Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed I f or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, I f in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4 ؊/؊ mice, cardiac cells with ''primitive'' pacemaker action potentials could be found. However, cardiac cells with ''mature'' pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node. Four genes encoding hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels have been identified and functionally expressed. All four HCN channels carry an inward current with the typical features of a current termed I h in the brain and I f in the heart (1-4). These currents have been implicated in a wide range of physiological functions including pacemaking activity of spontaneously firing brain and heart cells, control of resting membrane potential, response to sour taste, neuronal plasticity, and dendritic integration (reviewed in refs. 5-7).HCN4 is the predominant HCN transcript in the adult sinoatrial node (8-11). I f and HCN transcripts have also been identified in mouse embryonic hearts with HCN4 being the prevalent type at early stages (12). Little is known about the specific contributions of the individual HCN isoforms to the function of the heart. One of the main, but still controversially discussed hypotheses is that I f is one of the major currents contributing to the spontaneous diastolic depolarization of pacemaker cells and thereby to sinus node rhythm (5, 13). Furthermore, the -adrenergic up-regulation of sinus node rhythm has been attributed to the binding of cAMP to HCN channels resulting in an enhanced I f . However, it has been shown that diastolic depolarization is generated by multiple ionic currents with complex interactions (reviewed in ref. 14). The importance of I f has also been questioned because activation thresholds of I f vary and conflicting results were obtained with I f blockers (15-18).Direct evidence demonstrating the...
Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.
Pax6 is a key regulator of eye development in vertebrates and invertebrates, and heterozygous loss-of-function mutations of the mouse Pax6 gene result in the Small eye phenotype, in which a small lens is a constant feature. To provide an understanding of the mechanisms underlying this haploinsufficient phenotype, we evaluated in Pax6 heterozygous mice the effects of reduced Pax6 gene dosage on the activity of other transcription factors regulating eye formation. We found that Six3 expression was specifically reduced in lenses of Pax6 heterozygous mouse embryos. Interactions between orthologous genes from the Pax and Six families have been identified in Drosophila and vertebrate species, and we examined the control of Pax6 and Six3 gene expression in the developing mouse lens. Using in vitro and transgenic approaches, we found that either transcription factor binds regulatory sequences from the counterpart gene and that both genes mutually activate their expression. These studies define a functional relationship in the lens in which Six3 expression is dosage-dependent on Pax6 and where, conversely, Six3 activates Pax6. Accordingly, we show a rescue of the Pax6 haploinsufficient lens phenotype after lensspecific expression of Six3 in transgenic mice. This phenotypic rescue was accompanied by cell proliferation and activation of the platelet-derived growth factor ␣-R͞cyclin D1 signaling pathway. Our findings thus provide a mechanism implicating gene regulatory interactions between Pax6 and Six3 in the tissue-specific defects found in Pax6 heterozygous mice.homeodomain proteins ͉ transcription factors ͉ eye development
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