Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development

C2cd4c, encoded by a gene belonging to the C2cd4 family, contains a C2 domain conserved across species and is localized to the cytoplasm. To examine the role of C2cd4c in the pancreas, we studied its localization and generated C2cd4c knockout (KO) mice. C2cd4c was expressed in pancreatic endocrine progenitors at early embryonic stages. When endocrine cells arise from their precursors, C2cd4c is gradually confined to the insulin‐ and pancreatic polypeptide‐expressing cells of the endocrine. In the adult pancreas, C2cd4c is restricted to the beta cells. C2cd4c KO mice showed normal embryonic pancreatic development and adult pancreatic function. Thus, our results suggest that C2cd4c is dispensable for pancreatic development.

The pancreas is composed of acini that produce digestive enzymes for digestion of food, endocrine cells that produce hormones, and duct cells. All these lineages are derived from Pdx1-expressing multipotent pancreatic progenitor cells [1]. The endocrine compartment consists of hormone-producing endocrine cells, including insulinproducing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells, and pancreatic polypeptide-producing PP cells [2,3]. In mice, differentiation of pancreatic endocrine cells from the endocrine progenitor cells occurs approximately at embryonic day 14.5 (E14.5). Multipotent progenitors are known to exist in particular regions of the developing pancreas. Endocrine progenitor cells are located in the 'trunk region,' whereas acinar progenitor cells are located in the 'tip region' of the developing pancreas [4,5]. During this period, Nkx6.1-expressing early endocrine progenitor cells differentiate into Ngn3-expressing endocrine progenitor cells [1,6], which then give rise to insulin-expressing beta cells. After birth, pancreatic endocrine cells form the pancreatic islets [7]. Deletion of either Nkx6.1 or Ngn3 causes abnormal pancreatic endocrine development [8,9].
Previously we found that the gene C2 calcium-dependent domain-containing protein 4B (C2cd4b) is expressed in pancreatic b cells, as assessed by analyzing the gene expression profile of pancreatic progenitor cells derived from mouse ES cells. We confirmed the expression of C2cd4b in the embryonic pancreas by in situ hybridization [4]. C2cd4b and C2cd4a are members of the C2cd4 family, and the C2CD4A-C2CD4B locus has been identified as a risk factor for type 2 diabetes [10]. C2cd4c is another member of the C2cd4 family whose expression has not been reported. The C2cd4c gene product possesses the C2 domain with the Ca 2+ -binding motif, which is well conserved across species [11]. In contrast, C2cd4a and C2cd4b gene products do not have the C2 domain. In the present study, we focused on C2cd4c and generated C2cd4c/ LacZ knock-in (KI) mice to monitor its expression patterns and to investigate its functions.

Materials and methods
Reverse transcription and semiquantitative PCR analysis RNA was extracted from the cells using the RNeasy Micro-Kit (Qiagen, Heiden, Germany) and then treated with DNase I (Qiagen). Complementary DNA was synthesized from 3 lg of total RNA using ReverTra Ace (Toyobo, Osaka, Japan) and oligo(dT) primers. For the semiquantitative PCR analysis, DOD Dash (Toyobo) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 60°C for 2 s, and extension at 72°C for 45 s. RT-PCR products were separated by 5% nondenaturing PAGE, stained with SYBR Green I (Molecular Probes, Waltham, MA, USA), and visualized using KODAK 1D software (Eastman Kodak Company, Rochester, NY, USA). All primers used are listed in Table S1. Cell culture MIN6 and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Tokyo, Japan) supplemented with 10% FBS (Hyclone, Buckinghamshire, UK), 50 unitsÁmL À1 of penicillin, and 50 lgÁmL À1 of streptomycin (PS, Nacalai Tesque, Kyoto, Japan) in 5% CO 2 .
The expression vectors were transfected into MIN6 and COS-7 cells using Fugene HD (Promega, Madison, WI, USA), and analyzed with anti-FLAG or anti-HA antibodies. Fluorescent images of the transfected cells were acquired using a confocal microscopy (Leica TCS SP2 AOBS, Wetzlar, Germany).

Antibodies
For immunohistochemical analysis, rabbit anti-Pdx1

Measurement of blood glucose levels
Blood glucose levels were measured using the Life Check Sensor (Gunze, Osaka, Japan). For glucose tolerance testing, mice were fasted for 16 h, after which blood glucose levels were measured at indicated time points after intraperitoneal glucose administration at 2 mg per body weight (g) [17].

Results
C2cd4c is a C2 domain-containing protein localized to the cytoplasm Previously, we identified C2cd4b expression in pancreatic beta cells [4]. C2cd4b is a member of the C2cd4 family, consisting of C2cd4a, C2cd4b, C2cd4c, and C2cd4d. The C2cd4 family genes, except C2cd4d, were expressed in early stages in the embryonic pancreas and in the adult islets (Fig 1A,B). Among the C2cd4 family genes, C2CD4C and C2CD4D contain the C2 domain, which is well conserved across species (Fig 1C). Of note, C2CD4A and C2CD4B do not contain the C2 domain, and C2CD4D contains a shorter C2 domain.
A previous study suggested that C2CD4A and C2CD4B are localized in the nucleus in COS-7 cells [18]. We examined the intracellular localization of C2CD4C, by overexpressing Flag-tagged or HAtagged C2CD4C in MIN6 and COS-7 cells. With both N-terminal and C-terminal tags, C2CD4C was found to be localized in the cytoplasm, but not in the nuclei, of both cell types (Fig. 1D). These results suggested that C2CD4C functions might differ from those of C2CD4CA and C2CD4B. This encouraged us to study the role of C2cd4c.

Generation of C2cd4c/LacZ KI mice
To generate C2cd4c/LacZ KI mice, we obtained the JM8A3-A10 embryonic stem (ES) cell line from the KOMP Repository [12][13][14]. JM8A3-A10 ES cells with the LacZ gene inserted into the ORF of the C2cd4c locus were generated by homologues recombination (Fig. 2A). C2cd4c/LacZ KI ES cells were then injected into the blastocysts of C57/BL6 mice to generate C2cd4c/LacZ KI mice. To confirm the correct insertion of the LacZ gene into the C2cd4c locus, genomic DNA of the mutant or control wild-type mice was digested using EcoR1 restriction enzyme and processed for Southern blot analysis, using the 5 0 -or 3 0 -arm as the probes. In mutant mice, the 5 0 -probe and the 3 0 -probe detected products of 9.5 or 14.5 kb, respectively. In contrast, in wild-type mice, both probes detected  Fig. 2A,B). From these results, we confirmed the generation of C2cd4c/LacZ KI mice.

C2cd4c is expressed in the developing and in the adult pancreas
We then examined the expression of C2cd4c in the pancreas by visualizing LacZ activity. For this, we used Salmon-gal (S-gal; 6-chloro-3-indolyl-beta-D-galactopyranoside) in combination with Nitro Blue tetrazolium [15], or SPiDER-bGal, which is rendered fluorescent by the enzymatic reaction [16]. We visualized C2cd4c/LacZ activity during embryonic development (at E14.5-18.5) and in adult C2cd4c/LacZ KI heterozygous mice (Fig. 3). To clarify the localization of LacZ staining in the pancreas, we performed immunohistochemical analysis of pancreatic markers after S-gal or SPiDER-bGal staining (Fig. 3). At E14.5, LacZ activity visualized by S-gal staining was observed in Pdx1-expressing pancreatic epithelium. Particularly, LacZ staining overlapped with Pdx1-strong positive cells in the trunk region, which are known to give rise to the endocrine cells [5]. We stained with the endocrine progenitor markers Nkx6.1 and Ngn3 [8,9] and found that some LacZ-positive cells also expressed Nkx6.1 or Ngn3. The regions of LacZ-positive cells were smaller than those of Pdx1or Nkx6.1-expressing cells. However, Ngn3-expressing cells are fewer in number and seem to lie within the LacZ-positive region. Costaining for insulin, glucagon, or amylase revealed that the LacZ-positive cells overlapped with insulin-or glucagon-expressing endocrine cells, but not with the amylase-expressing acinar cells [3]. At E18.5, many SPiDER-bGal-positive cells overlapped with insulin-or pancreatic polypeptide-positive cells, but few overlapped with glucagon-or somatostatin-positive cells (Fig. 3B). We confirmed that S-Gal staining (Fig. 3C, upper panels) and SPiDER-bGal staining (Fig. 3C, lower panels) gave similar results in the adult islets. In the adults, LacZ-positive cells expressed insulin but none of the other endocrine    (Fig. 3C). Taken together, these results indicate that C2cd4c is expressed in early endocrine progenitors during the embryonic stages, then gradually localized to insulin-expressing b cells and PP cells, then solely in the insulin-expressing b cells in the islets of the adult pancreas.
C2cd4c KO mice were born following Mendelian distribution (Fig. 4B). The body weight of KO mice was slightly lower than that of C2cd4c +/À heterozygous mice at 2 months after birth (Fig. 4C). There seems no marked differences in food intake (H. Omori, unpublished). As C2cd4c was expressed in the endocrine region of the embryonic pancreas and in b cells of the adult, we examined pancreatic development of KO mice. At E14.5, the endocrine progenitor markers Ngn3 and Nkx6.1 were normally expressed in KO mice (Fig. 5A), and embryonic alpha or beta cells identified by glucagon or insulin expression were formed normally (Fig. 5B). Amylaseexpressing acinar cells and Dolichos biflorus Agglutinin (DBA)-expressing ductal cells were also normal in the KO mice (Fig. S1). We then examined the adult pancreas of KO mice. KO mice showed normal pancreas and islet morphology (Fig. 6A,B). We tested the pancreatic function of KO mice, because C2cd4c is expressed in b cells that function to maintain blood glucose homeostasis. Both nonfasted and fasting blood glucose levels in KO mice were normal (Fig. 7A,B). Intraperitoneal glucose tolerance test (IPGTT) also revealed that KO mice showed normal blood glucose tolerance after glucose challenge (Fig. 7A). We confirmed by semiquantitative RT-PCR analysis that no marked overexpression of C2cd4a or C2cd4b was observed in the C2cd4c KO mice (Fig. S2). These results therefore suggest that C2cd4c is dispensable for normal pancreatic development.

Discussion
Several groups have used comprehensive genome wide analysis to identify genes that play roles in the proliferation and differentiation of pancreatic beta cells [19]. Previously, we reported that C2cd4b is expressed in the embryonic pancreas [4]. Here, we focused on C2cd4c, which is well conserved across species and contains the C2 domain with the Ca 2+ -binding motif [20]. C2 domains are independently folded modules of about 130 residues, and form a compact b sandwich of two 4-stranded b-sheets [11,21]. Many of the C2 proteins are involved in membrane trafficking and fusion, and serve as Ca 2+ effectors for divergent Ca 2+ -mediated cellular processes [22]. The C2 domain-containing proteins such as synaptotagmin7, double C2-like domaincontaining proteins alpha and beta (Doc2a and Doc2b), and C2 domain-containing transmembrane protein 24 (Tmem24), are thought to bind to Ca2+ and function as Ca2+ sensors for the rapid phase of release in synaptic transmission or insulin exocytosis [21,[23][24][25]. In the pancreas, C2CD4C is the only C2CD4 family member that contains a C2 domain and is expressed from developing stages and in the adult islets. This suggests that C2cd4c might have some functions related to insulin exocytosis, which encouraged us to focus on C2cd4c.
C2cd4c is strongly expressed in the trunk region of the E14.5 embryonic pancreas, where endocrine progenitor cells are located, and in the adult islets. The period between E12.5 and E15.5 is known as the second transition of the embryonic pancreas, during which differentiation into the endocrine and exocrine cells is observed [7]; Nkx6.1 and Ngn3 are expressed in the trunk region [1,9]. We identified that C2cd4c expression overlapped with that of these endocrine progenitor markers. At E18.5, C2cd4c was expressed  in the insulin-expressing cells and in the pancreatic polypeptide-expressing cells, although only a few glucagon-expressing cells and somatostatin-expressing cells seemed to express C2cd4c. These results suggest that C2cd4c is gradually confined to the beta cells of the embryonic pancreas, and then becomes restricted to b cells in the adult.
C2cd4c KO mice were born following Mendelian distribution and were healthy. Fasting blood glucose levels and glucose tolerance were normal. However, the weight of KO mice was significantly less than that of heterozygous mice, which however seemed not due to less food intake by visual inspection. The reason for the lower body weight in the homozygous mice remains to be determined. Although C2cd4c is expressed during pancreatic development, the expression of pancreatic endocrine genes Ngn3, Nkx6.1, insulin, and glucagon was not affected in KO mice. Pancreatic islets also developed normally. Our results indicate that C2cd4c is not required for normal pancreatic development. However, C2cd4c might play a role in b cell regeneration and might show redundant roles with other members of the C2cd4 family, which remains to be investigated. Recently, Synaptagmin 4, lacking a C2 domain, was reported to have an inhibitory role for exocytotic activity [26]. Therefore, it might be useful to investigate further into the function of C2cd4c with respect to the other members of the C2cd4 family genes in the regulation of insulin exocytosis.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Acinar and duct cells are not affected in the knockout (KO) mice. Fig. S2. No marked increase in C2cd4a or C2cd4b expression in C2cd4c KO mice. Table S1. Primers used to detect gene expressions.