Dynamics of embryonic pancreas development using real-time imaging - PubMed (original) (raw)

Dynamics of embryonic pancreas development using real-time imaging

Sapna Puri et al. Dev Biol. 2007.

Abstract

Current knowledge about developmental processes in complex organisms has relied almost exclusively on analyses of fixed specimens. However, organ growth is highly dynamic, and visualization of such dynamic processes, e.g., real-time tracking of cell movement and tissue morphogenesis, is becoming increasingly important. Here, we use live imaging to investigate expansion of the embryonic pancreatic epithelium in mouse. Using time-lapse imaging of tissue explants in culture, fluorescently labeled pancreatic epithelium was found to undergo significant expansion accompanied by branching. Quantification of the real-time imaging data revealed lateral branching as the predominant mode of morphogenesis during epithelial expansion. Live imaging also allowed documentation of dynamic beta-cell formation and migration. During in vitro growth, appearance of newly formed beta-cells was visualized using pancreatic explants from MIP-GFP transgenic animals. Migration and clustering of beta-cells were recorded for the first time using live imaging. Total beta-cell mass and concordant aggregation increased during the time of imaging, demonstrating that cells were clustering to form "pre-islets". Finally, inhibition of Hedgehog signaling in explant cultures led to a dramatic increase in total beta-cell mass, demonstrating application of the system in investigating roles of critical embryonic signaling pathways in pancreas development including beta-cell expansion. Thus, pancreas growth in vitro can be documented by live imaging, allowing visualization of the developing pancreas in real-time.

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Figures

Figure 1. In vitro expansion of the mouse pancreatic explant

A. e10.5 dorsal pancreatic (dp) bud attached to stomach (s) and the duodenum (d). B. e10.5 explant in culture for 7 days undergoes significant expansion of the dorsal (dp) and ventral buds (vp), resembling acinar structures. Bar, 200μm. To evaluate the morphology of pancreatic growth in vitro, H&E staining was carried out on explants grown in Matrigel™ for 6 days (D). Pancreatic epithelial development closely resembles exocrine morphology at embryonic stage e15.5 (C). Immunohistochemical analysis of pancreatic explants grown in culture for 7 days reveals expression of the pancreatic transcription factor Pdx-1 (green) and the ductal marker mucin (red) (E), exocrine marker amylase (F), and endocrine markers insulin, glucagon (G), and pancreatic polypeptide (H). Bar, 75μm.

Figure 2. Pancreatic bud expansion in vitro mimics in vivo marker expression

A comparison of the expression profile of pancreas-specific markers during growth in Matrigel™ with in vivo embryonic growth using RT-PCR demonstrates a striking resemblance in expression pattern. Pancreatic explants dissected at e10.5 were grown in Matrigel™ for 6 days, and RNA harvested each day. The top row in each panel represents days in culture. RT-PCR on pancreatic RNA collected from embryos (e10.5 to e15.5) is shown in the lower row in each panel.

Figure 3. Expansion and branching of the embryonic pancreatic epithelium in real-time

A. Pancreatic explants were dissected at e10.5 from the Pdx1-GFP transgenic embryos and grown in culture for 4 days before imaging. B. Explants expressing Pdx1-Cre-Z/EG were dissected at e11.5 and grown in culture for 4 days followed by imaging. Still images from time-lapse movies show the overall expansion of the pancreatic epithelium (yellow branches) and branching by budding (red arrows). Time of imaging is shown in hours:minutes. Bar, 200μm.

Figure 4. Description of branching in the pancreatic epithelium during morphogenesis

A. Frames from a time-lapse movie of an e9.5 Pdx1-GFP pancreatic explant grown in culture for 4 days before imaging are shown. Bar, 200μm. B. A magnified view of a region of the growing pancreatic epithelium (indicated in the red box in A) depicts branching in the pancreas. Bar, 50μm. Time of imaging is shown in hours:minutes. Lateral buds appear (B, and C, 3a, labeled 2, 3 and 4) as the “primary” bud (labeled as 1) expands. In C, different modes of branching morphogenesis are represented- (1) type “1-2” terminal bifurcation, (2) type “1-3” lateral budding, and (3) lateral branching observed during pancreas growth. The order of appearance of buds exemplifies this lateral mode of expansion in the pancreas.

Figure 5. Lateral branching is the predominant mode of budding in the pancreatic epithelium during branching morphogenesis

Budding modules were identified and labeled as protrusions in the growing epithelium, and tracked over time for evidence of branching. Quantification of the mode of budding was carried out by counting the modules that underwent lateral budding or terminal bifurcation, and expressed as a percent of total. Bar, 200μm.

Figure 6. Real-time imaging of ß-cells during embryonic pancreatic development

A. Still frames from a time lapse movie depict the increase in the total fluorescence in the field, indicating an increase in ß-cells over time. Time of imaging is shown in hours:minutes. Bar, 100μm. B. Quantification of the fluorescent objects show an increase in the total area of fluorescence and the average size of objects (n=3).

Figure 7. Migration of ß-cells towards clusters of cells

A. Stills from a time-lapse series show migration of a ß-cell towards a larger cluster of cells. The distance between the single cell (red arrowhead) and the cluster of cells (yellow arrowhead) changed from 88.5μm to 49.9μm during imaging. Time of imaging is shown in hours:minutes. B. Migration of a single ß-cell occurred over 365μm (a to b, magenta line) at an average velocity of 11.4μm/hr.

Figure 8. Role of Hedgehog signaling in ß-cell specification in culture

A. Wide-field images depict the presence of insulin-GFP fluorescence in untreated and cyclopamine treated samples, with negligible GFP signal in the Shh treated sample. Corresponding bright field images show tissue morphology. B. The percentage of pancreatic explants positive for GFP after 6 days in culture were quantified [n= 57 (untreated), 53 (cyclopamine, 10μM), 11 (cyclopamine, 5μM) and 29 (Shh, 300ng/ml)]. C. Real-time quantitative PCR analysis of transcript expression in pancreatic explants in culture for 8 days was carried out (n=2 independent experiments, a total of 12 pancreatic explants for untreated or treated with cyclopamine or Shh). The effects of cyclopamine (5μM) or recombinant Shh (300ng/ml) treatment on pancreas-specific genes as well as components of the Hh signaling pathway are shown. No change in transcript level is indicated by a red dotted line. Treatment with cyclopamine led to a dramatic increase in insulin expression. ins, insulin; glu, glucagon; ss, somatostatin; amy, amylase; muc, mucin; ptc, patched.

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Dynamics of embryonic pancreas development using real-time imaging - PubMed (original) (raw)