embryonic pancreata were harvested from time - mated cd1 strain mouse intercrosses of ngn3 females and males and stored individually in rnalater ( ambion ) at 4c until genotyping by pcr ( 30 cycles ; ngn3 forward , 5-cctcttctggctttcactac-3 and reverse , 5-ggagcgagagtttgatgtgg-3 ; neomycin forward , 5-gtcttgtcgatcaggatgatctc-3 and reverse , 5-caatatcacgggtagccaacgc-3 ) . pancreatic islets were prepared from 6-month - old female cd1 mice by collagenase digestion and isolated by ficoll / histopaque gradients and handpicking ( each preparation : six mice/1,000 islets ) . tc1 - 6 , tc3 , min6- , and mpac - tumor cell lines ( 10 ) were grown to 60% confluency in rpmi medium . total rna was extracted from tissues with trizol reagent ( invitrogen , carlsbad , ca ) , purified by rneasy columns ( qiagen , valencia , ca ) . biotin - labeled crna was synthesized by the affymetrix protocol ( affymetrix , santa clara , ca ) and hybridized to mgu74av2 and/or moe 430 2.0 microarrays . data were analyzed with genespring 7.2 ( silicon genetics , redwood city , ca ) and gc robust multi - array average ( gc rma ) used for normalization . anova was applied to cell line data , and p values were corrected by benjamini and hochberg 's procedure . netaffx ( affymetrix , santa clara , ca ; http://www.affymetrix.com/analysis/index.affx ) was used to extract a common probe set between moe430 2.0 and mgu74av2 platforms . gene classification was performed with fatigo ( http://fatigo.bioinfo.cipf.es ) . quantitative rt - pcr ( qrt - pcr ) was performed on cdna ( 5 ng ) derived from total rna by iscript cdna synthesis kits ( bio - rad laboratories , hercules , ca ) . assays used fam dye  labeled taqman mgb probes and an abi 7000 pcr instrument ( applied biosystems , foster city , ca ) and were normalized to glyceraldehyde-3-phosphate dehydrogenase . mouse pancreata were fixed in 4% paraformaldehyde and embedded in optimal cutting temperature compound ( tissue tek ; sakura finetek , torrance , ca ) for immunofluorescence microscopy . sections ( 6 m ) were blocked in tyramide signal amplification blocking solution ( zymed , carlsbad , ca ) incubated overnight with guinea pig anti - insulin , mouse anti - glucagon ( sigma , st louis , mo ) , rat anti - somatostatin ( abcam , cambridge , ma ) , or rabbit anti - znt8 aa268 - 369 ( 9 ) . secondary antibodies conjugated to alexa 488 , 1-amino-1-methyl-3(4)-aminomethylcyclohexane , cyanine 5 , and cyanine 3 fluorophores ( jackson immunoresearch laboratories , west grove , pa ) were applied for 60 min and sections mounted in glycerol - based media . images were acquired with an olympus 1x70 microscope equipped with a photometrics quantix cooled monochromatic ccd camera ( kodak chip kaf1400 ) . pancreatic microarray analyses were performed on wild - type and ngn3 mice at e12.5 , e15.5 , and e18.5 . the majority of the endocrine fate allocations occur between e13.5 and e15.5 , when ngn3 is maximally expressed ( 11,12 ) ; endocrine markers ( insulin and glucagon ) subsequently appear along with neuroendocrine markers , e.g. , prohormone convertases and chromogranins . thus , comparison of e12.5 with e15.5 identifies transcripts that characterize precursor cells , responses to the activation of ngn3 expression , and the transition to the differentiated endocrine fate . at e12.5 , 203 gene transcripts were downregulated > threefold in ngn3 versus wild - type littermates and , at e15.5 , 54 ( table 1 and supplementary tables 2 and 3 , available in an online appendix at http://dx.doi.org/10.2337/db07-1126 ) . fewer genes were upregulated : 53 at > threefold in e12.5 and 17 in e15.5 ( data not shown ) . at e18.5 , 645 transcripts were downregulated > 3-fold in ngn3 versus wild - type animals ( supplementary table 4 ) , 290 > 4-fold , and 35 > 10-fold . validation of select transcripts by qrtpcr demonstrated that glucagon ( down 1,400-fold ) , insulin ( down 400-fold ) , and znt8 ( down 18-fold ) were readily detectable in wild - type but not ngn3 animals with 40 cycles of pcr ( data not shown ) . of the transcripts that were downregulated > fourfold , 46 mapped to transcripts highly expressed in adult islets ( raw value > 2,000 including znt8 ) and 28 to transcripts moderately expressed ( 5002,000 ) ( table 2 ) . comparison of tc1.6- and tc3-cell transcripts identified 1,554 transcripts that were > 2-fold enriched in tc3 versus tc1.6 and 403 that were > 10-fold enriched ( supplementary table 5 ) . conversely , 1,202 transcripts were > 2-fold enriched in tc1.6- versus tc3-cell lines and 212 > 10-fold ( supplementary table 6 ) . of the genes downregulated > fivefold in the ngn3 mice , 74 were enriched in tc - cells and 108 in tc - cell lines ( supplementary tables 7 and 8) . another 1,396 transcripts were expressed in both tc- and tc - cell lines and 774 not detected . f ) showed that insulin transcripts were enriched in adult islets versus tc- and not expressed in tc- or mpac - cell lines ( fig . 1d ) . by microarray analysis , insulin expression in islets and -cell lines did not differ since signals were saturated ( > 25,000 arbitrary units ) . glucagon transcript levels by qrtpcr were higher in islets than in tc - cells and present at a low level in tc3- and min6-cells but absent in mpac - cells ( fig . 1e ) . by microarray analysis , the glucagon signals saturated in islets and tc - cells but otherwise showed the same trend of expression . znt8 transcript levels by qrtpcr were 4- to 5-fold higher in -cell lines than in tc1.6-cells and 50- to 100-fold higher than in ductal cells ( fig . microarray analysis showed znt8 expression in islets and - and -cell lines but not in ductal cells . immunohistochemical localization of znt8 in wild - type adult pancreas showed its expression in the majority of islet cells and absence in acinar and ductal tissue . it was principally colocalized with insulin and also found in a subpopulation of somatostatin - positive cells . e18.5 ngn3 pancreas tissue did not show any znt8 , insulin , glucagon , or somatostatin expression ( data not shown ) . the loss of expression of genes in the pancreas of ngn3 mice provides a model to define the transcriptome of the endocrine pancreas and offers insight into the transcriptional and morphogenetic factors responsible for the patterning and differentiation of the endocrine lineage . genes responsive to paracrine signaling from endocrine cells adjacent to exocrine or ductal cells might also have been revealed , although there was little evidence of this . as previously observed ( 5 ) , expression of the pancreatic islet hormones ( insulin , glucagon , somatostatin , pancreatic polypeptide , and ghrelin ) were markedly diminished alongside genes known to be expressed in islets but not in acinar tissue ( iapp , chgra , chgrb , npy , pyy , pcsk1 , pcsk2 , and igrp / g6pc2 ) . several candidate transcriptional regulators of islet cell lineage development were likewise absent ( neurod , mafb , insm1 , myt1 , pax6 , and isl1 ) . at e12.5 , there are a few endocrine cells that are immunoreactive to glucagon and occasional insulin and glucagon double - positive cells . during this phase , robo1 ( dutt1 protein , orthologous to the drosophila roundabout ) and an expressed sequence tag 9430047l24rik encoding unc5c homolog are the two most endocrine - enriched transcripts . robo1 encodes a molecule of the neural - cell adhesion molecule family that interacts with the extracellular ligand slit ( 1315 ) that has been implicated in migration of axons , myoblasts , and leukocytes in vertebrates and in lung development ( 16 ) . at e15.5 , four transcripts notably absent in ngn3 pancreas were myt1 , crystallin 2 , secretogogin ef - hand , and 4930568n03rik ( genbank accession no . microarray data defining the pancreatic development kinetics from e12.5 to e18.5 ( k. juhl , s. sarkar , j. jensen , j. hutton , unpublished data ) suggest that myt1 is downstream of ngn3 , as previously documented ( 3,17 ) . the av323033 est cluster is a component of the unigene identification mm.209896 , which represents nucleolar protein four ( nol4 ) , which is expressed in libraries from brain , eye , thymus , pancreas , endocrine , spinal cord , and male genitalia . at e18.5 , genes notably absent in ngn3 pancreas included mafb , nkx6.1 , wbscr14 , nnat , syt13 , and pcsk1n  genes that function in mature islet cells as transcription factors ( mafb , nkx6.1 , and wbscr14 ) ( 18 ) or in stimulus secretion coupling . some transcripts downregulated at this point , however , are expressed at earlier times in wild - type and ngn3 pancreas , notably pitx2 , ramp2 , hmgn3 , and gtpbp4-pending , indicating that their cell specification is changing . ramp2 , for example , is a chaperone for the calcitonin receptor - like receptor that mediates adrenomedullin action on growth and differentiation where strong mesenchymal - epithelial interactions take place ( 19 ) . comparison of tc and tc expression data with the list of 2,352 genes that were downregulated at any age in ngn3 mice revealed 74 -cell candidates including glucagon , arx , brn4 , spp1 , irx2 , rbp4 , mafb , car2 , tfpi , vegfc , and fev - pending and 108 -cell candidates including ins1&2 , iapp , neuronatin , pdx1 , g6pcrs , npy , prcad , sepp1 , sytl4 , hpca , and atp2a3 . genes that were expressed in both cell types included genes classifed as neuroendocrine ( chgra and -b , scgn2 and -3 , scgn , cpe , and pcsk1 and -2 ) and transcription factors active in multiple pancreatic endocrine cells ( pax6 , isl1 , neurod1 , and nkx2.2 ) . in all , the ngn3 model did a remarkably good job of predicting endocrine - specific and neuroendocrine genes , especially given that it reports on 5% of the pancreatic tissue . at the time of the secondary transition , ngn3-downregulated transcripts are likely to reflect specific transcripts of the immediate precursors of endocrine cells and thus overlap with profiles derived from fluorescence - activated cell sorter  sorted ngn3enhanced green fluorescent protein cells ( supplementary tables 9 and 10 ) ( 3,20,21 ) . twenty - nine of the 190 transcripts of the mgu74av2 dataset of gu et al . ( 3 ) showed overlap including low - copy number transcription factors ( mafb , arx , brn4 , ngn3 , neurod1 , pax6 , myt1 , and zfp288 ) . of the remaining 161 transcripts , 112 were expressed at e12.5 and e15.5 irrespective of ngn3 gene status , and four ( gnao , gip , sgne1 , and nkx2.2 ) were not found . the above microarray datasets can be downloaded from an open access web site : http://www.cbil.upenn.edu/rad/php/displaystudy.php?study_id=1330 . while the current study focuses on embryological development of the pancreas , these datasets can potentially be analyzed in other ways to provide an understanding of islet function in health and disease . given the current interest in identifying biomarkers that could be used to isolate -cells and their precursors or to image islet mass , it was of interest to review how effective the selection criteria used here to ascertain -cell specificity would be in identifying a -cell marker of lower abundance . the example is the insulin granule zinc transporter znt8 ( slc30a8 ) , a type 1a diabetes autoantigen ( 9 ) and a gene associated with type 2 diabetes susceptibility ( 8) . znt8 transcripts were decreased 18-fold in ngn3 mice pancreas at e18.5 ( table 2 ) , in tc3- and min6-cells , and to a lesser extent in tc - cells ( fig . 1a and f ) but not in a mouse pancreatic ductal cell line ( mpac ) . immunofluorescence microscopy showed colocalization with insulin and with a minor islet cell population that coincides with the -cell . a lower level of expression in the -cell was indicated by qrtpcr and other studies ( 22 ) but could not be confirmed by immunohistochemistry . a similar strategy could apply the current datasets for the discovery of novel regulatory processes involved in islet metabolism , stimulus secretion coupling , gene transcription , and intracellular and intracellular signaling .