Pten -LRB- phosphatase and tensin homologue gene -RRB- haploinsufficiency promotes insulin hypersensitivity Aims/hypothesis Insulin controls glucose metabolism via multiple signalling pathways , including the phosphatidylinositol 3-kinase -LRB- PI3K -RRB- pathway in muscle and adipose tissue . The protein/lipid phosphatase Pten -LRB- phosphatase and tensin homologue deleted on chromosome 10 -RRB- attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K . The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake . Introduction Type 2 diabetes mellitus is a multifactorial disease with a complex pathophysiology that includes defects in insulin production and a failure in peripheral tissues to take up glucose in response to insulin . Signalling via phosphatidylinositol 3-kinase -LRB- PI3K -RRB- is a key pathway in the regulation of glucose uptake by insulin -LSB- 1 -RSB- . Activation of PI3K via insulin receptor substrate-1 causes PI3K to phosphorylate inositol-containing phospholipids at the 3 ′ - position of the inositol ring , increasing the levels of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate -LRB- PIP3 -RRB- , among others . PIP3 activate a phosphoinositide kinases , which in turn phosphorylate and activate protein kinase B -LRB- PKB -RRB- , also known as Akt , a key effector kinase for many of the downstream metabolic effects initiated by insulin via PI3K . The phosphatase and tensin homologue -LRB- PTEN -RRB- is a phosphatase which recognises both protein and lipid substrates . As a lipid phosphatase , PTEN dephosphorylates PIP3 at the 3 ′ - position of the inositol ring , thereby acting as an antagonist to PI3K signalling -LSB- 2 , 3 -RSB- . PTEN was originally identified as a tumour suppressor gene , and is one of the most commonly mutated genes in human cancer -LSB- 4 -- 6 -RSB- . Mutation or deletion of PTEN has been identified in many cancers including prostate , brain , breast , endometrium and skin -LSB- 4 -- 8 -RSB- . As many of the metabolic outcomes of insulin are achieved through recruitment of PI3K and the subsequent rise in PIP3 levels , PTEN may have a critical role in modulating sensitivity to insulin-stimulated glucose uptake . Recent studies in which Pten was ablated specifically in liver , adipose tissue and muscle in mice by Cre recombinase-based strategies have shown a role for Pten in the regulation of insulin sensitivity in those organs , and highlighted the contributions of those organs to glucose homeostasis of the whole animal -LSB- 9 -- 11 -RSB- . However , the effect of global reduction of Pten levels on glucose uptake and metabolism has not yet been shown in an in vivo mouse model . Unfortunately , experimental genetic inactivation of both Pten alleles -LRB- Pten − / − -RRB- results in early embryonic lethality -LSB- 6 , 12 -RSB- . Thus mice harbouring inactivation of one Pten allele -LRB- haploinsufficiency , Pten + / − -RRB- are an important experimental model for studying the role of Pten in vivo . Haploinsufficiency for Pten has important consequences for cell proliferation and tumorigenesis in mice -LSB- 6 , 12 -- 14 -RSB- . Pten + / − mice exhibit defective apoptosis in T cells , B cells and macrophages and spontaneous neoplasms in various tissues including prostate , liver , endometrium and others -LSB- 12 -- 14 -RSB- . Here we show that Pten haploinsufficiency results in insulin hypersensitivity and enhanced insulin-mediated glucose uptake . Materials and methods Experimental animals All laboratory animals were cared for and used according to guidelines of the Canadian Council on Animal Care . Pten + / − mice were generated by R. Parsons -LSB- 12 -RSB- . These mice were backcrossed with C57BL6 mice for more than ten generations . The genotypes of the mice were determined as described -LSB- 12 -RSB- . Blood glucose determination For determination of blood glucose levels , glucose levels in blood samples taken from tail vein were determined using a One Touch Ultra blood glucose monitor -LRB- LifeScan Inc. , Milpitas , CA , USA -RRB- . For the glucose tolerance test , mice were fasted overnight -LRB- 15 h -RRB- prior to i.p. injection of glucose -LRB- 2 mg/g body weight -RRB- . Blood glucose levels were then determined at the indicated time-points following glucose injection . For the insulin challenge test , mice were fasted for 15 h prior to i.p. injection of 0.6 mU insulin/g body weight . Blood glucose levels were then determined at the indicated time-points following insulin injection . Determination of insulin in plasma Mice were fasted for 15 h , then approximately 100 μl of blood was collected from the tail vein using pipette tips precoated with heparin . The mice were then injected i.p. with 2 mg glucose/g body weight , and a second blood sample was obtained 15 min following injection . The blood samples were centrifuged at 805 g in a table-top centrifuge , and insulin in the plasma fraction was determined using an insulin ELISA system -LRB- ALPCO Diagnostics , Salem , NH , USA -RRB- according to the manufacturer 's instructions . Isolation of pancreatic islets and glucose-stimulated insulin release Islets were isolated from pancreata of mice as described -LSB- 15 -RSB- . Pancreata from three mice were pooled . After 24 h in culture , islets were preincubated in Krebs -- Ringer bicarbonate buffer plus 0.1 % BSA -LRB- KRBB-BSA -RRB- and 1.67 mmol/l glucose . The islet cells were incubated for 1 h in 1 ml KRBB-BSA containing the desired concentration of glucose -LRB- 1.67 or 16.7 mmol/l -RRB- . The incubation medium was then collected , and centrifuged and the supernatant fractions were used for assay of insulin content by ELISA . Total extractable insulin in islets was determined by adding 0.5 ml lysis buffer -LRB- 1 mol/l acetic acid with 0.1 % BSA -RRB- to the islet pellet . Cell debris was pelleted by centrifugation , and the supernatant fraction was used for insulin assay . Fluorescent immunohistochemistry of pancreatic islets Immunocytochemistry has been described -LSB- 16 -RSB- . Pancreata from wild-type and Pten + / − mice were embedded in paraffin . Five-micron sections were incubated for 1 h at room temperature with guinea-pig anti-insulin antibody -LRB- 1:100 dilution ; Dako , Mississagua , ON , Canada -RRB- and rabbit anti-glucagon antibody -LRB- Dako ; 1:75 dilution -RRB- . Sections were visualised by incubation with Alexa -- Fluor 488 goat anti-guinea-pig -LRB- Invitrogen , Molecular Probes , Burlington , ON , Canada ; 1:100 dilution -RRB- and Texas Red donkey anti-rabbit antibodies -LRB- Jackson ImmunoResearch Laboratories , West Grove , PA , USA ; 1:100 dilution -RRB- . Sections were examined by fluorescence microscopy . Beta cell mass calculation Following insulin immunostaining , pancreatic sections were captured under an FITC filter using a Zeiss Axioplan 2 microscope equipped for epifluorescence and Pathvysion imaging software . The insulin immunopositive area -LRB- as a proportion of pancreatic area -RRB- was determined in at least six fields per section using Zeiss AxioVision software , performed on eight pancreatic sections per mouse . Beta cell mass was calculated as pancreatic mass times the percent beta cell area for each mouse . 2 - -LSB- 18F -RSB- fluoro-2-deoxyglucose uptake and microPET scanning Mice were fasted for 15 h prior to i.p. injection with 0.6 mU insulin/g body weight . Twenty minutes after insulin injection , mice were injected with 2 - -LSB- 18F -RSB- fluoro-2-deoxyglucose -LRB- 18FDG -RRB- -LRB- 4.1 MBq -RRB- via the tail vein , and whole-body distribution of 18FDG was monitored by positron emission tomography -LRB- PET -RRB- using a microPET R4 tomograph -LRB- Concorde Microsystems , Knoxville , TN , USA -RRB- . The image data were attenuation-corrected using a 68Ge rod source . Wild-type and Pten + / − mice were positioned side-by-side and scanned simultaneously for 60 min after 18FDG injection . Data were analysed using the manufacturer 's software , ASIPro VM 5.0.1.0 . For data analysis , regions of interest -LRB- ROIs -RRB- were specified in the coronal section of a hindlimb for each mouse . The 18FDG activities in each ROI were summed across all coronal planes , and expressed as a proportion of 18FDG in the whole mouse -LRB- total 18FDG activity was also taken as the sum of activity across coronal planes -RRB- . Primary myocyte culture Primary myocytes were cultured from fore - and hindlimbs from wild-type and Pten + / − mice according to a protocol adapted from Springer et al. -LSB- 17 -RSB- . Skeletal muscle was excised and minced and then incubated at 37 °C for 2 h with 2.4 units/ml dispase II . Dispase digestion was halted by addition of 10 ml F-10-based myocyte medium -LRB- 400 ml F-10 nutrient mixture , 100 ml fetal bovine serum , 25 μg basic fibroblast growth factor , penicillin/streptomycin -RRB- . The tissue slurry was then strained through a fine metal mesh and centrifuged at 500 g for 5 min . The supernatant fraction was removed to a fresh centrifuge tube and the pellet was resuspended and plated to a 5-cm collagen-coated culture dish in a total of 4 ml F-10-based medium . The supernatant fraction was re-centrifuged and the pellet was pooled with the first . The cells were passaged when they reached 70 -- 80 % confluence . To enrich for myocytes , the old medium was removed by aspiration , the cells washed with PBS , then incubated in a film of this buffer for 15 min . Myocytes were loosened from the dish by striking the dish several times sharply on the bench top . Cells were washed from the dish with PBS . The PBS incubation was repeated and the cells were pooled . Cells kept in culture for more than 5 -- 6 days began to exhibit the elongated morphology typical of myotubes -LSB- 18 -RSB- . Cells in these older cultures also exhibited spontaneous twitching . All experiments were performed with cells on days 2 -- 3 of culture . 2-Deoxy -LSB- 3H -RSB- glucose uptake Experiments were performed on wild-type or Pten + / − cells from passage two to eight , grown on 24-well -LRB- 1-cm diameter wells -RRB- plates . Cells were preincubated in serum-free medium for 12 h prior to experiments . The cells were washed twice with HEPES-buffered saline -LRB- 140 mmol/l NaCl , 20 mmol/l HEPES , 2.5 mmol/l MgSO4 , 1 mmol/l CaCl2 , 5 mmol/l KCl , pH 7.4 -RRB- , then incubated with 10 μmol/l 2-deoxy -LSB- 3H -RSB- glucose -LRB- 0.037 MBq/ml -RRB- for 5 , 10 , 20 or 30 min , in the absence or presence of 0.1 μmol/l insulin . At the end of the incubation period , the radioactive substrate was removed , and the cells were washed three times with ice-cold 20 mmol/l glucose . Cells were lysed with 0.2 ml 2 % SDS , and the lysates were used for scintillation counting . Non-specific diffusion of 2-deoxy -LSB- 3H -RSB- glucose was determined by incubating cells in the presence of 2-deoxy -LSB- 3H -RSB- glucose and 10 μmol/l cytochalasin B , a specific inhibitor of glucose uptake -LSB- 19 -RSB- . Determination of cell-surface GLUT1 and GLUT4 levels Primary myocytes were grown in 10-cm culture dishes . The cells were rinsed with PBS , and incubated with 0.5 mg/ml sulpho-NHS-LC-biotin -LRB- Pierce Biotechnology , Rockford , IL , USA -RRB- in PBS for 20 min . Cells are impermeable to this reagent and it labels the amine groups of cell-surface proteins . Following the incubation , the cells were washed three times with PBS , then lysed with 0.5 ml lysis buffer -LRB- 1 % NP-40 , 150 mmol/l NaCl , 50 mmol/l Tris pH 7.5 , including protease and phosphatase inhibitors -RRB- . Protein was quantified with a BCA protein assay kit -LRB- Pierce -RRB- . Streptavidin-agarose beads -LRB- Pierce -RRB- were pre-blocked by incubation with 2 % BSA in lysis buffer . Each sample -LRB- 1.2 mg protein -RRB- was incubated with the streptavidin beads -LRB- 100 μl , 50 % bead slurry -RRB- for 1 h at room temperature . The beads were washed five times , for 30 -- 60 min each wash , using lysis buffer containing 0.1 % SDS . After the final wash , 50 μl SDS sample-loading buffer were added , and heated at 95 °C for 10 min , then vortexed to elute the biotinylated proteins . The proteins in the supernatant fractions were analysed by SDS-PAGE , then transferred to nitrocellulose . Proteins were probed using polyclonal antibodies against GLUT1 and GLUT4 -LRB- also known as SCLA2A1 and SCLA2A4 , respectively -RRB- . Western immunoblotting for PKB , phospho-PKB and phospho-glycogen synthase kinase 3 -LRB- GSK3 -RRB- - α/β Total protein extracts of primary myocytes were prepared by lysing cells in NP-40-based lysis buffer . Thirty to fifty micrograms of protein per sample were analysed by SDS-PAGE , then transferred to nitrocellulose . Rabbit polyclonal anti-PKB , anti-phospho-PKB -LRB- Ser473 -RRB- and anti-phospho-GSK3-α / β -LRB- Ser21/9 -RRB- antibodies were obtained from Cell Signaling Technology -LRB- Beverly , MA , USA -RRB- . Mouse monoclonal anti-vinculin antibody was obtained from Sigma -LRB- St Louis , MO , USA -RRB- . Horseradish peroxidase-conjugated secondary antibodies were obtained from Dako . Protein bands were visualised using an enhanced chemiluminescence system and autoradiography by exposure to X-ray film . Statistical analysis Statistical analysis was performed using Student 's t test . Significance was assessed at the 95 % confidence level . Results As glucose metabolism may be altered due to a change in insulin sensitivity in Pten + / − mice , blood glucose levels in wild-type and Pten + / − mice were determined . Slight but statistically significant hypoglycaemia was observed in Pten + / − mice during ad libitum feeding -LRB- 11.3 % lower blood glucose in Pten + / − mice -RRB- -LRB- Table 1 -RRB- . Fasting caused a drop in glucose levels in both groups , with slight but significant hypoglycaemia in the Pten + / − group -LRB- 10.8 % lower blood glucose -RRB- relative to wild-type mice . An insulin challenge test revealed hypersensitivity to insulin in Pten + / − mice -LRB- Fig. 1a -RRB- . Following i.p. insulin injection -LRB- 0.6 mU/g body weight -RRB- , blood glucose levels in Pten + / − mice remained depressed at all time-points measured -LRB- up to 120 min -RRB- . In contrast , wild-type mice started to recover after 30 min , and glucose levels returned to fasting levels 120 min after injection . A glucose tolerance test showed that Pten + / − mice had greater glucose tolerance than wild-type mice -LRB- Fig. 1b -RRB- . After i.p. glucose injection , blood glucose levels in Pten + / − mice returned to fasting levels in less than 60 min , approximately twice as rapidly as in wild-type mice . Table 1Blood glucose levels in wild-type and Pten + / − mice during ad libitum feeding and after a 15-h fast Mean blood glucose ± SD -LRB- mmol/l -RRB- p value -LRB- independent Student 's t test -RRB- Wild-typePten + / − Ad libitum feeding7 .1 ± 0.4 -LRB- n = 5 -RRB- 6.3 ± 0.2 -LRB- n = 4 -RRB- 0.006 Fasting6 .5 ± 0.3 -LRB- n = 5 -RRB- 5.8 ± 0.3 -LRB- n = 5 -RRB- 0.008 Fig. 1Insulin hypersensitivity in Pten + / − mice . a Insulin challenge test . Mice were fasted for 15 h prior to i.p. injection of insulin -LRB- 0.6 mU/g body weight -RRB- . Blood glucose levels were determined at the indicated times . Means ± SEM for six wild-type mice -LRB- white circles -RRB- and six Pten + / − mice -LRB- black circles -RRB- . b Glucose tolerance test . Mice were fasted for 15 h prior to i.p. injection of glucose at 2 mg/g body weight . Means ± SEM for six wild-type mice -LRB- white circles -RRB- and seven Pten + / − mice -LRB- black circles -RRB- The observed difference in ability to normalise blood glucose levels could be due to differences in insulin sensitivity or islet function and insulin production . We therefore investigated insulin levels in the mice , as well as properties of pancreatic cell morphology and function . Fasting plasma insulin levels were slightly but significantly lower in Pten + / − mice -LRB- Table 2 -RRB- , which is consistent with increased peripheral insulin sensitivity . Fifteen minutes after i.p. glucose administration -LRB- 2 mg glucose/g body weight -RRB- , similar increases in plasma insulin were observed in the two groups -LRB- Table 2 -RRB- , suggesting that there is no marked difference in insulin production in the Pten + / − mice . Insulin released from islets isolated from Pten + / + and Pten + / − mice was also examined . Islets were cultured as described in Materials and methods , and incubated in the presence of low -LRB- 1.67 mmol/l -RRB- or high -LRB- 16.7 mmol/l -RRB- glucose . Insulin released into the medium was determined by ELISA and calculated as percentage of total insulin content in the islets . Per cent of total insulin release -LRB- presented as mean value ± SD -RRB- was similar under conditions of low glucose -LRB- Pten + / + , 3.3 ± 0.4 % vs Pten + / − , 2.8 ± 0.3 % ; p > 0.05 no significant difference , n = 3 per group -RRB- and high glucose -LRB- Pten + / + , 4.2 ± 0.7 % vs Pten + / − , 3.7 ± 0.2 % ; p > 0.05 no significant difference , n = 3 per group -RRB- . Table 2Plasma insulin levels in wild-type and Pten + / − mice after fasting , and 15 min after i.p. glucose administration -LRB- 2 mg glucose/g body weight -RRB- Mean plasma insulin ± SD -LRB- ng/ml -RRB- p value -LRB- independent Student 's t test -RRB- Wild-type -LRB- n = 7 -RRB- Pten + / − -LRB- n = 6 -RRB- Fasting0 .50 ± 0.040.36 ± 0.100.05Post-glucose challenge1 .8 ± 0.52.4 ± 0.4 > 0.05 -LRB- not significant -RRB- Immunofluorescence of pancreatic sections revealed similar morphology of islets of wild-type and Pten + / − mice -LRB- Fig. 2 -RRB- , suggesting Pten haploinsufficiency has no marked effect on islet morphology . Accordingly , beta cell mass was not found to be significantly different between wild-type and Pten + / − mice -LRB- Table 3 -RRB- . Fig. 2Pancreatic islet morphology and immunohistochemistry for insulin -LRB- green -RRB- and glucagon -LRB- red -RRB- in wild-type -LRB- a -RRB- and Pten + / − -LRB- b -RRB- mice . Pancreatic sections were immunostained as described in Materials and methods and visualised by fluorescence microscopy . Original magnification × 40Table 3Pancreatic beta cell mass of Pten + / + and Pten + / − littermates Mean beta cell mass ± SE -LRB- mg -RRB- Pten + / +1.24 ± 0.30 -LRB- n = 3 -RRB- Pten + / − 1.29 ± 0.15 -LRB- n = 3 -RRB- Skeletal muscle is an important tissue involved in insulin-stimulated glucose uptake -LSB- 10 , 20 -RSB- . In vivo glucose uptake was assessed by monitoring distribution of 18FDG into muscles in real-time by PET . 18FDG is transported into cells by glucose transporters and is phosphorylated by hexokinase , but is not a substrate for further metabolism in the glycolytic pathway -LSB- 21 -RSB- . To compare insulin-stimulated glucose uptake in wild-type and Pten + / − mice , the mice were fasted for 15 h , then injected i.p. with insulin -LRB- 0.6 mU/g body weight -RRB- 20 min prior to injection with 18FDG via the tail vein . Mice were imaged for 18FDG uptake into the hindlimb for 60 min -LRB- Fig. 3a -RRB- . 18FDG activity in hindlimbs was analysed as a proportion of total body activity , and was found to be higher in Pten + / − than in wild-type mice at all time-points -LRB- Fig. 3b -RRB- . Fig. 3Insulin-stimulated uptake of 18FDG in wild-type and Pten + / − mice . Mice were fasted for 15 h prior to i.p. injection of 0.6 mU insulin/g body weight . Twenty minutes after insulin injection , mice were injected with 18FDG via a tail vein , and whole-body distribution of 18FDG was monitored by microPET for 60 min . The experiment was repeated three times with similar results . Typical results are shown . a -- f Imaging of 18FDG distribution in coronal section ; wild-type mouse is on the left and Pten + / − mouse is on the right -LRB- a 5 min ; b 15 min ; c 25 min ; d 35 min ; e 45 min ; f 55 min -RRB- . Hindlimb muscles were highlighted as regions of interest , and 18FDG activity in each region of interest quantified across coronal planes at each time-point , as described in Materials and methods . g Graphical representation of relative 18FDG activity . Data are shown as proportion of 18FDG activity in hindlimbs relative to activity in whole body for wild-type mice -LRB- circles -RRB- and Pten + / − mice -LRB- triangles -RRB- -LRB- mean ± SD ; n = 3 per point -RRB- Tissues from Pten + / − mice exhibited slightly reduced levels of Pten protein relative to wild-type mice -LSB- 14 , 22 -- 24 -RSB- . In order to examine cellular glucose uptake and intracellular signalling events , primary myocytes were cultured from hindlimb muscle of wild-type and Pten + / − mice . In the absence of insulin , cells from Pten + / − mice showed increased 2-deoxy -LSB- 3H -RSB- glucose uptake at all time-points measured , to a maximum increase of twofold relative to wild-type cells at the 3 h time-point -LRB- Fig. 4a -RRB- . This result , together with the lower fasting glucose level of Pten + / − mice , suggest that the PI3K/PKB pathway was chronically activated in Pten + / − animals . In the presence of insulin , Pten + / − cells exhibited a further increase in glucose uptake , up to threefold greater than wild-type cells . This observation is consistent with the above results of the insulin and glucose challenge tests and 18FDG uptake experiment in showing a hypersensitivity to insulin due to Pten haploinsufficiency . Fig. 4a 2-Deoxy -LSB- 3H -RSB- glucose uptake in wild-type and Pten + / − myocytes . Myocytes were serum-starved for 12 h prior to incubation with 10 μmol/l 2-deoxy -LSB- 3H -RSB- glucose -LRB- 0.037 MBq/μmol/l -RRB- for the indicated times with 0 or 1 μmol/l insulin . Following incubation cells were lysed and aliquots of lysates were taken for determination of radioactivity by scintillation counting . Means ± SD for three separate determinations . White circles , wild-type cells 0 μmol/l insulin ; black circles , wild-type cells 1 μmol/l insulin ; white triangles , Pten + / − cells 0 μmol/l insulin ; black triangles , Pten + / − cells 1 μmol/l insulin . b Cell-surface GLUT1 and GLUT4 levels . Levels of cell-surface GLUT1 and GLUT4 proteins in wild-type -LRB- WT -RRB- and Pten + / − cells were determined as described in Materials and methods Glucose transport into skeletal muscle occurs by a facilitated diffusion process mediated primarily by the glucose transporter proteins , GLUT1 and GLUT4 . The level of GLUT4 at the plasma membrane is tightly regulated by insulin , whereas GLUT1 is found constitutively on the cell surface -LSB- 25 -RSB- . Since 2-deoxy -LSB- 3H -RSB- glucose uptake was elevated in Pten + / − cells even in the absence of insulin -LRB- Fig. 4a -RRB- , we analysed cell-surface GLUT4 and GLUT1 by western immunoblot as described in Materials and methods -LRB- Fig. 4b -RRB- . This analysis showed increased GLUT4 at the cell surface in Pten + / − cells , while similar GLUT1 levels were observed in the two cell types . As PKB is a key effector kinase that mediates many of the downstream cellular events induced by the PI3K signalling pathway , including insulin-stimulated glucose uptake -LSB- 26 , 27 -RSB- , we determined the phosphorylation status of PKB in wild-type and Pten + / − myocytes . Little to no phosphorylation was observed in the absence of insulin , and phosphorylation at Ser473 was observed upon addition of insulin -LRB- Fig. 5 -RRB- . Whereas PKB phosphorylation started to decrease after 4 h in wild-type cells in the presence of insulin , phosphorylation was sustained in Pten + / − cells during the course of the experiment -LRB- up to 6 h -RRB- . Immunoblotting for total PKB protein showed that PKB levels were consistent in Pten + / − and Pten + / + cells -LRB- Fig. 5 -RRB- . Immunoblotting for vinculin was included as a protein-loading control . An immediate downstream substrate of PKB is GSK3β . This enzyme becomes inactivated upon phosphorylation by PKB , in turn allowing its substrate , glycogen synthase , to mediate glycogen synthesis and thus promote glucose storage . Consistent with the observed enhancement of PKB phosphorylation in Pten + / − cells , these cells also exhibited a higher degree of GSK3β phosphorylation in comparison with Pten + / + cells -LRB- Fig. 5 -RRB- . Fig. 5Phosphorylation of PKB and GSK3β . Myocytes were serum-starved for 12 h prior to incubation with 1 μmol/l insulin for the indicated times . Cell lysates were prepared , and proteins were separated by PAGE followed by western immunoblot analysis for vinculin , total PKB , phospho-PKB -LRB- p-PKB S473 -RRB- and phospho-GSK3β -LRB- p-GSK3β -RRB- as indicated . This experiment was repeated three times with typical results shown . WT , wild-type Discussion Recent studies utilising tissue-specific deletion of Pten in liver , adipose and muscle have shown that these organs make important contributions to the glucose homeostasis of the animal and clearly establish Pten as a negative regulator of insulin signalling in vivo -LSB- 9 -- 11 -RSB- . In a separate report , in vivo antisense treatment resulted in 90 % reduction of Pten levels in the liver of diabetic mice , normalising blood glucose levels and improving performance in an insulin tolerance test -LSB- 28 -RSB- . While the above studies focused on the role of Pten in specific tissues , the current work defines the effects of partial systemic reduction of Pten on the regulation of insulin sensitivity in the context of the whole animal . PTEN expression/activity is tightly regulated by a complex mechanism that is currently poorly understood . PTEN is transcriptionally regulated by transcription factors such as p53 , Egr-1 , NFκB and SMADs , while protein levels and activity are modulated by phosphorylation , oxidation , subcellular localisation , phospholipid binding and protein stability -LSB- 29 -RSB- . Previous studies showed that Pten haploinsufficiency results in only a 15 -- 50 % reduction in Pten protein , leaving a substantial amount of Pten protein remaining in the tissues examined -LSB- 14 , 22 -- 24 , 30 -RSB- . In our hands , western blot analysis indicated similar decreases in tissue Pten levels -LRB- data not shown -RRB- . The present study provides the first evidence that Pten haploinsufficiency leads to insulin hypersensitivity in vivo , suggesting that even small changes in Pten levels or activity can lead to dramatic effects on insulin responses . Thus partial systemic inhibition of PTEN activity may represent a novel strategy to ameliorate the pathology of type 2 diabetes . The PI3K/PKB signalling axis is central to maintenance of normal glucose homeostasis -LSB- 1 -RSB- . Pten-deficient cells typically have elevated levels of intracellular PIP3 -LSB- 31 , 32 -RSB- . The sustained phosphorylation of PKB/Akt and a downstream effector , GSK3β , observed in the current study is consistent with previous reports of elevated PKB/Akt activation as a result of impaired desensitisation of PI3K-dependent signals under conditions of Pten inactivation or inhibition -LSB- 9 -- 11 , 28 -RSB- . A subtle reduction of Pten in the Pten + / − mice appears sufficient to achieve a prolonged and robust insulin signal . The roles of PIP3 and PKB/Akt in insulin-mediated glucose uptake have been well-established ; therefore , our data collectively suggest that sustained PI3K/PKB/Akt / GSK3 signalling may , in part , contribute to insulin hypersensitivity in Pten + / − mice . However , other PKB/Akt-independent signalling pathways , as well as additional signals delivered by the protein phosphatase and/or adaptor functions of Pten may also contribute to the insulin hypersensitivity phenotype manifested by Pten haploinsufficiency . GLUT1 glucose transporters are constitutively present at the cell surface , while GLUT4 cycles between intracellular vesicles and the cell surface in a manner controlled by insulin-mediated signalling mechanisms -LSB- 25 -RSB- . In unstimulated fat and muscle cells , the majority of GLUT4 exists in intracellular compartments , while the insulin signal stimulates the exocytosis of GLUT4 to the cell surface . Studies involving PKB/Akt-null animals and silencing of PKB/Akt by RNA interference have demonstrated that PKB/Akt is of primary importance in GLUT4 translocation -LSB- 33 -- 36 -RSB- . In the current study , we observed an enhancement of 2-deoxyglucose uptake in Pten + / − cells even in the absence of insulin stimulation , which was consistent with our observation of increased biotinylation of GLUT4 at the cell surface , while GLUT1 levels were unaffected . However , we detected no increase in phosphorylation of PKB/Akt in the absence of insulin in Pten + / − cells . Consistent with these observations , a recent study in which Pten was specifically knocked down by RNA interference reported slight but significant enhancement of deoxyglucose uptake in adipocytes , but no apparent increase in phosphorylation of PKB/Akt -LSB- 37 -RSB- . These results could reflect a mechanism -LRB- s -RRB- affecting glucose transport involving Pten/PIP3 but independent of PKB/Akt . Such a mechanism could involve the atypical protein kinases C-λ and - ζ -LRB- PKC-λ and PKC-ζ -RRB- , which , like PKB/Akt , are subject to control by PI3K via PIP3 and 3-phosphoinositide-dependent protein kinase-1 -LRB- PDK-1 -RRB- -LSB- 38 -- 40 -RSB- . A number of studies have implicated PKC-λ/ζ in insulin-stimulated glucose transport and GLUT4 translocation -LSB- 38 , 41 -- 43 -RSB- . These studies indicate a role for PKC-λ/ζ which appears to be complementary or parallel to that of PKB/Akt . The importance of the PI3K pathway in insulin signalling has raised interest in developing pharmaceutical antagonists targeting PIP3 phosphatases such as PTEN or Sh2-containing inositol 5 ′ - phosphatase 2 -LRB- SHIP2 ; also known as Inppl1 -RRB- in an effort to enhance insulin responsiveness in type 2 diabetes -LSB- 44 -RSB- . While a study on Ship2-null mice initially implicated Ship2 as a key regulator of glucose uptake -LSB- 45 -RSB- , it was subsequently recognised that these mice also had deleted a neighbouring gene , Phox2a , which may have contributed to the observed phenotype -LRB- corrigendum appears in Nature vol . 431 , p. 878 -RRB- . A new gene-targeted mouse has since been generated in which only the gene encoding Ship2 is deleted -LSB- 46 -RSB- . Interestingly , this mouse does not exhibit insulin hypersensitivity but is protected against diet-induced obesity , -LSB- 46 , 47 -RSB- . In the light of these new findings , our observations of dramatic enhancement of insulin sensitivity and glucose uptake resulting from Pten haploinsufficiency suggests that PTEN plays a role in the desensitisation of insulin signalling , and strengthens the hypothesis that PTEN is a key regulator of insulin-stimulated glucose uptake . Type 2 diabetes is associated with insulin deficiency and resistance to insulin signalling -LSB- 48 , 49 -RSB- . The current study provides further support for PTEN as a candidate for drug intervention in treating type 2 diabetes . However , enthusiasm for systemic PTEN inhibitors as a therapeutic approach for treating type 2 diabetes is tempered by the potential side-effects of such agents . Most notably , a modest reduction of Pten in Pten + / − mice promotes tumour formation in a wide variety of tissue , suggesting that long-term chronic administration of systemic PTEN inhibitors may lead to enhanced cancer development and progression . Furthermore , the PI3K pathway is an important regulator of numerous normal cellular processes and systemic PTEN inhibition may cause dysfunction of many cells and tissues , leading to alterations in liver , kidney , brain function , etc. . For example , hepatocyte-specific Pten-deficient mice exhibit hepatomegaly and liver steatosis -LSB- 9 -RSB- . Systemic inhibition of PTEN may therefore be unacceptable clinically because of these inherent potential toxicities -LSB- 44 -RSB- . However , targeted delivery of PTEN antagonists to muscle cells or adipocytes may overcome these limitations and they hold promise as clinically viable agents in treatment of type 2 diabetes -LSB- 44 -RSB- . This study shows that partial inhibition of Pten is sufficient to enhance insulin sensitivity . This observation is important from a therapeutic standpoint , as most drugs are unlikely to completely inhibit enzyme activity in vivo , nor would complete inhibition be desirable given the potential for cancer development resulting from chronic or overactivation of the PI3K/PKB pathway -LSB- 12 , 14 , 23 -RSB- . In conclusion , Pten haploinsufficiency results in insulin hypersensitivity , which underscores the importance of PTEN in attenuating insulin signals , and suggests that small-molecule drugs that antagonise PTEN activity may represent a novel class of insulin-sensitising agents and targeted delivery of these agents to muscle cells or adipocytes may have potential clinical utility in treating insulin resistance observed in type 2 diabetes .

O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

glucose uptake
insulin hypersensitivity
insulin sensitivity
pten haploinsufficiency


Redundancy and evolution of GATA factor requirements in development of the myocardium The transcription factors , GATA4 , 5 and 6 , recognize the same DNA sequence and are all expressed in the developing myocardium . However , knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium . Here we present evidence for redundancy in this family for the first time . Using morpholinos in both Xenopus and zebrafish embryos , we show that GATA4 knockdown , for example , only affects cardiac marker expression in the absence of either GATA5 or GATA6 . A similar situation pertains for GATA5 in Xenopus whereas , in zebrafish , GATA5 -LRB- faust -RRB- plays a major role in driving the myocardial programme . This requirement for GATA5 in zebrafish is for induction of the myocardium , in contrast to the GATA6 requirement in both species , which is for differentiation . This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling , suggesting that a mutual maintenance loop for GATA , BMP and Nkx expression is the evolutionarily conserved entity . Introduction The GATA factors are zinc finger transcriptional activators that bind to the consensus DNA sequence -LRB- A/T -RRB- GATA -LRB- A/G -RRB- . They have been identified throughout eukaryotes and been shown to play critical roles in both haematopoiesis and cardiogenesis in vertebrates and Drosophila -LRB- Fossett and Schulz , 2001 ; Nemer and Nemer , 2001 -RRB- . Of the six evolutionarily conserved GATA genes in vertebrates , GATA4 , 5 and 6 are expressed in the heart as it develops . Loss and gain of function studies in P19 embryonal carcinoma cells indicated a requirement for GATA4 in the differentiation of cardiac restricted cells to beating cardiomyocytes -LRB- Grepin et al. , 1997 , 1995 -RRB- . In addition , overexpression of GATA4 in Xenopus embryos and explants resulted in expression of cardiac differentiation markers and in some cases spontaneously beating tissue -LRB- Jiang and Evans , 1996 ; Latinkic et al. , 2003 -RRB- . However , in the GATA4 null mouse , normal amounts of myocardial tissue appeared to be formed -LRB- Holtzinger and Evans , 2005 ; Kuo et al. , 1997 ; Molkentin et al. , 1997 ; Narita et al. , 1996 -RRB- . Thus , even though cardia bifida and defects in looping morphogenesis were observed in the null mouse embryos , specification of the myocardium appeared to take place normally . A suggested explanation for this was the elevated expression of GATA6 -LRB- Holtzinger and Evans , 2005 ; Kuo et al. , 1997 ; Molkentin et al. , 1997 ; Narita et al. , 1996 ; Pu et al. , 2004 -RRB- . Consistent with this proposed redundancy of function within the family , GATA5 and 6 are also active in the P19 cell line and Xenopus explant assays described above . Thus , it appears that each of these three GATA family members possesses the capability of inducing cardiac differentiation in gain-of-function assays , however demonstration that they exhibit such redundancy in vivo awaits combinatorial loss-of-function assays . The GATA5 null mouse shows no cardiac phenotype , however it may not be a true knockout due to the potential formation of a truncated protein containing the DNA binding domain -LRB- Nemer and Nemer , 2002 -RRB- . In the zebrafish , a critical role for GATA5 in specification of the myocardium has been demonstrated by loss and gain of function assays -LRB- Reiter et al. , 1999 -RRB- . The fausttm236 mutant shows a severe reduction in expression of cardiac markers and injection of GATA5 RNA induces ectopic expression of the same markers . However , GATA4 expression in the zebrafish fausttm23 mutant is significantly reduced and overexpression of GATA5 results in ectopic expression of GATA4 . Thus , these studies raise the possibility that the GATA5 knockdown phenotype is due to the combined loss of GATA4 and 5 . The GATA6 null mouse is an embryonic lethal due to an extra-embryonic defect and chimeras have indicated that GATA6 is not required for specification of the myocardium -LRB- Kabrun et al. , 1997 ; Koutsourakis et al. , 1999 ; Kuo et al. , 1997 ; Molkentin , 2000 ; Molkentin et al. , 1997 ; Morrisey et al. , 1997 -RRB- . However , we have presented evidence that GATA6 is required for the maintenance and differentiation of cardiac progenitors in zebrafish and Xenopus embryos -LRB- Peterkin et al. , 2003 -RRB- . The likely resolution of these apparently contradictory data is that the major consequence of lost GATA6 function is non-cell autonomous and can therefore be rescued by surrounding wild type cells in mouse chimeras . The likely non-cell autonomous target for GATA6 is BMP -LRB- Peterkin et al. , 2003 -RRB- . However , this requirement for GATA6 is for differentiation of cardiac progenitors and not for their initial specification . In this study we use antisense morpholinos in Xenopus and zebrafish embryos to deplete combinations of GATA4 , 5 and 6 for the first time . This has allowed us to provide the first experimental support for redundancy in vivo . In addition , we show that the strong dependence of zebrafish on GATA5 is not mirrored in Xenopus , where this GATA factor plays only a redundant role , like GATA4 in both species . The requirement for GATA5 in the zebrafish is for the induction of the myocardial programme , whereas in Xenopus , GATA activity is only required for differentiation . We propose that the primary function for GATA factors in development of the myocardium is in creating a sub-circuit of the regulatory network , involving another critical transcription factor , Nkx , and a crucial signalling pathway , BMP . This mutually supportive sub-network is evolutionarily stable , even though where the network is initiated appears to be more flexible . Materials and methods In situ hybridisation of whole-mounted and sectioned embryos Xenopus and zebrafish were maintained and embryos were raised and staged using standard conditions -LRB- Nieuwkoop and Faber , 1967 ; Westerfield , 1993 -RRB- . In situ hybridisations on whole-mounted and sectioned embryos were carried out as previously described -LRB- Ciau-Uitz et al. , 2000 ; Jowett , 2001 ; Walmsley et al. , 1994 -RRB- . All RNA probes used were labelled with digoxigenin -LRB- DIG -RRB- except for MyoD and Krox20 which were used in double in situ hybridisations and labelled with fluorescein . Detection of the antibody -- alkaline phosphatase was done using BM purple -LRB- Roche -RRB- or Fast red -LRB- Sigma -RRB- . After in situ hybridisation , embryos were re-fixed in 4 % paraformaldehyde , zebrafish embryos were transferred into 75 % glycerol to be photographed . Cryostat sections were performed after in situ hybridisation , embryos were fixed as above and washed in 30 % sucrose . Embryos were transferred into embedding chambers in O.C.T Compound -LRB- Tissue-Tek -RRB- and 30 μm sections were cut on a Leica CM3050S . Morpholino -LRB- MO -RRB- injection The GATA4/5/6 antisense morpholinos were designed and manufactured by Genetools . Morpholino sequences : Xenopus GATA4 MO 5 ′ ctggcaactcaatccacaaaatcca3 ′ -LRB- data shown here -RRB- , a second morpholino designed against the same pseudo-allele as described by Afouda et al. -LRB- 2005 -RRB- -LRB- data not shown , 5 ′ agctatactctgatacatcctgatc3 ′ -RRB- , and a third GATA4 MO designed to block both pseudo-alleles -LRB- a kind gift from Todd Evans -RRB- -LRB- data not shown -RRB- gave the same results . Zebrafish GATA4 MO 5 ′ gccatcgttacaccttgatacatat3 ′ or a second splice morpholino as described by Holtzinger and Evans -LRB- 2005 -RRB- . For Xenopus GATA5 MO 5 ′ gctacaaacctcacagctcc3 ′ see Afouda et al. -LRB- 2005 -RRB- . Zebrafish splice GATA5 MO 5 ′ tgttaagatttttacctatactgga3 ′ . For Xenopus and zebrafish GATA6 MOs see Peterkin et al. -LRB- 2003 -RRB- . MOs were diluted in deionised water and injected as described -LRB- Peterkin et al. , 2003 -RRB- . For zebrafish , 25 ng GATA4 MO , 25 ng GATA5 MO and/or 5 ng GATA6 MO were injected into single-cell embryos individually and in combination . For Xenopus embryos , a total of 20 ng of GATA4 MO or GATA5 MO and 10 ng of GATA6 MO were injected individually or in combination . Results GATA6 is the only essential GATA activity in Xenopus myocardium We have reported previously that GATA6 is required for the maintenance and maturation of cardiomyocytes in Xenopus -LRB- Peterkin et al. , 2003 -RRB- . The phenotypes consequential upon depletion of GATA4 or 5 in Xenopus , however , have not been previously reported . In the case of GATA5 , the need to know is made greater because of its major contribution in zebrafish , and the inability to determine if this is a general requirement in vertebrates by comparison with the mouse knockout , because the reported mutation in the mouse appears not to be a null -LRB- Nemer et al. , 1999 -RRB- . Therefore , before examining depletion of combinations of GATA factors , we examined the individual loss of GATA4 and 5 in comparison to the already known GATA6 phenotype . The design and quality control of MOs against Xenopus GATA4 , 5 and 6 have been reported previously -LRB- Peterkin et al. , 2003 ; Afouda et al. , 2005 -RRB- . For GATA4 , as well as the MO reported previously , two other MOs , one against both pseudo-alleles -LRB- Todd Evans , personal communication -RRB- , were tested and gave the same results . For GATA5 and 6 , MOs were designed to target both pseudo-alleles of the Xenopus laevis genes . The optimal amount of each MO injected was determined by titration to ensure that the maximum dose without non-specific effects was used . The extent of knockdown by these ATG MOs was determined by co-injection of tagged reporter RNAs followed by Western blotting -LRB- Afouda et al. , 2005 ; Peterkin et al. , 2003 -RRB- . Very little residual protein was detected in each case . When MOs against individual GATA factors were injected separately into the presumptive heart field , the dorsolateral marginal zone , of 4-cell Xenopus embryos , cardia bifida was induced in each case -LRB- Fig. 1A , visualised by staining the cells for expression of Myosin Light Chain 2 -LRB- MLC -RRB- -RRB- . Cardia bifida has been reported previously for the GATA4 knockout mouse -LRB- Kuo et al. , 1997 ; Molkentin et al. , 1997 -RRB- , the GATA5 mutant zebrafish , faust -LRB- Reiter et al. , 1999 -RRB- , and GATA6 morphant Xenopus and zebrafish embryos -LRB- Peterkin et al. , 2003 -RRB- , but this is the first direct comparison in a single species showing that all three GATA factors are required for the timely migration of cardiac precursors to the midline for fusion of the heart tube . This is in contrast to requirements in the differentiation of the myocardium -LRB- see below -RRB- , where only one member of the family is essential . It seems likely that the requirement for GATA4 , 5 and 6 in midline migration of myocardial precursors is actually in the underlying endoderm , where they are all expressed and which has been shown to be essential in mouse and zebrafish for heart tube fusion -LRB- Afouda et al. , 2005 ; Alexander et al. , 1999 ; Molkentin et al. , 1997 ; Narita et al. , 1997 ; Reiter et al. , 1999 ; Weber et al. , 2000 -RRB- . To determine the effects of the GATA MOs on programming of the myocardial cells , as opposed to their morphological movements , the levels of expression of the transcription factors , Nkx2 .5 and Tbx5 , and of the contractile machinery genes , cardiac actin -LRB- CA -RRB- , and MLC , were monitored by whole mount in situ hybridisation . In contrast to the GATA6 MO , which causes a profound reduction in the expression of these genes -LRB- Peterkin et al. , 2003 -RRB- -LRB- Fig. 1D -RRB- , GATA4 and GATA5 MOs had minimal effects -LRB- Figs. 1B , C ; for all three MOs and for each marker n was 30 -- 50 -RRB- . Despite the cardia bifida at tailbud stages -LRB- stages 28 -- 32 -RRB- -LRB- Fig. 1A -RRB- , the gross morphology of the hearts at later stages -LRB- stage 43 -RRB- in GATA4 and GATA5 morphants looked similar to those in wild type embryos , i.e. the cardia bifida was only transient -LRB- data not shown -RRB- . In contrast , as previously described -LRB- Peterkin et al. , 2003 -RRB- , little or no cardiac tissue was observed in embryos depleted of GATA6 -LRB- data not shown -RRB- . Thus , it would appear that , apart from the transient bifida , the loss of GATA4 or GATA5 has little effect on cardiogenesis in Xenopus . To ensure that the GATA4 and 5 morpholinos were properly functional , they were injected vegetally at the single-cell stage , and were shown to reduce the expression of Sox17α during gastrulation -LRB- data not shown -RRB- -LRB- Afouda et al. , 2005 -RRB- . Furthermore , the gut of GATA5 morphants failed to coil properly , as previously reported -LRB- Afouda et al. , 2005 -RRB- . In addition , for these and several of the combination experiments described below , all three GATA4 MOs gave the same results . We therefore conclude that for development of the myocardium in Xenopus embryos , GATA6 is the only essential GATA factor . GATA factor redundancy in Xenopus myocardium On the basis of slightly increased expression of GATA6 in GATA4 knockout mice , redundant roles for the GATA factors in the myocardium have been suggested -LRB- Kuo et al. , 1997 ; Molkentin et al. , 1997 ; Narita et al. , 1996 ; Watt et al. , 2004 -RRB- . In Xenopus , expression of neither GATA5 nor GATA6 was significantly increased in GATA4 MO injected embryos -LRB- data not shown -RRB- . Similarly , in GATA5 and GATA6 MO injected embryos : in neither case was an increase in expression of the other two GATA factors observed -LRB- data not shown -RRB- . However , redundancy does not necessarily depend on an increase in expression of the redundant family member : continued expression could suffice , and that is what we see in all three cases . Therefore to formally test redundancy within the GATA family , we injected combinations of MOs into the presumptive heart field of 4-cell Xenopus embryos , and monitored MLC and Nkx2 .5 expression by whole mount in situ hybridisation -LRB- Fig. 2 -RRB- . Embryos were classified as unaffected -LRB- wild type , + -RRB- , mildly -LRB- − -RRB- or strongly -LRB- − − -RRB- down regulated , or displaying no expression at all -LRB- − − − -RRB- -LRB- Fig. 2A -RRB- . Numbers of embryos in each category were scored and the results displayed in histograms -LRB- n = 31 -- 94 -RRB- -LRB- Figs. 2B , C -RRB- . The greater effects of the GATA6 MO are immediately apparent , with clear increases in the affected categories at the expense of the wild type category compared to both control embryos and also to GATA4 or GATA5 MO injected embryos . When combinations of two MOs were injected , evidence for redundancy was revealed -LRB- Figs. 2B , C -RRB- . Despite having little effect on their own , both GATA4 and GATA5 MOs made the phenotype of GATA6 MO injected embryos more severe when injected with it . Furthermore , the phenotype observed when GATA4 and 5 MOs were injected in combination was significantly worse than either alone , suggesting that the minimal phenotype for the single injections relied on the continued presence of the other GATA factor . When all three MOs were injected together , the phenotype was the most extreme of all with the vast majority of embryos having no expression of MLC at all . We therefore conclude that , while GATA6 is the only individually essential player in driving the myocardial programme in Xenopus , the other two GATA factors are responsible for the residual expression of cardiac genes . Furthermore , in the absence of GATA6 , their roles are increased . This is evident from their significantly greater effects on embryo phenotypes when combined with GATA6 MO compared to on their own . GATA activity is required for differentiation but not induction of the myocardium in Xenopus We have shown previously that GATA6 is required for the maintenance/maturation of the myocardium rather than its induction in both Xenopus and zebrafish embryos -LRB- Peterkin et al. , 2003 -RRB- . As expected , based on the absence of a late phenotype , embryos injected with GATA4 or GATA5 MOs had no effect on early Nkx2 .5 expression , as seen for GATA6 MO -LRB- n = 60 , 72 , and 89 , respectively -RRB- -LRB- Fig. 3A -RRB- . In order to determine if the lack of an early effect , even for GATA6 which has a strong late phenotype , was the result of redundancy within the GATA family , we examined Nkx2 .5 expression at neurula stages in embryos injected with all three MOs -LRB- n = 102 -RRB- -LRB- Fig. 3A -RRB- . Expression was unaffected , as seen with each of the MOs on their own . Although Nkx2 .5 is also expressed in the underlying endoderm at this time , we showed by examining sections that the signal in the cardiac mesoderm is unaffected -LRB- Fig. 3B , territory delineated by dashed lines -RRB- . Furthermore , a similar result was obtained for Nkx2 .3 -LRB- n = 55 -RRB- -LRB- Fig. 3C -RRB- , which is not expressed or is very weak in the endoderm at this time -LRB- Fig. 3D -RRB- . The expression of eHAND was also unaffected at this stage -LRB- Fig. 3E , territory delineated by dashed lines -RRB- . We therefore conclude that , despite their earlier expression , GATA factors are not required for induction of the myocardial programme in Xenopus , as seen by the continued expression of the other early regulators , Nkx2 .5 , Nkx2 .3 and eHAND , and their own continued expression , but rather for its maintenance/maturation . GATA4 is not essential for induction or differentiation of zebrafish myocardium GATA5 -LRB- faust -RRB- mutant zebrafish have profound defects in the myocardium , displaying reduced expression of several myocardial genes -LRB- Reiter et al. , 1999 -RRB- . In addition , GATA6 has been shown to be required for maintenance/maturation of the myocardial programme in zebrafish as seen in Xenopus -LRB- Peterkin et al. , 2003 -RRB- . In order to determine the relative effects of these two GATA factors , and to determine the contribution of GATA4 , we separately injected into zebrafish embryos MOs against each of these GATA factors . The GATA4 MO was shown to specifically block translation of a co-injected GATA4 RNA and not GATA5 or GATA6 RNAs -LRB- Supplementary Figs. 1A , B , C -RRB- . The GATA5 MO was designed to block splicing between exons 1 and 2 of the GATA5 gene , which was confirmed in injected embryos by RT -- PCR -LRB- Supplementary Figs. 1D , E -RRB- . This splice blocking morpholino was designed upstream of the exons encoding the zinc fingers to prevent any protein produced binding DNA . However , the creation of a dominant negative GATA5 via splicing from an upstream cryptic site is formally possible -LRB- see Supplementary Fig. 1D -RRB- but the ability of GATA4 and 6 morpholinos to enhance the cardiac phenotype in combinations -LRB- see below -RRB- makes this unlikely . Furthermore , the GATA5 morphant heart phenotype was indistinguishable from that seen in the faust mutant , both in single and combination experiments -LRB- Supplementary Fig. 1F and see below -RRB- . The GATA6 MO has been reported previously -LRB- Peterkin et al. , 2003 -RRB- . The effects of the three MOs injected separately into zebrafish embryos were determined by monitoring expression of the transcription factor , nkx2 .5 , and the contractile machinery genes , ventricular myosin heavy chain -LRB- vmhc -RRB- and cardiac myosin light chain 2 -LRB- cmlc2 -RRB- -LRB- Fig. 4 -RRB- . GATA5 and 6 MOs induced cardia bifida as described previously for the faust mutant and the GATA6 MO -LRB- Peterkin et al. , 2003 ; Reiter et al. , 1999 -RRB- . In contrast , in GATA4 MO injected embryos , the myocardial cells appeared to have migrated and fused normally at the midline . We therefore conclude that , in zebrafish , only GATA5 and 6 are required for the proper migration of cardiac precursors . In contrast to mice and Xenopus , GATA4 appears to be uninvolved in this process . The previously reported effects on myocardial gene expression of GATA5 or GATA6 knockdown -LRB- Peterkin et al. , 2003 ; Reiter et al. , 1999 -RRB- were immediately evident in these MO injected embryos -LRB- Fig. 4 -RRB- . GATA5 MO injection led to substantially reduced expression of nkx2 .5 -LRB- 19/19 -RRB- , vmhc -LRB- 42/43 -RRB- and cmlc2 -LRB- 40/41 -RRB- , as seen for the faust mutant . GATA6 MO injection also resulted in reduced expression of these markers -LRB- 6/6 , 60/60 and 42/44 -RRB- but to a lesser extent . In contrast , GATA4 MO injection had little or no effect on cardiac marker gene expression levels -LRB- n = 28 , 69 and 53 -RRB- . Spatially the expression of the markers in the GATA4 morphants looks altered compared with the controls due to defects in late cardiac morphogenesis , consistent with those described by Holtzinger and Evans -LRB- 2005 -RRB- . We therefore conclude that for laying down the myocardial programme in zebrafish , GATA5 has the greatest effect with a significant contribution from GATA6 . In contrast , GATA4 makes little or no contribution , at least to the expression of the markers tested . GATA factor redundancy in zebrafish myocardium To determine if , as in Xenopus , there is redundancy within the GATA family in zebrafish , we injected the MOs in combinations -LRB- Fig. 4 -RRB- . Morphant embryos were classified into three types , unaffected -LRB- type + -RRB- , down regulated -LRB- type − -RRB- or absent -LRB- type − − -RRB- . Both the GATA5 and the GATA6 MO phenotypes were made worse by the co-injection of the GATA4 MO -LRB- Figs. 4B , D and F -RRB- , as seen in Xenopus , and despite the fact that the GATA4 MO had little or no effect when injected on its own -LRB- n = 18 -- 39 -RRB- . We therefore conclude that a significant proportion of the residual cardiac gene expression in GATA5 or GATA6 MO injected embryos is driven by GATA4 , even though the consequences of its loss in the presence of GATA5 or GATA6 are minimal . Thus , redundancy within the GATA family is apparent in the zebrafish myocardium as in Xenopus . The level of residual cardiac marker expression in the GATA4 and 5 MO combination or the GATA4 and 6 MO combination at 26 hpf was very low -LRB- Fig. 4 -RRB- . The level for the GATA5 and GATA6 MO combination was undetectable with 100 % of the embryos losing expression , suggesting that , while GATA4 can cover for the absence of either GATA5 or GATA6 , it can not cover for the absence of both , which seems unlikely . We therefore monitored the expression of GATA4 in flat-mounted -LRB- Fig. 5A -RRB- MO injected 10-somite embryos to determine if it was still expressed -LRB- Fig. 5C -RRB- . We found that GATA5 MO on its own caused a reduction in GATA4 expression -LRB- 22/36 embryos -RRB- , and residual expression was removed completely by the addition of the GATA6 MO -LRB- n = 35 -RRB- -LRB- Fig. 5C -RRB- . The GATA4 expression seen in GATA4 morphants reflects the use of a translation-blocking morpholino rather than a splice-blocker . In the same experiment the expression of nkx2 .5 was affected in the same way as already described -LRB- Fig. 5B -RRB- . We therefore conclude that the complete absence of cardiac marker expression in GATA5 plus GATA6 MO injected embryos results from the simultaneous absence of GATA4 expression . Thus , as seen for Xenopus embryos , the absence of all three GATA factors completely abolished cardiac marker expression . GATA activity is required for induction of the myocardial programme in zebrafish We have shown that GATA activity is only required for the maintenance/maturation of the myocardial programme in Xenopus . While we have shown that the GATA6 requirement in zebrafish is also late -LRB- Peterkin et al. , 2003 -RRB- , nkx2 .5 expression at 6 somites has been shown to be affected in zebrafish faust mutants -LRB- Reiter et al. , 1999 -RRB- , suggesting that an additional difference between the species might be the timing of requirements for GATA activity . We therefore tested this earlier requirement with more markers and to determine if it is subject to redundancy . Firstly , we examined nkx2 .5 expression in MO injected embryos at 5 somites when it is first expressed -LRB- Fig. 6A -RRB- . For the GATA5 MO , we found a major reduction in expression totally consistent with the reductions seen later and with those reported for the faust mutant -LRB- Reiter et al. , 1999 -RRB- . We also observed very little effect for the GATA4 or 6 MOs on their own , but both made the GATA5 MO phenotype more severe , consistent with their back-up roles being active at this early stage . Similar observations were made for GATA4 and hrt expression in 5-somite embryos and for tbx5 expression in 10-somite embryos -LRB- tbx5 expression is first detected at ∼ 7 somites -RRB- -LRB- Figs. 6B , C , D -RRB- . In contrast , nkx2 .7 expression was unaffected even by triple knockdown -LRB- Fig. 6E -RRB- . We therefore conclude that , for the markers studied and in contrast to Xenopus , establishing the full early myocardial programme in zebrafish depends on GATA activity . The continued presence of cells expressing nkx2 .7 suggested that apoptosis had not yet occurred , and this was confirmed by TUNEL and acridine orange assays -LRB- data not shown -RRB- . Furthermore , re-specification to more anterior or more posterior mesodermal fates was not observed , as judged by the domains of expression of anterior lateral plate and pronephric markers -LRB- data not shown -RRB- . We therefore conclude that in the absence of GATA activity , the cells remain undifferentiated at least up to the 10-somite stage . Discussion Redundancy GATA4 , 5 and 6 are an example of a gene family co-expressed in a specific tissue , in this case the myocardium . Although some differences in their binding site preferences have been detected -LRB- Sakai et al. , 1998 -RRB- , all three bind to canonical GATA sites with high affinity . Because of this and the relatively mild phenotypes generated in loss of function experiments , they have been suggested to act redundantly -LRB- Jiang et al. , 1998 ; Kuo et al. , 1997 ; Molkentin et al. , 1997 ; Narita et al. , 1996 ; Watt et al. , 2004 -RRB- . Here for the first time we present evidence in support of this with respect to laying down the genetic programme of the myocardium . The redundancy is particularly striking for GATA4 , whose individual loss has essentially no effect on induction or maturation of the myocardium in either zebrafish or Xenopus , in contrast to assumptions of its importance in much of the literature . For this member of the family , its contribution is only revealed in the absence of GATA5 or 6 , thereby constituting a formal demonstration of redundancy . Similar demonstrations are evident for both Xenopus GATA5 and in early heart induction for zebrafish GATA6 , where they are not the essential players . These redundant GATA activities thus most likely account for the residual expression of cardiac markers in the absence of the essential GATA factor . Indeed little change was observed in expression of the remaining GATA factor in double morphant embryos compared to wild type siblings in either zebrafish or Xenopus -LRB- data not shown -RRB- . The one exception was GATA5 and 6 double morphant zebrafish embryos where the complete loss of GATA4 was used to effect a triple knockout -LRB- Fig. 5C -RRB- . GATA factors are an ancient family and in vertebrates have existed with three family members in the heart at least since fish -LRB- Patient and McGhee , 2002 -RRB- . Thus , the redundancy reported here would appear to be evolutionarily very stable . Maynard Smith and colleagues have developed simple genetic models to analyse selection pressures on redundant genes and have concluded that evolutionary stability can be achieved if the two -LRB- or more -RRB- genes perform the same function , but with slightly different efficacies , as seen here -LRB- Nowak et al. , 1997 -RRB- . The less efficient family member comes into its own when paired with a mutant form of the more efficient family member . Another evolutionarily stable model can be achieved where two -LRB- or more -RRB- genes perform more than one function : the redundancy occurring only with respect to one specific function . GATA4 , 5 and 6 have an ever-growing list of functions in other tissues , so this scenario is more than adequately satisfied as well -LRB- Afouda et al. , 2005 ; Capo-Chichi et al. , 2005 ; Ketola et al. , 2004 ; Molkentin , 2000 ; Yang et al. , 2002 -RRB- . The evolutionary stability of this model depends on random mutations being more likely to render the genes inactive for all functions rather than just for one of their functions . Finally , yet another model suggests that redundancy should be more common in genes displaying specific spatio-temporal expression patterns during development , as is the case for GATA4 , 5 and 6 . For this model , the developmental error rates applicable to these genes need to be higher than their germ line mutation rates : a requirement that is currently unknown . The primary GATA factor An unexpected finding was that the member of the family whose loss has the biggest effect differs between Xenopus and zebrafish . For single knock downs , GATA6 has the strongest effect on myocardial gene expression in Xenopus whereas GATA5 does so in zebrafish . Although at first glance this might suggest a switch in roles for GATA5 and 6 , a consideration of the timing of their actions suggests an alternative view . The action of GATA6 in Xenopus is after the initial expression of other early markers such as Nkx2 .5 , suggesting a role in differentiation of the myocardium -LRB- Peterkin et al. , 2003 -RRB- . GATA6 knockdown in zebrafish has a very similar effect . Thus , in both organisms , knockdown of GATA6 leaves early marker expression initially intact but decaying with time , whereas when GATA5 was knocked down in zebrafish , expression of Nkx2 .5 and other early markers was compromised from the outset -LRB- Reiter et al. , 1999 ; this study -RRB- . The difference between the two organisms therefore can be characterised as the gain or loss of an early function for GATA5 . The early role for GATA activity in zebrafish appears not to be masked by redundancy in Xenopus because even triple knockdown of GATA4 , 5 and 6 leaves early expression of myocardial markers intact . The role of GATA5 in myocardial induction in mouse and chick embryos is currently unclear . Although in P19 embryonal carcinoma cells induced to differentiate into cardiomyocytes , GATA5 up-regulation occurs after Nkx2 .5 , precluding an early function during induction -LRB- Alexandrovich et al. , 2006 -RRB- , the mouse knockout of GATA5 retained the capacity to synthesise a truncated form of the protein containing both zinc fingers , which would likely have significant activity , preventing a definitive conclusion -LRB- Nemer and Nemer , 2002 -RRB- . Likewise , the attempts to date to knock down GATA5 activity in the chick were only partial and , in addition , attempted after induction of the myocardium -LRB- Jiang et al. , 1998 -RRB- . It is therefore not yet clear if the early role for GATA5 has been acquired by zebrafish or lost by Xenopus . In Drosophila , the GATA factor , pannier , is required both upstream and downstream of the Nkx2 .5 homologue , tinman -LRB- Gajewski et al. , 2001 ; Klinedinst and Bodmer , 2003 -RRB- . In the nematode , the GATA factors , Med1 and 2 , are expressed in the mesendodermal precursor to the mesoderm giving rise to part of the pharynx , an organ that has homologies to the heart , and upstream of the Nkx2 .5 homologue , ceh22 , suggesting that an early role for GATA factors may be ancestral -LRB- Broitman-Maduro et al. , 2006 ; Maduro et al. , 2001 ; Rodaway and Patient , 2001 -RRB- . Mesendodermal expression is seen for both GATA5 and GATA6 in zebrafish , while in Xenopus , mesendodermal expression is seen for GATA4 and 6 -LRB- Fletcher et al. , in press ; Rodaway et al. , 1999 ; J. Broadbent , A. Gibson and R. Patient , unpublished observations -RRB- . Thus , the early role for GATA5 in zebrafish may reflect its early expression in the lineage of cells leading to the myocardium whereas , in Xenopus , early expression of GATA5 is restricted to the endoderm , appearing in the cardiac mesoderm at a later stage -LRB- Weber et al. , 2000 -RRB- . That GATA6 is also expressed early in this lineage in both species , and GATA4 likewise in Xenopus , and yet neither plays a role in induction of the myocardium , suggests that GATA5 , at least in zebrafish , is alone in containing the requisite amino acid sequence for this function . All three GATA factors recognise the same DNA sequence therefore , in the absence of known binding site preferences for GATA5 , activities specific to GATA5 are likely to include protein -- protein interactions . Thus , GATA5 may be more suited to the necessary interactions in the early mesendoderm than either GATA4 or 6 . Feedback loops and timing Genetic regulatory networks -LRB- GRNs -RRB- consist of functionally linked regulatory genes encoding transcription factors and their controlling extra-cellular signals -LRB- Davidson et al. , 2002 -RRB- . They contain motifs , or recurring wiring patterns , which occur with frequencies far greater than in randomised networks -LRB- Lee et al. , 2002 ; Milo et al. , 2002 ; Shen-Orr et al. , 2002 -RRB- . One such motif is a feedback loop . Mathematical modelling of positive feedback loops indicate that they promote the persistence of signals and have the potential to store information , such that , for example , signalling can readily flip the system from one state to another -LRB- Bhalla and Iyengar , 1999 -RRB- . The observation in Drosophila that pannier is both upstream and downstream of tinman , raises the possibility that the establishment of a mutual regulatory loop for these two key regulators is critical evolutionarily -LRB- Gajewski et al. , 2001 ; Klinedinst and Bodmer , 2003 -RRB- . Evidence for a similar feedback loop exists in mice , where a cardiac GATA gene has been shown to be Nkx dependent and vice versa -LRB- Brewer et al. , 2005 ; Davis et al. , 2000 ; Lien et al. , 1999 ; Molkentin et al. , 2000 -RRB- . Davidson and Erwin have recently proposed that regulatory motifs of this type are evolutionarily stable components of GRNs , which they called kernels -LRB- Davidson and Erwin , 2006 -RRB- . They highlight a heart-field specification kernel , which is conserved from Drosophila to vertebrates . Strikingly the Nkx2 .5 / tinman GATA/pannier feedback loop is central to this kernel . Our work supports this hypothesis and further suggests that the establishment of this kernel is more critical in evolution than where the loop is initiated . Thus , GATA activity is required to initiate Nkx2 .5 expression in zebrafish but not in Xenopus , nevertheless both establish the loop -LRB- Fig. 7A -RRB- . Interestingly , as seen for GATA activity , the requirement for BMP signalling differs between zebrafish and Xenopus . Thus , in zebrafish , BMP signalling is required to initiate expression of cardiac markers including GATA5 -LRB- Reiter et al. , 2001 -RRB- , whereas in Xenopus , it is only required for their maintenance -LRB- Walters et al. , 2001 -RRB- . In view of the links between BMP and GATA factors in several different tissues , including the myocardium , it seems likely that the early requirement for GATA activity in zebrafish is linked to the early requirement for BMP , and likewise the later requirement in Xenopus -LRB- Fig. 7B -RRB- -LRB- Peterkin , 2003 -RRB- . The cascade of events in Drosophila predicts that the Drosophila BMP signal , Decapentaplegic -LRB- Dpp -RRB- , activates tinman , and they then act in concert to initiate the expression of pannier . Subsequently tinman and pannier maintain each other 's expression , whilst pannier -LRB- in the ectoderm -RRB- maintains Dpp expression . Dpp signalling feeds back to maintain expression of tinman and pannier thus completing the loop -LRB- Fig. 7C ; for review see Sorrentino et al. , 2005 -RRB- . Thus , in summary , the data imply that the initiating factor and the direction in which the loop flows are not important . Ultimately it is the establishment of the loop that is essential and failure to do so leads to the loss of differentiated myocardium .

B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O B O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

bmp
nkx
xenopus
zebrafish
redundancy
evolution
heart
feedback loops