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First published online 3 December 2003
doi: 10.1242/dev.00921

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Global expression analysis of gene regulatory pathways during endocrine pancreatic development

Guoqiang Gu^1,*,, James M. Wells^1,,, David Dombkowski², Fred Preffer², Bruce Aronow and Douglas A. Melton^1,

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

View larger version (49K): [in a new window]   Fig. 1. Scheme of the temporal and spatial gene expression analysis for four stages of islet development. The red box highlights the temporal gene expression comparison and the green boxes highlight the spatial expression comparisons. (1) At E7.5, endoderm (black arrowhead) and mesectoderm (red arrowhead) were manually separated and analyzed. (2) At E10.5, the eGFP+ cells from pancreatic buds (red dashed lines), stomach/duodenum (white dashed lines; two solid white lines indicate the approximate borders of dissection), and eGFP– cells from Pdx1-eGFP transgenic animals were separated by FACS and analyzed. (3) At E13.5, endocrine progenitor cells expressing Ngn3-eGFP were separated by FACS from eGFP– cells (yellow arrowhead, primarily exocrine and ductal cells) and both eGFP+ and eGFP– cells were analyzed. Adult islets were hand picked and used for direct analysis (green dashed lines. The blue staining within the islet is from Pdx1-lacZ). cRNA probes for each sample were hybridized to Affymetrix microarrays Mu11K, Mu74Av1 or Mu74Av2 (Materials and methods). Temporal analysis (the red box) compared the gene expression patterns of endoderm, pancreatic progenitors (Pdx1-eGFP+), endocrine precursors (Ngn3-eGFP+), and adult islets. Spatial analysis (green boxes 1, 2 and 3) compared different samples from the same stages, e.g. genes expressed in E7.5 endoderm versus mesoderm + ectoderm.

View larger version (45K): [in a new window]   Fig. 2. Clustering of genes expressed at each stage of endocrine development. These clustered genes are also grouped according to their biological function. (A) A GeneSpring clustering analysis that identified four groups of genes that were specifically expressed in each stage of development: E7.5 endoderm, E10.5 PDX1+ pancreas cells, E13.5 NGN3+ endocrine progenitors, and adult islets. For each group of genes, the X-axis represents the stage and the Y-axis represents the normalized abundance (intensity) of each transcript. Each line shows the normalized expression level of one gene during each stage of development. (B) Pie charts of genes that were expressed at each stage of development, clustered by biological function. For a more comprehensive comparison of genes in each biological class, we have combined the temporally and spatially regulated genes from the supplemental tables into one pie chart per stage of development. The absolute number of genes in each biological class is shown next to each pie sector. For a complete list of genes either temporally or spatially enriched in each cell sample, see Supplementary Tables.

View larger version (38K): [in a new window]   Fig. 3. Genes expressed in endoderm. (A) Venn diagram illustrating genes that were at least threefold enriched in unspecified endoderm as compared to adjacent mesoderm and ectoderm. 203 genes were enriched in the endoderm, whereas 262 genes were enriched in the mesectoderm. (B) The endodermal expression of four genes, Apo AIV, Dkk1, Prss12 and Eya2, identified from our microarray analysis, was verified by RT-PCR (end, endoderm; mec, mesoderm + ectoderm; HPRT is used as a control for RT-PCR) and whole mount ISH. These genes were not previously shown to be expressed in endoderm. The endodermal expression of Eya2 was verified by sectioning the stained embryo. ect, ectoderm; end, endoderm; mes, mesoderm.

View larger version (74K): [in a new window]   Fig. 4. A summary of genes expressed in the pancreatic Pdx1-eGFP+ cells and non-pancreatic cells at E10.5 (including Pdx1-eGFP+ cells of the stomach + duodenum as well as PDX1-GFP– cells). (A) Venn diagram showing that 158 genes (green) were enriched in the eGFP+ cells within the pancreatic region whereas 208 (red) genes were enriched in the PDX1+ cells in the duodenum and stomach or eGFP-cells. (B-E) Expression analyses of four genes by RT-PCR and in situ hybridization. ß actin expression was used as a control. (B) Spintz1(Af010499) was highly expressed in the pancreas and duodenum but not the stomach. (C) Tm4sf3 (AA571115) was expressed at a high level in the stomach and duodenum but not the pancreas. (D) Nkx2.2 was expressed at a higher level in the pancreas. (E) Osteopointin was only expressed in the pancreas. Black dashed lines, stomach; green dashed lines, duodenum; red dashed lines, pancreas.

View larger version (91K): [in a new window]   Fig. 5. Genes expressed in the Ngn3-eGFP+ and Ngn3-eGFP– cells at E13.5. (A) Venn diagram showing that 204 genes (green) were enriched at least threefold in the Ngn3-eGFP+ cells as compared to 256 (red) genes that were enriched in the Ngn3-eGFP– cells. (B-E) Expression analysis of four genes by ISH. Go (B), Foxa3 (C), Myt1 (D), and Rbp (E) were only detected at high levels in a set of scattered pancreatic cells, similar to Ngn3. Black dashed lines, stomach; green dashed lines, duodenum; red dashed lines, pancreas.

View larger version (110K): [in a new window]   Fig. 6. Functional characterization of Myt1 during mouse endocrine development. (A-D) In situ hybridization showing Ngn3 and Myt1 expression at several stages of embryogenesis. A is a control showing Ngn3 expression in the developing pancreatic region (red dashed lines) at E8.5. (B) At E8.5 Myt1 is expressed at a low level in the prospective pancreatic region in a manner similar to Ngn3 (red dashed line). The prospective pancreatic region is recognized by its position below somite 8-10 (total of 15 somites), and the position of the dorsal aorta (green arrow). Strong Myt1 expression was also detected in the neural tube (black arrow). (C) At E10.5, Myt1 was detected in a scattered fashion in the pancreatic bud, within or close to duct-like structures (red arrowheads). (D) At E15.5, Myt1 expression was much reduced (see text). (E-H) Transgenic expression of dnMyt1 in endocrine progenitor cells inhibits endocrine differentiation. (E) Insulin+ cells in a representative wild-type pancreas. (F) Insulin+ cells in a representative pancreas section of a Ngn3-dnMyt1 littermate. Note fewer insulin-expressing cells in F. Similarly, the number of glucagon+ cells is reduced in the Ngn3-dnMyt1 expressing embryo (H) compared with that of wild type embryo (G). In E-H, Cy3-conjugated antibodies were used. Scale bar: 25 µm.

View larger version (197K): [in a new window]   Fig. 7. Myt1 is involved in generation of glucagon and somatostatin-expressing cells in chicken embryonic endoderm and may be in the same pathway as Ngn3. (A-C) In normal chicken embryos (A) glucagon (brown staining, black arrow) and somatostatin-expressing cells (data not shown) are absent in chicken stomach and duodenum. When Nzf2b was ectopically expressed in the chicken embryonic gut endoderm around stage 18, glucagon expression was induced in the stomach (B) and duodenum (C). A small, yet significant, number of somatostatin-expressing cells were also induced (D, red arrowhead). (E,F) dnMYT1 inhibits NGN3-mediated induction of glucagon+ cell formation in chicken gut endoderm. Ngn3 + Myt1a (E) or Ngn3 + dnMyt1 (F) were co-electroporated into chicken gut endoderm respectively. Glucagon expression (brown antibody staining) and Myt1 or dnMyt1 expression (blue, in situ hybridization) was measured. In E, co-expression of Ngn3 and Myt1a (data not shown) (Grapin-Botton et al., 2001) resulted in induction of glucagon+ cells (brown and blue color overlap resulting in dark brown/purple). In F, cells that co-express Ngn3 and dnMyt1 almost never express glucagon (brown and blue cells do not overlap) suggesting that the presence of dnMYT1 inhibited the ability of NGN3 to promote the generation of glucagon-expressing cells. Black dashed line, stomach; green dashed line, duodenum; red dashed line, pancreas.