BMC Developmental Biology BioMed Central Research article Open Access Identification of known and novel pancreas genes expressed downstream of Nkx2.2 during development Keith R Anderson1, Peter White3,4, Klaus H Kaestner3 and Lori Sussel*1,2 Address: 1Department of Biochemistry and Program in Molecular Biology, University of Colorado Health Science Center, Denver, CO, 80045, USA, 2Department of Genetics and Development, Columbia University, New York, NY 10032, USA, 3Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania, 752B CRB, 415 Curie Blvd, Philadelphia, Pennsylvania 19104, USA and 4The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive, Columbus, OH 43205, USA Email: Keith R Anderson - [email protected]; Peter White - [email protected]; Klaus H Kaestner - [email protected]; Lori Sussel* - [email protected] * Corresponding author Published: 10 December 2009 Received: 27 May 2009 Accepted: 10 December 2009 BMC Developmental Biology 2009, 9:65 doi:10.1186/1471-213X-9-65 This article is available from: http://www.biomedcentral.com/1471-213X/9/65 © 2009 Anderson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing β cells and glucagon-expressing α cells are replaced by ghrelin- expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas. Results: Microarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2-/- embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2-/- pancreas. Conclusions: Expression profiling of Nkx2.2-/- mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step. Background many of the enzymes required for digestion of carbohy- The pancreas is a multifunctional organ that is critical for drates, lipids and proteins. To carry out these diverse func- maintaining glucose homeostasis and for producing tions, the pancreas contains three major tissue types: the Page 1 of 17 (page number not for citation purposes) BMC Developmental Biology 2009, 9:65 http://www.biomedcentral.com/1471-213X/9/65 exocrine acinar cells, the endocrine cells that comprise the Nkx2.2 is a homeodomain containing transcription factor islet of Langerhans, and the ductal epithelial cells. critical for endocrine pancreas specification [19]. Prior to Although each of these pancreatic components performs e15.5, the pancreas of Nkx2.2-/- mice appears overtly nor- unique functions, all are derived from a defined set of mal and contains wild type numbers of undifferentiated endodermally-derived progenitors [1]. Subsequent pan- Pdx1+ pancreatic epithelium and Ngn3+ endocrine pro- creatic morphogenesis and differentiation of these pro- genitor cells [12,19,33]. As development proceeds, the genitor populations is dependent on the concerted action Nkx2.2-/- mice form the normal numbers of total endo- of multiple transcriptional regulators. Early during pan- crine and exocrine cells; however, within the endocrine creatic bud evagination, Pancreatic duodenal homeobox compartment all β cells, most α cells and a subset of PP 1 (Pdx1) and Pancreatic transcription factor 1a (Ptf1a) are cells fail to differentiate and are replaced by a ghrelin-pro- co-expressed in the pancreatic progenitor population ducing cell population, which persists to comprise the [1,2]. Ptf1a, a basic helix-loop-helix (bHLH) transcription majority of a misshapen islet. Nkx2.2-/- mice die within factor, becomes restricted to the exocrine cell population, days after birth with severe hyperglycemia [19,34]. Nkx2.2 where it is essential for exocrine cell formation and func- expression is initiated at e8.75 in the dorsal pancreas and tion [2,3]. Pdx1 expression is maintained throughout the e9.5 in the ventral pancreas, shortly after the activation of early pancreatic epithelium and becomes mostly restricted Pdx1 in each anlage [5]. High level Nkx2.2 expression to β and δ cells after the secondary transition, although subsequently becomes restricted to the centralized epithe- low levels of Pdx1 persist in some acinar cells into adult- lium at e11.5, where it is upregulated in a large number of hood [4-6]. Pdx1 has distinct functions at each develop- the Ngn3+ endocrine progenitor cells [5,12]. Cells express- mental stage and in each of the cell types where it is ing low levels of Nkx2.2 are also apparent throughout the expressed, and itself is critically dependent on the winged undifferentiated pancreatic epithelial population until helix transcription factors Foxa1 and Foxa2 [7-9]. Ngn3, a after the secondary transition [5]. Multiple in vitro and in bHLH transcription factor, is required downstream of vivo studies suggest that Nkx2.2 mediates early islet cell Pdx1 to activate the endocrine differentiation program fate decisions by functioning both as a transcriptional [10-13]. Further islet cell fate determination in the Ngn3+ repressor and activator, depending on temporal and cellu- cells then depends on a number of additional transcrip- lar context [35-39]. To date, however, MafA and insulin tion factors including Pdx1, Nkx2.2, Pax4, Pax6, Isl1, (Ins2) are the only two verified direct targets identified NeuroD1, Arx, and Nkx6.1, each of which has been iden- downstream of Nkx2.2 in the pancreas [35,38]. MafA and tified and characterized through genetic deletion or over- Ins2 both represent mature β cell genes; the molecular expression studies [14-19]. These and other transcription mechanisms by which Nkx2.2 controls early endocrine factors are then necessary for appropriate neogenesis, dif- specification remain largely unknown. In an effort to ferentiation, and maturation of the five islet cell types identify downstream direct and indirect targets of Nkx2.2 (recently reviewed in [5,20-22]). that promote or inhibit appropriate islet cell differentia- tion, we performed microarray analyses at e12.5 and Although much is known about the identity, expression e13.5 during pancreas development. This analysis has and function of many of the intrinsic regulatory proteins resulted in the identification and characterization of involved in pancreatic development and differentiation, expression differences for several known and previously the identification of downstream targets of these tran- uncharacterized pancreatic genes. These studies clarify the scription factors to mediate these developmental events position of Nkx2.2 within the known transcription factor has proven to be more challenging. In efforts to identify network that regulates islet specification. Furthermore, we downstream functional players during pancreagenesis, have identified a novel role for Nkx2.2 in the regulation of multiple groups have determined pancreatic gene expres- several exocrine-specific genes, suggesting that Nkx2.2 sion profiles of wild type and transcription factor knock- may also function to influence gene expression in the out models, including Pdx1, Ngn3, NeuroD1, and FoxA2 early Pdx1+ Ptf1a+ pancreatic progenitor cell. [23-30]. Alternatively, temporal comparisons between transcription factor positive cell populations, such as Results and Discussion Pdx1+ or Ngn3+ [31,32], by microarray analysis have also Gene expression differences between wild type and uncovered important downstream factors. Here we Nkx2.2-/- pancreata present the first study comparing gene expression altera- To identify genes that function downstream of Nkx2.2 tions between wild type and Nkx2.2-/- mouse pancreata at during early pancreas development, we compared gene e12.5 and e13.5, the stages marking the onset of the sec- expression profiles of e12.5 or e13.5 wild type and ondary transition when the major wave of islet cell type Nkx2.2-/- pancreata. We restricted our analysis to the onset specification events occur for both endocrine and exo- of the secondary transition, when