Expression pattern of protein kinase C ? during mouse embryogenesis.

1.4 MB·English

by  Carracedo, Sergio

δ
Expression pattern of protein kinase C during
mouse embryogenesis
Carracedo et al.
Carracedoetal.BMCDevelopmentalBiology2013,13:2
http://www.biomedcentral.com/1471-213X/13/2
Carracedoetal.BMCDevelopmentalBiology2013,13:2
http://www.biomedcentral.com/1471-213X/13/2
RESEARCH ARTICLE Open Access
δ
Expression pattern of protein kinase C during
mouse embryogenesis
Sergio Carracedo*, Ursula Braun and Michael Leitges
Abstract
Background: The members of the protein kinase C(PKC) family consist of serine/threonine kinases classified
according to their regulatory domain. Those that belong to thenovel PKC subfamily, such as PKCδ, are dependent
ondiacylglycerol but not Calcium when considering theircatalytic activity. Although several studies have shown
theimportanceof PKCδ in different cellular events inhealth and disease,the overall in vivo distribution of this PKC
isoform during development is still lacking. Through Lac Zand antibody staining procedures, weshow here the
invivo expression ofPKCδ during mouseembryogenesis.
Results: Ganglia were thedomains with most prominent expression of PKCδ inmost of the stages analysed,
although PKCδ could also be detected inheart and somites atearlier stages, and cartilage primordium and skin
among other sites inolder embryos.
Conclusions: The strong expression of PKCδ ingangliaduring murine development shown in this study suggests a
significant role ofthis isoform as well as redundancy with other PKCs within thenervous system,since PKCδ
deficient mice develop normally.
Keywords: Novel protein kinase C,PKC delta, Mouse embryogenesis, Lac Z, Ganglia
Background migration [13] or transcription [14]. Also, although PKCδ
Inmammals,thePKCfamilyconsistsofatleast10serine/ deficiency does not prevent mouse viability [15], this iso-
threoninekinasesgroupedintothreesubfamiliesattending form appears important in different contexts in health, as
totheirregulatorydomainandrequirementsfortheiracti- in the immune system [16] or insulin sensitivity [17], and
vation. Unlike conventional PKCs, novel PKCs (nPKC), disease, as for example in arteriosclerosis [15]. However,
such as PKCδ, are not dependent on calcium but on dia- dataregardingthegeneralPKCδexpressionpatternduring
cylglyceroltochangefromtheirself-inhibitedstatetotheir mouse embryogenesis are still missing. This study shows
active conformation [1]. PKCδ activity can be regulated in the spatiotemporal expression of PKCδ during midgesta-
different manners, including phosphorylation of its activa- tion by immunostaining wild type mouse embryo sections
tionloopbykinases[2]andbyautophosphorylationofdif- andbyusingPKCδdeficientembryosexpressingtheLacZ
ferent sites throughout its regulatory domain and hinge reporter gene under the control of the endogenous PKCδ
region [3]. In addition, it can become a lipid-independent promoter. These results could be helpful when addressing
enzyme with altered substrate specificity under certain functional redundancy or exclusiveness of this nPKC dur-
conditions [3] and show altered cofactor requirements [4]. ingmurinedevelopment.
PKCδ interacts with different proteins, such as Shc [5] or
p23[6],andinitsactivestate isabletophosphorylatedif- Results and discussion
ferent substrates, such asSTATs [7] or ERK [8].Function- PKCδexpressionfromE8.5toE9.5
ally, PKCδ regulates different processes, such as cell cycle PKCδ analysis was mainly performed through LacZ
(byeitherslowing[9,10]orpromoting[8,11]cellprolifera- staining.Antibody staining with the correspondingnega-
tion, depending on the context), apoptosis [12], cell tive controls was also performed at E9.5 and E13.5 to-
gether with the LacZ staining method as a control for
*Correspondence:[email protected]
the specifity of the LacZ signal. To confirm the absence
TheBiotechnologyCentreofOslo,UniversityofOslo,Gaustadalleen21,Oslo
N-0349,Norway of endogenous β-galactosidase activity in our stainings,
©2013Carracedoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
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wild type littermates underwent the same protocol in PKCδexpressionfromE10.5toE12.5
parallel(Figures 1A-C). At E10.5, novel β-galactosidase activity was observed at
At E8.5, whole mount LacZ staining showed signal the roof of the hind brain, third branchial pouch, fourth
mainlyatthe primitive heartandyolk sac(Figure 1C). branchial pouch and mandibular component of the first
WholemountLacZ stainingofPKCδdeficientembryos branchial arch (Figures 2A and B). The signal observed
at E9.5 showed signal in rostral extremity of the noto- at E9.5 in trigeminal (V) neural crest tissue became
chord, trigeminal (V) neural crest tissue, dorsal aorta, more prominent at E10.5 (Figures 2A and B). E11.5 was
pericardium, and developing heart. A closer look to the the earliest developmental stage at which ganglia started
heart allowed for visualization of walls of the primitive to show Lac Z reporter signal. Thus, dorsal root ganglia,
ventricle, atrium and truncus arteriosus (Figures 1E-H). facio-acoustic (VII-VIII) ganglion complex and trigem-
ImmunostainingofwildtypeembryosectionsatE9.5con- inal (V) ganglia all displayed β-galactosidase activity
firmedexpressionofPKCδinsomites and the sameareas (Figure 2C). In 12.5 dpc embryos, dorsal root ganglia
of the heart and pericardium (Figures 1I-L). The specifi- showed increased LacZ staining, and the trigeminal (V)
city of the signal given by the antibody was confirmed by ganglion became also prominently stained (Figures 2D
using PKCδ deficient embryo sections corresponding to and E). In addition, novel signal was detected at this
thesamedevelopmentalstage(Figure1J). stage in the vestibulocochlear ganglion (Figure 2D) and
Figure1PKCδ expressionatE8.5andE9.5.A-C,lackofsignalduetoendogenousβ-galactosidasewasconfirmedbyapplyingthesameLacZ
stainingprotocolsonwildtypeembryos(controls).D,atE8.5,X-Galstainingisdetectedintheyolksacandprimitiveheart(ysandph,
respectively).E-H,atE9.5,trigeminal(V)neuralcresttissue(tnc),rostralextremityofthenotochord(not),dorsalaorta(dor),somites(som),
pericardium(per)anddevelopingheartallshowedLacZsignal.Inthelatter,reporteractivitywasdetectedatthewallsoftheprimitiveventricle
(ven),atrium(atr)andtruncusarteriosus(tru).I-L,Antibodystainingof4μmE.9.5embryosectionsconfirmedtheexpressionofPKCδinheartand
somites.E9.5PKCδdeficientembryosections(J)wereusedasanegativecontrolfortheantibody.
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Figure2PKCδexpressioninwholemountembryosfromE10.5toE12.5.AandB,atE10.5,roofofthehindbrain(rhb),thirdbranchial
pouch(tbp),fourthbranchialpouch(fbp)andmandibularcomponentofthefirstbranchialarchshownovelLacZreporteractivity.Signalatthe
trigeminal(V)neuralcresttissue(tnc)becamemoreprominentthanatE9.5.FigureBisaclose-upoftheinsetfoundinfig.A.C,AtstageE11.5,
stainingofthetrigeminal(V)ganglion(trg),facio-acoustic(VII-VIII)ganglioncomplex(fag),anddorsalrootganglia(drg)appearedallLacZstained.
D-G,12.5dpcembryosshowincreasedsignalindorsalrootganglia(drg),strongLacZactivityinthetrigeminalganglion(trg),andnovelactivity
atvestibulocochlearganglion(ves),neuraltube(neu)andprecartilageprimordiaofbonesatforelimbs(for)andhindlimbs(hin).
neural tube (Figure 2E). The broad and strong LacZ re- plexus (Figures 3A-D). However, LacZ signal in the
porter activity detected in ganglia suggests that PKCδ latter two domains was not detectable in PKCδ+/−
may have a significant role within the nervous system in embryos (Figure 2B). At this stage, LacZ-stained
mice. However, there is no obvious defect within the embryos were also embedded in paraffin blocks to gen-
nervous system during murine development and adult- erate sections that could let us better identify domains
hood. We believe this is due to functional redundancy where β-galactosidase activity occurred. Given the low
among members of the PKC family. For example, over- signal observed in the 4 μm-thick sections, 15 μm sec-
lapping expression of PKC δ [18] and ε [19] within the tions were used instead in order to obtain a more prom-
nervous system can be already found as part of a recent inent LacZ staining signal. Unfortunately, sections of
study that uses an in situ hybridization approach to such thickness affected somewhat the quality of the cor-
show the expression pattern of a high number of tran- responding photographs. However, we were still able to
scripts in the mouse embryo [20]. Thus, a redundancy identify domains that could also be observed in whole
where these two isoforms compensate each other could mount embryos, such as dorsal root ganglia, trigeminal
be a reason why no obvious phenotype is observed in (V) ganglion, vestibulocochlear ganglion, neural tube or
the nervous system of PKC δ or ε single deficient mice cartilage primordium at limbs (Figures 3E-K), as well as
duringmouseembryogenesis. new areas that we could not see in whole embryos, such
At 12.5 dpc, embryos also showed novel reporter as loop of midgut within physiological umbilical hernia,
activity at the precartilage primordia of bone at dorsal part of tongue and lower border of nasal septum
forelimbs and hindlimbs, such as femur and radius (Figures 3L-M). At this stage, there seemed to be pro-
(Figures 2FandG). blems with penetration of X-Gal in the embryo and
therefore proper detection of signal in several domains,
PKCδexpressionatembryonicstagesE13.5andE14.5 such as trigeminal ganglion (Figure 3G). Furthermore,
At E13.5 (Figure 3), dorsal root ganglia showed approxi- sites such as stomach, which appeared stained at E12.5
mately the same strong LacZ signal observed in trigem- (data not shown), was not detectable at E13.5, possibly
inal (V) ganglia (Figures 3A-D). New domains with due to the same problem. We also performed immunos-
β-galactosidaseactivityatthisstageofdevelopmentwere taining of PKCδ in wild type and PKCδ deficient (nega-
the caudal part of the medulla oblongata, inferior gan- tive control) mouse embryo sections at E13.5, which
glion of glossofaringeal (XI) nerve, skin, and choroid confirmed its expression at sites already identified in
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LacZ stained embryos: dorsal root ganglia, inferior gan- at limbs (Figures 3N-W). In addition, antibodies to
glion of glossofaringeal (XI) nerve, vestibulocochlear PKCδ applied on cross sections also revealed expression
ganglion, trigeminal (V) ganglion, loop of midgut within in the stomach and metanephros (Figure 3X). Sagittal
physiological umbilical hernia dorsal part of tongue, sections reported the atrium of the heart, which could
lower border of nasal septum, and cartilage primordium not be seen in LacZ stained embryos or sections,
Figure3(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure3PKCδexpressionishighestwithinthenervoussystematE13.5.A-D,atembryonicstageE13.5,wholemountstainingallowedfor
thedetectionofLacZsignalinskin(ski)inhomozygousbutnotheterozygousembryos.Choroidplexus(cho),neuraltube(neu),caudalregionof
themedullaoblongata(cmo),inferiorganglionofglossofaringeal(XI)nerve(inf),trigeminal(V)ganglion(trg),anddorsalrootganglia(drg)all
showednovelβ-galactosidaseactivityinwholeembryos.E-M,15μmsagittalsectionswereobtainedfromLacZstainedembryos.Reporter
activityobservedinmostcellsofdorsalrootganglia(drg),trigeminal(V)ganglion(trg),inferiorganglionofglossofaringeal(XI)nerve(inf),
vestibulocochlearganglion(ves)andneuraltubeconfirmedthesignalobservedinLacZ-stainedwholeembryos.Also,LacZreporteractivitywas
detectedatcartilageprimordiumatlimbs(pre),theloopofmidgutwithinphysiologicalumbilicalhernia(loo),dorsalpartoftongue(ton),and
lowerborderofnasalseptum(sep).Areaslabelledas*couldbedetectedonlyviaLacZstainingandthuswerenotreported.NandO,atE13.5,
PKCδdeficientmousesections(δ−/−)wereusedasnegativecontrolsforimmunohistochestristry(AandB).P-Y,atE13.5,antibodiestoPKCδ
confirmedLacZstainingatdorsalrootganglia(drg),trigeminal(V)ganglion(trg),inferiorganglionofglossofaringeal(XI)nerve(inf),
vestibulocochlearganglion(ves),loopofmidgutwithinphysiologicalumbilicalhernia(loo),dorsalpartoftongue(ton)andlowerborderofnasal
septum(sep).CrosssectionsallowedforimmunodetectionofPKCδincartilageprimordiumatlimbs(pre),metanephros(met)andwalland
mucosalliningofthestomach(sto).Inaddition,antibodystainingwasdetectedintheatriumoftheheart(atr).
possibly due to penetration issues of X-Gal (Figure 4Y), Conclusions
as earlier mentioned. There were some areas detected Our expression pattern for PKCδ during mouse midges-
through Lac Z staining that could not be detected via tation suggests that several domains, such as cartilage
immunostaining. In these areas, the Neo cassette that primordium or skin, express this novel PKC isoform.
was used to generate PKCδ deficient mice might have However, the nervous system is the main site of expres-
influenced the expression of PKCδ [21], although PKCδ sion for PKCδ. More specifically, dorsal root ganglia and
might instead be too lowly expressed to see immuno- trigeminal (V) ganglia are the domains where PKCδ
signal withthe protocolwe used. seems to be most prominently expressed. Thus, these
Consistent with previous mRNA studies at E14.5 [20], data suggest that PKCδ may have an important role
our LacZ staining of embryos at this stage showed signal within the nervous system in mice, as already suggested
in brain, neural tube and ganglia (Figures 4A and B). by studies in other species [22,23]. The fact that there is
However, we also found β-galactosidase activity at sites no reported phenotype or functional deficiency in the
that we already described at earlier stages in this study, nervous system suggest the existence of functional re-
but whose patterns have not been reported before in dundancy among members of the PKC family. Thus, the
such work at the mRNA, such as skin (Figure 4A, which expression pattern of PKCδ may contribute to address
also appeared LacZ stained in heterozygous embryos, such redundancy in function as well as to identify
unlike at E13.5) or cartilage primordia of bones (mainly domains causing potential lethality in mice lacking sev-
at limbs, Figure 4C). The staining in bone was more eralPKCisoforms.
prominent and more defined than when identified at
E12.5. Here, it could be readily observed in precartilage Methods
primordia of digit, precartilage primordial of phalangeal Animalsandembryocollection
bone, and metatarsal bone (Figure 4C). In addition, 14.5 Generation of mice (129/SvPasCrl) carrying the mutated
dpc embryos displayed LacZ rerporter activity at whis- alleleforPKCδhasbeenpreviouslydescribed[15].Allani-
kers and precartilage primordium of nasal septum mal work was approved by the Folkehelse Institute, Oslo
(Figure 4D). (Norway) and performed according to its institutional
Figure4PKCδexpressionatE14.5.A-D,in14.5dpcembryos,increasedLacZactivitywasdetectedinchoroidplexus(cho),caudalpartof
medulaoblongata(cmo),skin(ski),dorsalrootganglia(drg)andneuraltube(neu).Moredefinedsignalthanatpreviousstageswasalsofoundin
precartilageprimordiaofdigit(dig),precartilageprimordialofphalangealbone(pha)andmetatarsalbone(met).LacZreporteractivitywasalso
observedinwhiskers(whi)andprecartilageprimordiumofnasalseptum(nas).
Carracedoetal.BMCDevelopmentalBiology2013,13:2 Page6of7
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guidelines and to the rules and regulations of the Fe- Competinginterests
deration of European Laboratory Animal Science Associ- Theauthorsdeclarenocompetinginterests.
ation´s (FELASA). Pregnancy stages were assigned upon
observation of vaginal plug at approximately midday, Authors'contributions
SCacquired,analysedandinterpretedthedata,andellaboratedthe
whichwasconsideredasE0.5.
manuscript.UBhelpedperformingsomeexperiments.MLandUBgenerated
PKCδdeficientmice.MLparticipatedinthedesignandinterpretationofthe
LacZstaining experiments,andhelpedtowritethemanuscript.Allauthorsreadand
approvedthefinalversionofthemanuscript.
Steps corresponding to fixation (4% paraformaldedyde in
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doi:10.1186/1471-213X-13-2
Citethisarticleas:Carracedoetal.:Expressionpatternofproteinkinase
Cδduringmouseembryogenesis.BMCDevelopmentalBiology201313:2.
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