The expression of diacylglycerol kinase theta during the organogenesis of mouse embryos
© Ueda et al.; licensee BioMed Central Ltd. 2013
Received: 7 February 2013
Accepted: 27 September 2013
Published: 1 October 2013
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© Ueda et al.; licensee BioMed Central Ltd. 2013
Received: 7 February 2013
Accepted: 27 September 2013
Published: 1 October 2013
Diacylglycerol kinase (DGK) is a key enzyme that regulates diacylglycerol (DG) turnover and is involved in a variety of physiological functions. The isoform DGKθ has a unique domain structure and is the sole member of type V DGK. To reveal the spatial and temporal expression of DGKθ we performed immunohistochemical staining on paraffin sections of mouse embryos.
At an early stage of development (E10.5 and 11.5), the expression of DGKθ was prominently detected in the brain, spinal cord, dorsal root ganglion, and limb bud, and was also moderately detected in the bulbus cordis and the primordium of the liver and gut. At later stages (E12.5 and 14.5), DGKθ expression persisted or increased in the neocortex, epithalamus, hypothalamus, medulla oblongata, and pons. DGKθ was also evident in the epidermis, and nearly all epithelia of the oropharyngeal membrane, digestive tract, and bronchea. At prenatal developmental stages (E16.5 and E18.5), the expression pattern of DGKθ was maintained in the central nervous system, intestine, and kidney, but was attenuated in the differentiated epidermis.
These results suggest that DGKθ may play important physiological roles not only in the brain, but also in diverse organs and tissues during the embryonic stages.
Many hormones and growth factors stimulate phospholipase C by activating its receptor. This activation results in the production of diacylglycerol (DG) at the plasma membrane, which triggers the activation of several enzymes, such as chimerins , Ras guanyl nucleotide-releasing protein (RasGRPs) [2, 3], transient receptor potential cation channel C (TRPC) , and both conventional and novel PKCs [5, 6]. DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), resulting in the inhibition of DG-mediated intracellular signal transduction. Additionally, PA is a lipid second messenger that regulates various target proteins, including atypical PKC, mTOR, and phosphatidylinositol 4-phosphate 5-kinase [7, 8]. Thus, DGK plays pivotal roles in various intracellular signaling pathways by regulating the DG and PA levels [9–12]. Ten DGK isoforms have been identified in mammal, and classified into five subtypes, from I to V, based on similarities in the domain structures.
The physiologic roles of DGK isoforms have been partially elucidated by analyses of DGK gene expression patterns and of genetically modified organisms. For example, DGKβ is a major isoform expressed in the brain, and the disruption of this isoform impairs memory and causes antidepressant-like effects in mice . In contrast, DGKα enhances interleukin 2-induced T cell proliferation , and knockout (KO) mice exhibit impaired induction of T cell anergy . Another isoform, DGKζ is a type IV DGK that is ubiquitously expressed in most mouse tissues. DGKζ KO mice exhibit abnormalities in multiple tissues, including a decrease in the number of dendritic spines, and an impairment of the immune response [4, 16, 17]. In regard to nervous system, a large number of studies have shown the roles of DGKs in neuronal spine density, synaptic activity, epileptogenesis and neuronal plasticity in mammals [18, 19] and C. elegans used as a genetic model . It is not clear the relationships of ten DGK isoforms in mammal. To better understand the role of each DGK and its redundancy among the DGK family, further studies in other subtypes of DGKs are necessary.
DGKθ was originally cloned from the rat brain and identified as the sole type V DGK . DGKθ contains three C1 domains and a Ras-association (RA) domain in the central region. Studies have shown that DGKθ is enriched in the nuclear matrix of various cultured cell lines , is negatively regulated by its interaction with the small GTPase RhoA , and translocate to the plasma membrane following phosphorylation by PKCϵ . The optimal activation of DGKθ may require both polybasic protein and acidic phospholipid cofactors, which have been shown to stimulate DGKθ synergistically in vitro [25, 26]. These multiple regulatory mechanisms enable DGKθ to mediate signals from a wide variety of extracellular ligands [24, 27] including epidermal growth factor , nerve growth factor , α-thrombin , and adenosine.
DGKθ is highly expressed in rat brain  and is present in a variety of cultured cell lines [22, 24]. However, there is limited information regarding this enzyme’s cell type-specific functions or its physiological roles in mammals. In this study, we examined the distribution and changes in expression from E10 to E17 to reveal the spatial and temporal expression of DGKθ protein in mouse embryos.
To determine the distribution of DGKθ, we performed immunohistochemistry (IHC) on paraffin sections of mouse embryos. We used two individual antibodies that target the C-terminal region of the protein, anti-DGKθ antibodies #1 and #2, to validate immunostaining patterns. Both antibodies detected a major 110-kDa band of DGKθ on an immunoblotting membrane containing the extract from whole brain (Additional file 1: Figure S1 C), in which DGKθ had been detected with RT-PCR (data not shown). The observed molecular size coincided with the size of endogenous protein (110 kDa) in HEK293 and HeLa cell lysates by immunoblotting [16, 23]. The specificity of this antibody was further confirmed by the transient transfection. These antibodies successfully detected exogenously expressed recombinant DGKθ protein by immunofluorescence in HeLa cells, but not other subtypes (Additional file 2: Figure S2 and data not shown).
In the developing central nervous system region at E14.5, DGKθ immunoreactivity was widely distributed in all brain regions. Particularly dense immunoreactivity was observed in the neocortex (nx), epithalamus (epi), medulla oblongata (mo), and the gray horn of spinal cord (Figure 2F-I, K and N). However, DGKθ was not detected in the ganglionic eminence (ge), which localizes to the medial side of the lateral ventricle (Figure 2H). In other areas, dense immunoreactivity was continually detected within various types of epithelia, including the tongue (to), tooth bud (tb), nasal cavity (nc), bronchus (br), midgut (mg), and urogenital sinus (us) (Figure 2F, I, J, M, O and P). In contrast to the immunoreactivity predominantly observed in the epithelia classified as simple or stratified epithelium, DGKθ protein was undetectable in the endothelial cells of the arterial trunk (Figure 2A and F). Beginning at E14.5, the fetal skin increased in thickness and formed the layer of the squamous epithelium. At the facial and dorsal surfaces we found a significant increase of immunoreactivity in the squamous epithelial cells, which successively differentiate into epidermis (Figure 2J and N). IHC without the DGKθ antibody failed to detect any signal in the sections from E10.5 to E14.5 (data not shown).
To examine the change in DGKθ expression during development, we analyzed the expression in dorsal skin from E14.5 until adulthood. In the developmental skin, the expression of DGKθ was significantly higher at E14.5 than at E17.5 and during adulthood (Figure 4A and B).
To examine the regional distribution of DGKθ during the embryonic period, we performed immunohistochemistry (IHC) on whole mouse embryos. The immunostaining patterns indicated DGKθ protein was widely expressed in multiple organs and abundantly expressed in the brain and dorsal skin of mouse embryos. These results were confirmed by the analysis of DGKθ mRNA using RT-PCR and in situ hybridization (Figure 4 and Additional file 1: Figure S1B). The IHC results demonstrated that the expression of DGKθ significantly increased in the neuroepithelium surrounding the neural tube and ventricles in the brain. Prominent immunoreactivity of DGKθ was observed in a variety of neurons in the gestational brain over the period examined (E10.5-18.5). Especially, DGKθ was detected in the marginal zone of the neocortex, but not in the medial side of the lateral ventricle at E14.5 and E16.5. Since developing mammalian telencephalon is known to require atypical PKC , these results may suggest that DGKθ is associated with differentiation of the neuronal lineage or the locomotion of immature neurons . During the prenatal period (E18.5), DGKθ was expressed in the developing cerebral cortex, hippocampus, and cerebellum. This distribution pattern is consistent with the in situ hybridization data for the adult rat brain . Other DGK isoforms are known to be expressed in the postnatal brain [34–36] and are needed for various roles of mature neurons [37–39]. C.elegans DGK-1, which has 39% identity to mouse DGKθ, regulates DAG levels generated by heterotrimeric G protein signaling in response to the neurotransmitters in nematode . Genetic analysis of C.elegans, dgk-1 shows the role of dopamine controlled locomotion and serotonin-controlled egg-laying behavior . The presence of DGKθ throughout the prenatal brain at E18.5 suggests either that DGKθ is involved in a common neuronal process, or that it provides a level of redundancy for other DGK isoforms in neurons [29, 39].
In the periphery, IHC results revealed that DGKθ is ubiquitously expressed in the layer of multiple organs during the embryonic period. In the intestine and kidney, the expression of DGKθ was prominent and persisted from E12.5 up to E18.5, while DGKθ expression in the lung, liver, and oropharyngeal membrane surrounding the tongue and nasal cavity was transient and attenuated before birth. Since DGKθ enhances the activation of EGF receptor stimulated with EGF via the counteraction of PKC activity in epidermoid cells , DGKθ may notably contribute to the development of epithelial cells during organogenesis (E10.5-E17.5). RT-PCR analysis demonstrated that the DGKθ mRNA was expressed in abundance throughout the developmental process, with highest expression in multiple key organs. Thus, DGKθ may have persistent roles in diverse organs and tissues during the embryonic stages.
We revealed for the first time the distribution of DGKθ during the embryonic period. These results suggest that DGKθ may play important physiological roles not only in the brain, but also in diverse organs and tissues during the embryonic stages, and will serve as a basis for future in vivo genetic, functional, and mutational analyses of DGKθ.
Eight-week-old mice were purchased from Nippon SLC, Inc. (Hamamatsu, Japan). The animal works were reviewed and approved by the Care and Use of Laboratory Animals of Kobe University. All procedures using experimental animals were performed according to the Guidelines for the Care and Use of Laboratory Animals of Kobe University.
Most chemical reagents were purchased from Wako Pure Chemical Inc. (Oosaka, Japan). C57BL/6 J mice were anesthetized and transcardially perfused with cold PBS(-) and fixed in 4% paraformaldehyde in PBS (-) for 10 min. Mouse embryos were isolated from pregnant mice and fixed in 4% paraformaldehyde in PBS (-) overnight . The mouse embryos were paraffin-embedded and sectioned at a 5 μm thickness by Kyoudoubyouri, Inc. (Kobe, Japan) or Genostaff, Inc. (Tokyo, Japan). The serial paraffin sections were deparaffinized in xylene, and rehydrated using a graded series of ethanol from 99% to 50% and washed in distilled water. The sections were then incubated in 0.03% H2O2 in PBS (-) for 1 hr, and permeabilized with 0.3% Triton X -100 in PBS (-) for 30 min. Subsequently, the sections were washed with 0.1% Tween 20 in PBS (-), and incubated in 0.1% Tween 20 in PBS (-) supplemented with goat serum (Dako, Glostrup, Denmark), serving as blocking reagent, for 1 hr. Next, the sections were treated with an anti-DGKθ antibody diluted (antibody #1, 1:50 ; antibody #2, 1:100) in Can Get Signal immunostain solution A (Toyobo, Tokyo, Japan) overnight. The antibodies against DGKθ, antibody #1 (immunogen of 691–820 aa) and antibody #2 (immunogen of 677–883 aa), were purchased from Santa Cruz Biotechnology (California, USA), and BD Biosciences (California, USA), respectively. The control samples were probed with antibody that had been pre-incubated with a DGKθ blocking polypeptide. The bound antibodies were visualized using EnVision + System-HRP (Dako). The stained sections were dehydrated using a graded series of ethanol and mounted using a coverslip and Mount-Quick (Daido Sangyo, Gunma, Japan). The specimens were then photographed using an All-In-One Microscope system (BZ-8000 and BZ9000, Keyence, Japan), as reported previously .
The specific blocking polypeptide of DGKθ (677–883 aa) was generated by PCR using the following primers, with pEGFP-N3-DGKθ as template: 5′-AGTCGGATCCGGCACAGGGAATGACCTTG-3′ and 5′-CGAGTCACGCTCCTCAAGTGATGAGGATCCAGTG-3′. The amplified PCR fragment was subcloned into the pQE30 vector (Qiagen, Tokyo, Japan) for expression and purification from E. coli.
Reverse transcription-PCR (RT-PCR) was performed as described previously . Briefly, organs or tissues were dissected from 15 embryos of mice under a stereomicroscope and frozen immediately on dry ice. The total RNA was isolated using a High Pure RNA Tissue Kit (Roche, Mannheim, Germany). For each tissue, equal amounts of RNA were reverse-transcribed with ReverTra Ace (Toyobo). The resulting cDNA was appropriately diluted and used to perform PCR with KOD-FX Neo (Toyobo).
The primer pair for mouse DGKθ was purchased from Takara-Bio Inc. (Otsu, Japan) and used to amplify the specific sequence : 5′-GGTTCAGAACAGGGCCAGGTAG-3′ and 5′-AGGGCTGTAGGCAGGCAAAC-3′ (NCBI : NM_199011, nt.3577-3706). RT-PCR was performed according to the manufacture′s specification. Reactions for DGKθ were performed for 35 cycles at 98°C for 10 s, 60°C for 30 s, and 68°C for 1 min. After electrophoretic separation, the amplified fragments were quantified using the image-analysis software Image J.
This investigation was supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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