Licenciado, Universidad De Buenos Aires (2007)
Doctor of Philosophy, Universidad De Buenos Aires (2013)
The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic ? cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human ? cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing ?-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal ? cells, adult ? cells, and other non-? cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, ? cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human ? cells should advance ?-cell replacement and other therapeutic strategies for diabetes.
View details for DOI 10.1101/gad.342378.120
View details for PubMedID 33446570
The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of human epithelial cells HEK293 and HEK293 T, deficient in cGAS or in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted more relevant in the production of interferon (IFN)-beta and IFN-lambda1 in response to baculovirus infection. In human epithelial cells, IFN-lambda1 production was also induced in a cGAS-independent and DNA-PK-dependent manner. Finally, we demonstrated that these cellular responses towards baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are non-pathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.
View details for DOI 10.1128/JVI.01555-20
View details for PubMedID 32796076
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ?-, ?- and ?-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ?-cell functional maturation, including evidence of fetal ?-cell GLP-1 production and signaling to ?-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
View details for DOI 10.1242/dev.186213
View details for PubMedID 32108026
CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.
View details for DOI 10.1530/REP-19-0517
View details for PubMedID 32240977
In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3?h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21?h with 1 (Cys 1) or 0.1?mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ?3?h with 16?×?106 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ? 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ? 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ? 0.05). The relative abundance of mRNAs coding for INF?, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p???0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ? 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p???0.05). In conclusion, the use of 1?mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.
View details for DOI 10.1016/j.theriogenology.2018.05.017
View details for Web of Science ID 000436055900004
View details for PubMedID 29807255