Gerald M. Kidder

A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

Altered gap junctional communication, intercellular signaling, and growth in cultured astrocytes deficient in connexin43.

Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831–1834, 1995). Using anti‐Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild‐type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild‐type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth. J. Neurosci. Res. 49:528–540, 1997.

Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis.

We have employed immunofluorescence with a rat kidney Na+/K+-ATPase polyclonal antibody to investigate the cellular distribution and timing of appearance of this enzyme during preimplantation development. The enzyme is first detected in the late morula within the cytoplasm of each blastomere. When cavitation begins this distribution changes dramatically to a ring encircling the blastocoel, restricted to the basolateral cell margins. Using this enzyme as a marker for cavitation, we examined its expression in embryos that had been treated with wheat germ agglutinin (WGA), which causes cleavage arrest and was reported to trigger premature compaction- and cavitation-like events in early cleavage stages (L. V. Johnson, 1986, Dev. Biol. 113, 1-9). Although WGA-treated 2-,4-, and 8-cell embryos quickly underwent compaction- and cavitation-like events, no Na+/K+-ATPase expression was observed. Thus the WGA effect does not likely involve acceleration of the developmental program for cavitation. Embryos arrested at the 8-cell stage but cultured overnight to Day 4, however, expressed the enzyme in the typical blastocyst pattern (around each fluid-filled cavity). We conclude that Na+/K+-ATPase expression is initiated or increases dramatically in the late morula and is independent of cytokinesis. The enzyme assumes a distribution during cavitation consistent with its presumed role in transtrophectodermal fluid transport.

Timing of transcription and protein synthesis underlying morphogenesis in preimplantation mouse embryos

During preimplantation development of the mouse, embryos pass through a series of morphogenetic events: compaction, fluid accumulation to form the blastocoele (cavitation), and escape from the zona pellucida (hatching). We have used the inhibitors alpha-amanitin and cycloheximide to investigate the timing of transcriptional and translational events underlying these morphogenetic stages. Groups of embryos were transferred from a common pool into medium containing one or the other inhibitor at regular time intervals, and then were scored over the ensuing 24 or more hours for their ability to reach a particular morphogenetic end point. By comparing the time when the control population reached an end point with the time at which embryos had to be transferred into the inhibitor in order to prevent them from reaching that end point, we could determine when in advance of each event the necessary transcription or protein synthesis has been completed. Our results suggest that compaction (as well as cleavage to the eight-cell stage) is an embryonically, rather than maternally, programmed event, although the necessary transcription is completed well in advance, at least by the early four-cell stage. The transcriptional and translational events underlying fluid accumulation, on the other hand, appear to be completed within a few hours of the start of this process. For hatching, there is once again a long delay between the apparent time of completion of the necessary transcriptional events and the process itself, with protein synthesis being completed just a few hours in advance. Our results raise the possibility that post-transcriptional regulatory mechanisms play an important role in the timing of morphogenetic events in early mouse embryos.

Failure of Spermatogenesis in Mice Lacking Connexin43

Abstract Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a “Sertoli cell only” phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.

Bidirectional communication between oocytes and follicle cells: ensuring oocyte developmental competence

Female fertility is determined to a large extent by the quality (developmental competence) of the oocyte as reflected in its ability to undergo meiosis, be fertilized, and give rise to a healthy embryo. Growth of the mammalian oocyte is coordinated with that of the follicle that encloses it by the actions of signals that pass in both directions between the germline and somatic components. This review summarizes what is known about the roles played by 2 different modes of intrafollicular signalling in oogenesis: paracrine factors activating receptors on the opposite cell type, and direct sharing of small molecules throughout the follicle via gap junction channels. Recent evidence indicates that these 2 modes of signalling interact to regulate oocyte growth and granulosa cell proliferation and that defects in either can contribute to female infertility.

MULTIPLE MEMBERS OF THE CONNEXIN GENE FAMILY PARTICIPATE IN PREIMPLANTATION DEVELOPMENT OF THE MOUSE

The connexin gene family, of which there are at least 12 members in rodents, encodes the protein subunits intercellular membrane channels (gap junction channels). Because of the diverse structural and biophysical properties exhibited by the different connexins, it has been proposed that each may play a unique role in development or homeostasis. We have begun to test this hypothesis in the preimplantation mouse embryo in which de novo gap junction assembly is a developmentally regulated event. As a first step, we have used reverse transcription-polymerase chain reaction (RT-PCR) to determine the connexin mRNA phenotype of mouse blastocysts, and have identified transcripts of connexins 30.3, 31, 31.1, 40, 43, and 45. Quantitative measurements indicated that all six of these connexin genes are transcribed after fertilization. They can be divided into two groups with respect to the timing of mRNA accumulation: Cx31, Cx43, and Cx45 mRNAs accumulate continuously from the two- or four-cell stage, whereas Cx30.3, Cx31.1, and Cx40 mRNAs accumulate beginning in the eight-cell stage. All six mRNAs were found to co-sediment with polyribosomes from their time of first appearance, indicating that all six are translated. The expression of Cx31.1 and Cx40 was examined by confocal immunofluorescence microscopy; whereas both could be detected in compacting embryos, only Cx31.1 could be seen in punctate membrane foci indicative of gap junctions. Taken together with other results (published or submitted), our findings indicate that at least four connexins (Cx31, 31.1, 43 and 45) contribute to gap junctions in preimplantation development. The expression of multiple connexin genes during this early period of embryogenesis (when there are only two distinct cell types) raises questions about the functional significance of connexin diversity in this context.

Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface

Studies of mice with targeted disruptions of specific connexin genes have revealed that at least two connexins, connexin37 (Cx37) and connexin43 (Cx43), play essential roles in ovarian follicle development. To explore the respective roles of these two connexins in gap-junctional communication between the developing murine oocyte and its surrounding cumulus granulosa cells, we used confocal immunofluorescence microscopy and oocyte preloading functional assays. Immunofluorescence microscopy located Cx37 within gap-junction plaques between granulosa cells and the oocyte, and Cx43 between surrounding granulosa cells. Preloading assays combining denuded oocytes and cultured granulosa cells expressing or lacking Cx37 or Cx43 revealed that Cx37 must be present in both cell types for the establishment of heterocellular gap-junctional coupling. Furthermore, immunofluorescence microscopy of cultured granulosa cells after incubation with denuded oocytes showed that the oocyte induces the formation of gap junctions containing Cx37 at the surface of granulosa cells. Continuous Cx37 expression in granulosa cells was confirmed using RT-PCR. Together, these results indicate that the growing murine oocyte is functionally coupled with granulosa cells by homotypic gap junctions composed of Cx37, and that the formation and/or stabilization of Cx37 junctions is selectively induced at the oocyte-granulosa interface by cell contact.

Control of gap junction formation in early mouse embryos

Intercellular communication via gap junctions begins in the eight-cell stage in early mouse embryos. We have studied the timing of this event in relation to compaction, and have begun to explore some of the possible control mechanisms underlying it. Gap junction formation was inferred by measuring ionic coupling as well as by observing the intercellular transfer of fluorescent dye. Embryos were obtained early on Day 3 of pregnancy by flushing the oviducts of HA/ICR mice that had been mated with CB6F1/J males. Gap junctions were detected only in those embryos which had achieved the fully compacted state. Inhibition of protein synthesis by cycloheximide treatment beginning as early as the late four-cell stage failed to block compaction or the acquisition of gap junctions, demonstrating that the necessary proteinaceous components are present in advance of these events. In order to test the possibility that gap junctions could be induced to form prematurely, fully compacted, communication-competent eight-cell embryos were aggregated with two- or four-cell embryos. Even after 10 hr of aggregation, no interembryonic gap junctions could be detected. Fully compacted eight-cell embryos when aggregated with each other, however, became ionically coupled within 3-5 hr. The number of interembryonic junctional channels was judged to be effectively small, since the aggregated embryos exhibited obvious ionic coupling but very weak dye coupling. In contrast to gap junction formation within embryos, junction formation between embryos was blocked by cycloheximide. These results demonstrate that gap junction formation in early mouse embryos is under precise temporal control, involving the assembly or mobilisation of preexisting components. This stockpile of components is either unavailable or insufficient to allow the formation of additional gap junctions between aggregated communication-competent embryos without new protein synthesis.

Differential contributions of connexin37 and connexin43 to oogenesis revealed in chimeric reaggregated mouse ovaries.

The gap junction proteins connexin37 and connexin43 are required for ovarian folliculogenesis in the mouse. To define their respective roles in oogenesis, chimeric ovaries containing either null mutant oocytes and wild-type granulosa cells or the reverse combination were grafted to the renal capsules of immunodeficient female mice. After three weeks, the oocytes were tested for meiotic competence and fertilizability in vitro. Ovaries composed of connexin43-deficient oocytes and wild-type granulosa cells produced antral follicles enclosing oocytes that could develop to at least the two-cell stage, demonstrating that oocytes need not express connexin43 to reach maturity. Conversely, both follicle development and oocyte maturation were impaired in ovaries containing either wild-type oocytes and connexin43-deficient granulosa cells or connexin37-deficient oocytes and wild-type granulosa cells. Thus absence of connexin43 from granulosa cells or connexin37 from oocytes is sufficient to compromise both oocyte and follicle development. Wild-type oocytes paired with connexin37-deficient granulosa cells generated antral follicles containing oocytes that developed to at least the two-cell stage. Therefore, connexin37 absence from granulosa cells need not impair fertility in mice. Dye transfer experiments revealed persistent oocyte-granulosa cell coupling in those follicles, indicating functional compensation by another connexin. The results indicate that mouse oocytes do not need to express connexin43 in order to develop into meiotically competent, fertilizable gametes, but must express connexin37 for communication with granulosa cells, a requirement for oogenesis.