Meagan N. Esbin

The human placenta is built by trophoblast cells that fuse together, secrete hormones, and invade the uterus, and defects in these processes contribute to pregnancy disorders such as preeclampsia. Because cell-cell fusion and hormone secretion are inherently non-cell-autonomous processes, their regulators have remained inaccessible to conventional pooled CRISPR screens. Here, we developed an arrayed CRISPR screen in fusogenic BeWo trophoblasts that simultaneously quantifies fusion and hCG secretion across 412 gene perturbations. The screen revealed that these two hallmark functions of trophoblast differentiation are genetically separable. We characterized the strongest novel hit, ZBTB10, in trophoblast stem cells, organoids, and placental tissue and find that ZBTB10 is an essential regulator of human trophoblast differentiation. ZBTB10 is required for invasive extravillous trophoblast differentiation and supports syncytiotrophoblast maturation, establishing it as a cross-lineage regulator that both activates and represses distinct trophoblast fate programs. Together, these findings provide a genetic platform and phenotypic dissection of how regulatory networks control human placental development.

The human placenta is built by trophoblast cells that fuse together, secrete hormones, and invade the uterus, and defects in these processes contribute to pregnancy disorders such as preeclampsia. Because cell-cell fusion and hormone secretion are inherently non-cell-autonomous processes, their regulators have remained inaccessible to conventional pooled CRISPR screens. Here, we developed an arrayed CRISPR screen in fusogenic BeWo trophoblasts that simultaneously quantifies fusion and hCG secretion across 412 gene perturbations. The screen revealed that these two hallmark functions of trophoblast differentiation are genetically separable. We characterized the strongest novel hit, ZBTB10, in trophoblast stem cells, organoids, and placental tissue and find that ZBTB10 is an essential regulator of human trophoblast differentiation. ZBTB10 is required for invasive extravillous trophoblast differentiation and supports syncytiotrophoblast maturation, establishing it as a cross-lineage regulator that both activates and represses distinct trophoblast fate programs. Together, these findings provide a genetic platform and phenotypic dissection of how regulatory networks control human placental development.

During human development, a temporary organ is formed, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure lined by syncytialized trophoblasts, a specialized cell type that forms via cell-cell fusion of underlying progenitor cells. Genetic and functional studies have characterized the membrane protein fusogens Syncytin-1 and Syncytin-2, both of which are necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression have been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we found that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. Although TFEB appears to play a critical role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting a previously uncharacterized role for TFEB in controlling the expression of human syncytins.

During human development, a subset of differentiating fetal cells form a temporary organ, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure which arises via cell-cell fusion of underlying fetal trophoblast stem cells. Genetic and functional studies have characterized the membrane protein fusogens, Syncytin-1 and Syncytin-2, that are both necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression has been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we find that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. While TFEB appears to play an important role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting that TFEB plays an alternative role in placental cells.

As scientific projects and labs benefit from increasingly interdisciplinary expertise, students and trainees find themselves navigating a myriad of academic spaces, each with its own workplace culture and demographics. A clear example is the interdisciplinary field of optics and biological microscopy which bridges biology, physics, and engineering. While Biology PhDs are now >50% women, men in physics and engineering fields still significantly outnumber women, resulting in an imbalance of gender representation among microscopists and other "tool innovators" in the interdisciplinary field of biological microscopy and biomedical optics. In addition to the cultural and cognitive whiplash that results from disparate representation between fields such as Biology, Engineering, and Physics, indifference from institutional leaders to implement equity-focused initiatives further contributes to cultures of exclusion, rather than belonging, for women. Here we elaborate on the motivation, structure, and outcomes of building a specific affinity-based bootcamp as an intervention to create an inclusive, welcoming learning environment for women in optics. Considering the presence of nonbinary, trans, and other gender minoritized scientists, we recognize that women are not the only gender group underrepresented in biological microscopy and biomedical optics; still, we focus our attention on women in this specific intervention to improve gender parity in biological microscopy and biomedical optics. We hope that these strategies exemplify concrete paths forward for increasing belonging in interdisciplinary fields, a key step towards improving and diversifying graduate education.

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP₆) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r² = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the “gold standard” test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the “gold-standard” CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.