in the PP lineage rather than transitioning to the Endo lineage.

Sox32's Role: Sox32 is a key transcription factor that promotes endodermal fate. The expression of Sox32 is negatively regulated by both Gsc and Ripply1. The interplay between these factors and Fgf signaling creates a regulatory network that finely tunes the balance between PP and Endo cell fates, ensuring proper lineage diversification during gastrulation.

In summary, the specific interactions between Fgf signaling and transcriptional regulators like Gsc, Ripply1, and Sox32 are pivotal in determining the fate of PP and Endo lineages. By modulating the expression and activity of these factors, Fgf signaling influences the delicate balance between mesodermal and endodermal differentiation.

What are the potential mechanisms that trigger the transcriptional difference of chromatin remodeling factors like Srcap in PP and Endo cells in response to Nodal signaling?

The transcriptional differences of chromatin remodeling factors such as Srcap in PP and Endo cells are likely influenced by several mechanisms in response to Nodal signaling:

Nodal Signaling Levels: Nodal signaling is known to induce chromatin remodeling and can enhance the transcription of target genes. The differential levels of Nodal signaling in PP and Endo cells may lead to distinct transcriptional responses. Higher Nodal activity in PP cells could promote the expression of Srcap, while lower levels in Endo cells may result in reduced Srcap expression.

Chromatin Accessibility: The chromatin state in PP and Endo cells is likely influenced by Nodal signaling, which can alter chromatin accessibility. The SWI/SNF complex, of which Srcap is a member, is involved in remodeling chromatin to facilitate transcription. Variations in Nodal signaling may lead to differential chromatin accessibility at the Srcap locus, thereby affecting its transcriptional activation.

Epigenetic Modifications: Nodal signaling may also induce specific epigenetic modifications, such as histone acetylation or methylation, that can influence the expression of chromatin remodeling factors. These modifications can create a permissive or repressive environment for the transcription of Srcap in PP and Endo cells.

Transcription Factor Interactions: The presence of other transcription factors that interact with Nodal signaling could also play a role in modulating Srcap expression. For instance, factors that are activated by Nodal signaling may bind to regulatory elements near the Srcap gene, enhancing its transcription in PP cells while being less effective in Endo cells.

Feedback Mechanisms: There may be feedback loops involving Srcap and other transcriptional regulators that respond to Nodal signaling. For example, Srcap could influence the expression of other genes that, in turn, modulate Nodal signaling levels, creating a dynamic regulatory network that maintains the transcriptional differences between PP and Endo cells.

In conclusion, the transcriptional differences of chromatin remodeling factors like Srcap in response to Nodal signaling are likely triggered by a combination of Nodal signaling levels, chromatin accessibility, epigenetic modifications, transcription factor interactions, and feedback mechanisms. These factors collectively contribute to the distinct cellular identities of PP and Endo lineages during development.

Besides Gsc and Ripply1, are there any other unidentified transcriptional repressors involved in the cooperative regulation of PP and Endo specification?

Yes, besides Gsc and Ripply1, there are likely other unidentified transcriptional repressors that play a role in the cooperative regulation of PP and Endo specification. The complexity of gene regulation during embryonic development suggests that multiple layers of transcriptional control are involved in determining cell fate. Here are some potential avenues for identifying additional repressors:

Gene Expression Profiling: High-throughput sequencing techniques, such as single-cell RNA-seq, can be employed to identify differentially expressed genes in PP and Endo cells. This approach may reveal novel transcriptional repressors that are enriched in one lineage over the other, indicating their potential roles in regulating cell fate.

Functional Genomics: Loss-of-function studies using CRISPR/Cas9 or morpholino knockdown techniques can help identify additional transcriptional repressors. By systematically knocking down candidate genes that are co-expressed with Gsc and Ripply1, researchers can assess their impact on PP and Endo specification.

Chromatin Accessibility Studies: Integrating ATAC-seq data with RNA-seq can provide insights into the regulatory landscape of PP and Endo cells. Identifying regions of differential chromatin accessibility may point to transcriptional repressors that bind to these regions and regulate the expression of key lineage-specific genes.

Network Analysis: Computational approaches can be used to construct gene regulatory networks based on known interactions and expression data. This can help predict additional transcriptional repressors that may cooperate with Gsc and Ripply1 in regulating PP and Endo specification.

Comparative Studies: Investigating other model organisms or developmental systems may provide insights into conserved transcriptional repressors that play similar roles in mesendoderm specification. This comparative approach can help identify candidates that warrant further investigation in zebrafish.

Previous Literature: Existing studies have suggested the involvement of other transcriptional repressors, such as Osr1, which has been shown to be enriched in PP cells. The potential for redundancy among transcriptional repressors means that multiple factors may work together to prevent endoderm specification.

In summary, while Gsc and Ripply1 are key players in the regulation of PP and Endo specification, there are likely other unidentified transcriptional repressors that contribute to this process. Future research employing various genomic and functional approaches will be essential to uncover these additional factors and elucidate their roles in mesendodermal cell fate regulation.