We have previously shown that the MED-1,2 divergent GATA factors act apparently zygotically to specify the fates of the MS (mesoderm) and E (endoderm) sister cells, born at the 7-cell stage of C. elegans embryogenesis. In the E cell, MED-1,2 activate transcription of the endoderm-promoting end-1 and end-3 genes. We demonstrate by in situ hybridization that med transcripts accumulate both in the EMS cell and in the maternal germline in a SKN-1-dependent manner. Removal of zygotic med function alone results in a weakly impenetrant loss of endoderm. However, med-1,2(-) embryos made by mothers in which germline med transcripts have been abrogated by transgene cosuppression fail to make endoderm 50% of the time, similar to the phenotype seen by RNAi. We also find that reduction of Med or End activity results in aberrant numbers of intestinal cells in embryos that make endoderm. We further show that regulation of the paralogous end-1 and end-3 genes consists of both shared and distinct inputs, and that END-3 activates end-1 expression. Our data thus reveal three new properties of C. elegans endoderm specification: both maternal and zygotic activities of the med genes act to specify endoderm, defects in endoderm specification also result in defects in gut cell number, and activation of the paralogous end-1 and end-3 genes differs qualitatively in the relative contributions of their upstream regulators.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs—and consequently the profile of individual membrane currents active during that action potential—differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to “normalize” the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4–6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near −80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen–Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.