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First published online 26 June 2003
doi: 10.1242/jcs.00631

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Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA
² Departments of Paediatrics and Physiology and Pharmacology, Child Health Research Institute, University of Western Ontario, London, Ontario N6C 2V5, Canada

View larger version (67K): [in a new window]   Fig. 1. Mist1KO acinar cells have greatly reduced levels of connexin32 protein. (A,B) Adult pancreas sections from wild-type (WT) and Mist1KO mice were processed for Cx32 immunofluorescence (green). WT pancreas sections (A) show a high number of Cx32-containing gap junction plaques (arrows) whereas Mist1KO sections (B) exhibit no gap junction staining. Nuclei are stained with the DNA fluorochrome DAPI (blue). (C) WT and Mist1KO pancreas protein samples were subjected to immunoblot analysis using antibodies against Mist1, ß-gal, Cx32 and ß-actin (as a control). Cx32 protein levels are greatly reduced in Mist1KO samples. (D-I) The loss of Cx32 is not coupled with the loss of other cell junctions. One-month-old pancreas samples were co-stained with antibodies against Cx32 (green) and amylase (D,E), ß-catenin (F,G), or occludin (red) (H,I). In all cases, Cx32 protein is absent in Mist1KO sections (E,G,I) and present in WT sections (D,F,H). Amylase staining reveals the disorganization in Mist1KO acinar cells, while no difference is observed between WT and Mist1KO samples for occludin or ß-catenin staining.

View larger version (91K): [in a new window]   Fig. 2. Cx32 protein is absent in Mist1KO pancreas acinar cells at birth. (A-F) Mist1 heterozygous (Mist1Het) and Mist1KO pancreas samples were subjected to Cx32 immunofluorescence. Cx32-containing gap junctions (green, arrows) are absent in all Mist1KO samples, regardless of age. Nuclei are stained with the DNA fluorochrome DAPI (blue). (G) Total RNA was isolated from wild type (WT) and Mist1KO pancreas samples from postnatal day 1 (PN1), 3-week old, 4-month and 8-month-old animals and then subjected to RNA blot analysis using a mouse Cx32 probe. In all cases, Cx32 mRNA levels are significantly lower in the Mist1KO samples than in the WT control mice. 12S rRNA hybridization was used as an internal control.

View larger version (63K): [in a new window]   Fig. 3. Mist1KO acinar cells are defective in intercellular communication. (A,B) Isolated acinar cells from wild type (WT) and Mist1KO mice were processed for Cx32 immunofluorescence (green). Similar to pancreatic sections, WT acinar cultures (A) show Cx32 plaques (arrows) on lateral cell borders whereas Mist1KO samples (B) exhibit no gap junction staining. Nuclei are stained with the DNA fluorochrome DAPI (blue). (C-H) Pancreatic acini were isolated from WT and Mist1KO mice, individual acinar cells (arrows) were injected with 10 mM 6-carboxyfluorescein and adjacent cells were monitored for the transfer of dye. Dye transfer to neighboring cells occurs rapidly in WT samples (1 minute, C-D) whereas no transfer is evident in Mist1KO acinar cells (E-H), even after extended times (20 minutes). C,E,G; phase contrast; D,F,H; fluorescence. (I,J) Pancreatic acini from WT and Mist1KO mice were analyzed for electrophysiology coupling. Single cells (Cell 1) were injected with 1 M KCl (arrows) and the spread of current was assessed using a recording electrode positioned on adjacent cells (Cell 2). WT (I) acinar cells exhibit normal electrophysiological coupling whereas no coupling is detected in the Mist1KO cells (J).

View larger version (89K): [in a new window]   Fig. 4. Cx26 mRNA, but not protein, is maintained in adult Mist1KO acinar cells. (A-C) Immunohistochemical analysis of wild type (WT) pancreatic acinar cells reveals that Cx32 (red) and Cx26 (green) are localized to the same gap junction plaques. (D) WT and Mist1KO pancreas RNA samples were subjected to RNA blot hybridization with the indicated nucleic acid probes. WT and Mist1KO samples express equivalent levels of Cx26 mRNA. (E) Immunoblot analysis of Cx26 protein in 3-week old mice reveals that WT and Mist1KO samples contain equivalent amounts of Cx26. However, samples from older Mist1KO animals no longer contain detectable levels of Cx26 protein. (F-I) Cx26 immunofluorescence (green, arrows) was performed on 3-week-old pancreas sections from Mist1Het and Mist1KO mice. Whereas Mist1Het mice exhibit normal, membrane bound Cx26 in the gap junctions (F,H), Cx26 protein in Mist1KO samples is localized diffusely within the cell (G,I). Note that the boxed regions in F and G are shown at higher magnification in H and I. Adult acini from WT (J) and Mist1KO (K) mice were also processed for Cx26 immunofluorescence (green, arrows). Adult Mist1KO acinar cells do not show significant Cx26-containing gap junctions. Nuclei are stained with the DNA fluorochrome DAPI (blue).

View larger version (39K): [in a new window]   Fig. 5. Mist1 and Cx32 are co-expressed in most secretory exocrine cell types. (A) RT-PCR analysis of several different tissues from wild type (WT) mice reveals that Mist1 and Cx32 transcripts are co-expressed in all serous exocrine cell types. The liver, which is not a serous exocrine organ, expresses the Cx32 gene but not the Mist1 gene. (B) RNA blot hybridization reveals that all serous exocrine tissues show a large decrease in Cx32 mRNA levels from Mist1KO mice when compared to WT control samples. (C-J) Cx32 (green, arrows) and ß-gal (red) immunofluorescence on Mist1Het and Mist1KO samples from the pancreas (C,D), lacrimal gland (E,F), submandibular gland (G,H) and liver (I,J). In all Mist1KO secretory exocrine cells in which the Mist1 locus is transcriptionally active (indicated by the presence of ß-gal protein in C-H, red), Cx32 immunoreactivity is greatly reduced. Mist1KO liver cells, which do not express Mist1, maintain high levels of Cx32. Nuclei in the liver panels are stained with the DNA fluorochrome DAPI (blue).

View larger version (18K): [in a new window]   Fig. 6. Mist1 activates expression of a Cx32 reporter gene. (A) AR42J exocrine pancreatic cells were electroporated with the Cx32 luciferase reporter (Cx32p-Luc) gene and an expression plasmid encoding the Mist1 cDNA. Mist1 protein is able to induce Cx32p-Luc expression approximately 15- to 20-fold. (B) A variety of other bHLH factors (both Class A and Class B) were examined for their ability to activate expression of the Cx32p-Luc gene. Only Mist1 is capable of activating this reporter gene. (C) Transfection of AR42J cells with the Cx32p-Luc and different Mist1 gene constructs confirms that DNA binding mutants (Mist1mut basic, mbasic) and protein dimerization mutants (Mist1mut helix 1, mhelix 1) are unable to activate expression of the Cx32p-Luc gene. Error bars indicate the standard error of the mean for a minimum of three independent transfections per gene construct.

View larger version (77K): [in a new window]   Fig. 7. Pancreatic acinar cells expressing a dominant negative Mist1 protein lack Cx32-containing gap junctions. Transgenic mice expressing the Mist1mut basic protein in acinar cells were analyzed for Mist1mut basic expression as well as for expression of Cx32. (B,D) The Mist1mut basic protein contains a myc epitope tag and can be detected using a myc antibody. (C,D) Acinar cells expressing the Mist1mut basic protein (red, arrowhead) do not accumulate Cx32-containing gap junctions (green, arrows), whereas acinar cells lacking the Mist1mut basic protein retain Cx32 expression. Nuclei in A are stained with the DNA fluorochrome DAPI (blue).