Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets

We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding ‘global’ cellular organization at 15–20 nm resolution. Two insulin-secreting beta cells—deemed ‘functionally equivalent’ by virtue of their location at the periphery of the same pancreatic islet—were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure–function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells’ individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.     

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones. Received: 22 June 1999 / Accepted: 1 September 1999     

Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.