Arrayed single-gene perturbations identify drivers of human anterior neural tube closure
Curation statements for this article:-
Curated by eLife
eLife Assessment
This study is a fundamental advance in the field of developmental biology and transcriptional regulation that demonstrates the use of hPSC-derived organoids to generate reproducible organoids to study the mechanisms that drive neural tube closure. The work is exceptional in its development of tools to use CRISPR interference to screen for genes that regulate morphogenesis in human PSC organoids. The additional characterization of the role of specific transcription factors in neural tube formation is solid. The work provides both technical advances and new knowledge on human development through embryo models.
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Abstract
Abstract
Genetic studies of human embryonic morphogenesis are constrained by ethical and practical challenges, restricting insights into developmental mechanisms and disorders. Human pluripotent stem cell (hPSC)–derived organoids provide a powerful alternative for the study of embryonic morphogenesis. However, screening for genetic drivers of morphogenesis in vitro has been infeasible due to organoid variability and the high costs of performing scaled tissue-wide single-gene perturbations. By overcoming both these limitations, we developed a platform that integrates reproducible organoid morphogenesis with uniform single-gene perturbations, enabling high-throughput arrayed CRISPR interference (CRISPRi) screening in hPSC-derived organoids. To demonstrate the power of this platform, we screened 77 transcription factors in an organoid model of anterior neurulation to identify ZIC2, SOX11, and ZNF521 as essential regulators of neural tube closure. We discovered that ZIC2 and SOX11 are required for closure, while ZNF521 prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of ZIC2 and SOX11 and an opposing role for ZNF521, suggesting that these transcription factors jointly govern a gene regulatory program driving neural tube closure in the anterior forebrain region. Our single-gene perturbation platform enables high-throughput genetic screening of in vitro models of human embryonic morphogenesis.
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eLife Assessment
This study is a fundamental advance in the field of developmental biology and transcriptional regulation that demonstrates the use of hPSC-derived organoids to generate reproducible organoids to study the mechanisms that drive neural tube closure. The work is exceptional in its development of tools to use CRISPR interference to screen for genes that regulate morphogenesis in human PSC organoids. The additional characterization of the role of specific transcription factors in neural tube formation is solid. The work provides both technical advances and new knowledge on human development through embryo models.
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Reviewer #1 (Public review):
Summary:
This is a wonderful and landmark study in the field of human embryo modeling. It uses patterned human gastruloids and conducts a functional screen on neural tube closure, and identifies positive and negative regulators, and defines the epistasis among them.
Strengths:
The above was achieved following optimization of the micro-pattern-based gastruloid protocol to achieve high efficiency, and then optimized to conduct and deliver CRISPRi without disrupting the protocol. This is a technical tour de force as well as one of the first studies to reveal new knowledge on human development through embryo models, which has not been done before.
The manuscript is very solid and well-written. The figures are clear, elegant, and meaningful. The conclusions are fully supported by the data shown. The methods are …
Reviewer #1 (Public review):
Summary:
This is a wonderful and landmark study in the field of human embryo modeling. It uses patterned human gastruloids and conducts a functional screen on neural tube closure, and identifies positive and negative regulators, and defines the epistasis among them.
Strengths:
The above was achieved following optimization of the micro-pattern-based gastruloid protocol to achieve high efficiency, and then optimized to conduct and deliver CRISPRi without disrupting the protocol. This is a technical tour de force as well as one of the first studies to reveal new knowledge on human development through embryo models, which has not been done before.
The manuscript is very solid and well-written. The figures are clear, elegant, and meaningful. The conclusions are fully supported by the data shown. The methods are well-detailed, which is very important for such a study.
Weaknesses:
This reviewer did not identify any meaningful, major, or minor caveats that need addressing or correcting.
A minor weakness is that one can never find out if the findings in human embryo models can be in vitro revalidated in humans in vivo. This is for obvious and justified ethical reasons. However, the authors acknowledge this point in the section of the manuscript detailing the limitations of their study.
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Reviewer #2 (Public review):
Summary:
This manuscript is a technical report on a new model of early neurogenesis, coupled to a novel platform for genetic screens. The model is more faithful than others published to date, and the screening platform is an advance over existing ones in terms of speed and throughput.
Strengths:
It is novel and useful.
Weaknesses:
The novelty of the results is limited in terms of biology, mainly a proof of concept of the platform and a very good demonstration of the hierarchical interactions of the top regulators of GRNs.
The value of the manuscript could be enhanced in two ways:
(1) by showing its versatility and transforming the level of neural tube to midbrain and hindbrain, and looking at the transcriptional hierarchies there.
(2) by relating the patterning of the organoids to the situation in vivo, in …
Reviewer #2 (Public review):
Summary:
This manuscript is a technical report on a new model of early neurogenesis, coupled to a novel platform for genetic screens. The model is more faithful than others published to date, and the screening platform is an advance over existing ones in terms of speed and throughput.
Strengths:
It is novel and useful.
Weaknesses:
The novelty of the results is limited in terms of biology, mainly a proof of concept of the platform and a very good demonstration of the hierarchical interactions of the top regulators of GRNs.
The value of the manuscript could be enhanced in two ways:
(1) by showing its versatility and transforming the level of neural tube to midbrain and hindbrain, and looking at the transcriptional hierarchies there.
(2) by relating the patterning of the organoids to the situation in vivo, in particular with the information in reference 49. The authors make a statement "To compare our findings with in vivo gene expression patterns, we applied the same approach to published scRNA-seq data from 4-week-old human embryos at the neurula stage" but it would be good to have a more nuanced reference: what stage, what genes are missing, what do they add to the information in that reference?
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