Biology subjects

Nieto, M. A.

Publications and source records attributed to Nieto, M. A..

4 recordsLinked to original sources

bioRxiv2024

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

developmental biology↗

bioRxiv2024

Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

Uveal melanoma (UM) can remain in clinical dormancy for decades only to later produce lethal metastases. Using Gq/11mut/BAP1wt UM xenograft models and human metastatic samples, we identified NR2F1 as a key inducer of UM disseminated cancer cell (DCC) dormancy. Dormant UM DCCs upregulate NR2F1, neural crest genes and, along with suppression of proliferation programs, NR2F1 silences YAP1/TEAD1 transcription by altering histone H3 activation marks. YAP1 can reciprocally repress NR2F1, but inhibiting Gq/11 signaling or activating NR2F1 can arrest UM growth. NR2F1 knockout led to dormant DCC awakening and liver metastatic growth. NR2F1 and YAP1 inverse expression was confirmed in human livers carrying UM solitary, small DCC clusters as well as large metastases. Intriguingly, RNA-seq and Cut&Run analysis revealed that NR2F1 short-circuits oncogene signaling by repressing multiple G-protein signaling components. Our work provides previously unrecognized mechanistic insight into UM DCC dormancy and potential pathways for interception. Statement of significanceNR2F1 epigenetically suppresses genes associated with G-protein signaling, cell cycle, and YAP1/TEAD1 pathways, inducing dormancy in uveal melanoma (UM) disseminated cancer cells. This study unveils novel markers for UM dormancy and reactivation, positioning NR2F1 as a promising target for intercepting residual and UM metastatic disease.

cancer biology↗

bioRxiv2021

The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation

The pronephros is the first renal structure in the embryo, arising after mesenchymal to epithelial transition (MET) of the intermediate mesoderm, where Pax2 induces epithelialisation of the mesenchyme. Here we show that, in the early embryo, Snail1 directly represses Pax2 transcription maintaining the intermediate mesoderm in an undifferentiated state. Reciprocally, Pax2 directly represses Snail1 expression to induce MET upon receiving differentiation signals. We also show that BMP7 acts as one such signal by downregulating Snail1 and upregulating Pax2 expression. This, together with the Snail1/Pax2 reciprocal repression, establishes a regulatory loop in a defined region along the anteroposterior axis, the bistability domain within the transition zone, where differentiation of the neural tube and the somites are known to occur. Thus, we show that the antagonism between Snail1 and Pax2 determines the epithelial/mesenchymal state during the differentiation of the intermediate mesoderm and propose that the bistability zone extends to the intermediate mesoderm, synchronizing the differentiation of tissues aligned along the mediolateral embryonic axis. SummaryThe antagonism between Snail and Pax2 factors in the embryonic differentiation zone, tightly regulates the timing of mesodermal epithelialisation

developmental biology↗

bioRxiv2019

The unjamming transition is distinct from the epithelial-to-mesenchymal transition.

Every organ surface and body cavity is lined by a confluent collective of epithelial cells. In homeostatic circumstances the epithelial collective remains effectively solid-like and sedentary. But during morphogenesis, remodeling or repair, as well as during malignant invasion or metastasis, the epithelial collective becomes fluid-like and migratory1-4. This conversion from sedentary to migratory behavior has traditionally been understood as a manifestation of the epithelial-to-mesenchymal transition (EMT) or the partial EMT (pEMT)5-8. However, in certain contexts this conversion has been attributed to the recently discovered unjamming transition (UJT), in which epithelial cells move collectively and cooperatively9-11. UJT and pEMT share certain aspects of collective cellular migration, but the extent to which these processes are distinct, overlapping or perhaps even identical has remained undefined. Using the confluent layer of well-differentiated primary human bronchial epithelial (HBE) cells, here we triggered UJT by exposing the sedentary layer to mechanical compression9-12. Cells thereafter migrated cooperatively, aligned into packs locally, and elongated systematically. Nevertheless, cell-cell junctions, apico-basal polarity, and barrier function remained intact in response, and mesenchymal markers remained unapparent. As such, pEMT was not evident. When we triggered pEMT and associated cellular migration by exposing the sedentary layer to TGF-{beta}1, metrics of UJT versus pEMT diverged. To account for these striking physical observations a new mathematical model attributes the effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Together, these findings establish that UJT is sufficient to account for vigorous epithelial layer migration even in the absence of pEMT. Distinct gateways to cellular migration therefore become apparent - UJT as it might apply to migration of epithelial sheets, and EMT/pEMT as it might apply to migration of mesenchymal cells on a solitary or collective basis, activated during development, remodeling, repair or tumor invasion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, living tissue is therefore seen to comprise an active engineering material whose modules for plasticity, self-repair and regeneration, are far richer than had been previously appreciated.

biophysics↗