Intracardial embryonic delivery of developmental modifiers in utero.

Our knowledge of organ ontogeny is largely based on loss-of-function (knockout) or gain-of-function (transgenesis) approaches. However, developmental modulators such as proteins, mRNAs, microRNAs(miRNAs), small interfering RNAs, and other small molecules may complement the above DNA-modifying technologies in a much more direct way. Unfortunately, their use is often limited by the ability of these compounds to cross the placenta and reach physiologically relevant concentrations when administered systemically to the mother. The design of safe and effective techniques to deliver these compounds into the embryo is therefore an area of great scientific potential. In this article we report a new method for introducing developmental modulators into murine embryos by means of direct injection into the heart. Unlike other reported methods that require surgical exposure of the uterus, our percutaneous ultrasound-guided approach allows for the intracardial injection of mouse embryos as early as embryonic day 10.5 (e10.5) and throughout gestation in a minimally invasive manner that largely preserves embryo viability. This system offers a critical advantage over in vitro settings because the effects of any given treatment can be observed without disturbing the native environment of the developing organ. Procedures are described for the delivery and detection of transducible proteins as well as morpholinos designed to block the expression of specific miRNAs within the living embryo.

Antisense miR-7 impairs insulin expression in developing pancreas and in cultured pancreatic buds.

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased ß-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to ß-cell death and generation of ß-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a ß-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.

TAT-mediated transduction of MafA protein in utero results in enhanced pancreatic insulin expression and changes in islet morphology.

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero.