Insm1promotes the transition of olfactory progenitors from apical and proliferative to basal ...

19 Pages·2011·4.4 MB·English

Insm1 promotes the transition of olfactory
progenitors from apical and proliferative to basal,
terminally dividing and neuronogenic
Rosenbaum et al.
Rosenbaumetal.NeuralDevelopment2011,6:6
http://www.neuraldevelopment.com/content/6/1/6(1February2011)
Rosenbaumetal.NeuralDevelopment2011,6:6
http://www.neuraldevelopment.com/content/6/1/6
RESEARCH ARTICLE Open Access
Insm1 promotes the transition of olfactory
progenitors from apical and proliferative to basal,
terminally dividing and neuronogenic
Jason N Rosenbaum1†, Anne Duggan1*†, Jaime García-Añoveros1,2*
Abstract
Background: Insm1 is a zinc-finger transcription factor transiently expressed throughout the developing nervous
system in late progenitors and nascent neurons. Insm1 is also highly expressed in medulloblastomas and other
neuroendocrine tumors.
Results: We generated mice lacking the Insm1 gene and used them to elucidate its role in neurogenic
proliferation of the embryonic olfactory epithelium. We found that deletion of Insm1 results in more apical cells
and fewer nascent and mature neurons. In the embryonic olfactory epithelium of Insm1 mutants we detect fewer
basal progenitors, which produce neurons, and more apical progenitors, which at this stage produce additional
progenitors. Furthermore, in the mutants we detect fewer progenitors expressing NEUROD1, a marker of terminally
dividing, neuronogenic (neuron-producing) progenitors (immediate neuronal precursors), and more progenitors
expressing ASCL1, a marker of the transit amplifying progenitors that migrate from the apical to the basal edges of
the epithelium while dividing to generate the terminal, neuronogenic progenitors. Finally, with timed
administration of nucleoside analogs we demonstrate that the Insm1 mutants contain fewer terminally dividing
progenitors at embryonic day 12.5.
Conclusions: Altogether, these results suggest a role for Insm1 in promoting the transition of progenitors from
apical and proliferative to basal, terminal and neuronogenic. This role appears partially conserved with that of its
nematode ortholog, egl-46. The similar effects of Insm1 deletion on progenitors of embryonic olfactory epithelium
and cortex point to striking parallels in the development of these neuroepithelia, and particularly between the
basal progenitors of olfactory epithelium and the subventricular zone progenitors of cortex.
Background divide terminally and produce neurons [1]. Despite its
During nervous system development, progenitors divide importance, we have limited knowledge of which genes
to produce more progenitors, glia and/or neurons. Deci- control progenitor transitions from proliferative to
sions to stop proliferating and produce neurons or glia neuronogenic.
determine shape, size and cellular composition of every In Caeneorhabditis elegans, the zinc-finger protein
part of the nervous system. These decisions can involve EGL-46 is transiently expressed in certain progenitors
transitions between apical and basal sides of a proliferat- about to divide terminally (neuronogenic, N/N divisions)
ing neuroepithelium. In embryonic olfactory epithelium and in their two nascent neurons, but not in the mature
(OE), apical progenitors generate sustentacular glia and neurons or in earlier progenitors that divide to produce
more progenitors, some of which transition basally, one or two progenitors (proliferative, P/P or P/N divi-
sions) (see discussion below). In egl-46 mutants, some of
these presumptive N/N progenitors fail to terminally
*Correspondence:[email protected];anoveros@northwestern. divide and generate additional progenitors [2-4] (see dis-
edu
†Contributedequally cussion below). Hence, EGL-46 regulates terminal neu-
1DepartmentofAnesthesiology,NorthwesternUniversityFeinbergSchoolof ronogenic divisions. We hypothesize that a mammalian
Medicine,303E.ChicagoAvenue,Ward10-070,Chicago,IL60611,USA homolog of EGL-46 also regulates transitions of
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Rosenbaumetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
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progenitors from proliferative to terminally dividing and mRNA of Insm1, detected in wild type embryos in the
neuronogenic. expected neuronogenic areas (including OE) [15], was
In mammals, egl-46 has two orthologs, Insm1 and missing in KO embryos (Figure 1D). These results, in
Insm2 [3,5]. Insm1 was originally identified as highly addition to confirming the functional KO of the Insm1
expressed in neuroendocrine tumors [6-13], but during gene, also demonstrate that the distribution reported
normal development it is transiently expressed through- with this same probe, in late progenitors and nascent
out the embryonic and adult developing nervous system neurons ([15] and data not shown), does indeed repre-
[14-18]. In particular, Insm1 mRNA is expressed in late sentthemRNAofInsm1,notofahomolog.
(not early) progenitors and nascent (not mature) neu- To avoid potentially confounding effects of other
rons [15]. Thus, the pattern of expression of Insm1 in genetic variables in our phenotypic assessment, we
mice is reminiscent of that of egl-46 in nematode neuro- placed the Insm1tm1.1Jga allele (from now on referred to
nal lineages, suggesting that both genes share a con- as Insm1-) into an isogenic background by performing
served function in regulating neuronal progenitor ten backcrosses into either the inbred C57BL/6 line or
proliferation. Insm1 has been implicated in the develop- the outbred CD1 line.
ment of the pancreas [19-22], sympatho-adrenal lineages
[23], cortex [16], and hindbrain [24]. We tested the Deletion of Insm1 causes embryonic lethality
function of this gene in the embryonic OE. Mating between heterozygotes did not result in any
The OE provides a simple neurodevelopmental model. Insm1-/- pups, which indicates that this genotype causes
This placode-derived neuroepitheliumcontainsonly one embryoniclethality.Thislethalitydoesnotseemtoresult
typeofneuron,whichoriginatesfromprogenitorslocated in or from developmental delay, as KO, wild type and
within the epithelium. Embryonic OE is spatially segre- heterozygous embryos of the same age did not differ in
gated: early progenitors and sustentacular cells localize weight (Additional file 1) or in anatomically defined
apically,lateprogenitorsbasally,andneuronsinbetween developmental hallmarks (Additional file 2). In order to
[1,25]. In embryonic OE, Insm1 mRNA is expressed in determinetheembryonicstageoflethality,we generated
intermediatecells(bothprogenitorsandnascentneurons) timed pregnancies and collected embryos at set days
andbasal(butnotapical)progenitors[15].Wegenerated post-coitus(fromhereonwardsdenotedasEforembryo-
mice entirely lacking Insm1 and used them to conclude nicday, withthefirst dayafter anightofmatingconsid-
thatthisgeneregulatesprogenitortransitionsfromapical ered E0.5). At E11.5, Insm1-/- embryos appeared at the
andproliferativetobasalandneuronogenic. expected Mendelian ratio (1/4). At E12.5 to E15.5,
Insm1-/- embryos appeared at somewhat lower ratios
Results than expected and by E16.5 there was a severe drop in
In order to determine the role of Insm1 in neuronal viability(Additionalfile3).Hence,Insm1-/-embryossuf-
development,we generatedknockout(KO)micelacking fer lethality around E16. In an attempt to obtain older
thisgene.The Insm1mRNAistranscribedfromasingle embryos, we administered L-DOPA (1 mg/ml) to preg-
exon(2,912bplong)containing155bpof5’UTR,1,566 nant dams starting no later than 7.5 days post-coitus
bpofcodingsequenceand1,206bpof3’UTR.Byhomo- (dpc), as it has been reported that this treatment
logous recombination in HM1-M embryonic stem (ES) increases viability of a similar KO [23,26]. Although in
cells (derived from Sv129 mice), we replaced 3.4 kb of ourhandsthistreatment didnotimprovetheviabilityat
genomic DNA containing the entire Insm1 exon with a earlier (E11.5 to E14.5) stages, we were able to obtain a
neomycin cassette surrounded by loxP sites for future fewembryosasoldasE20.5(Additional file3).Weused
excision(Figure1A).SeveraloftheserecombinedEScell these embryos to examine the effects of Insm1 deletion
cloneswereinjectedintoC57BL/6blastocystsandmulti- on neural development but we did not explore the rea-
ple chimeras were generated. Two (out of 17) chimeras sons for this lethality, which others attribute to a cate-
generatedfromoneoftheseEScellclones(11G9)passed cholamine deficiency resulting from developmental
the Insm1tm1Jga allele (which bears a ‘floxed’ neomycin defectsofthesympathoadrenallineage[23].
cassette in place of Insm1) through the germ line and
producedheterozygousprogeny.We generated the final Deletion of Insm1 results in more apical and fewer neuro-
KO allele, Insm1tm1.1Jga, by crossing these heterozygotes basal cells in the embryonic OE
with an E2a-Cre transgenic mouse, which express the After close examination of paired sections of KO and
Crerecombinaseubiquitously,thusremovingtheneomy- wild-type littermate embryos, we focused our attention
cin cassette and leaving only a loxP site in place of the on the OE, which appeared altered in the KO. The
wildtypealleleofInsm1(Figure1A).Weconfirmeddele- embryonicOEatE14.5andlaterstagescontainstwocel-
tion of Insm1 by Southern blot and by PCR (Figure 1B, lularlayersthatareeasilydistinguishedbasedonnuclear
C). Furthermore, in situ hybridization revealed that the morphology: apical and neuro-basal (Figure 2D, H).
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Figure1Generation ofInsm1knockoutmice.(A)Schematicoftherecombinationstrategyused togeneratetheInsm1knockout.Using
homologousrecombinationinembryonicstem(ES)cells,wereplacedagenomicDNAfragmentof3.4kb(inblue/cyan)containingtheentire
singleexonofInsm1plus927bpofupstreamand955bpofdownstreamsequencewitha‘floxed’neomycincassette(inred/pink).After
generatingchimericmiceand,fromthese,heterozygotes,theneomycincassettewasremovedbycrossingwithE2a-Cretransgenicmice.Probes
(5’and3’)usedforSouthernblotgenotypingandDNAfragmentsobtainedbyPCRgenotypingofthewildtype,knockout+neomycinand
knockoutallelesaredepicted.(B)SouthernblotsofBglIIdigestsofgenomicDNAfromrecombinedEScellswith5’and3’probesrevealthe
expectedbandsfromtheknockout(KO)andwildtypeallelesandconfirmthatrecombinationwascomplete.(C)GenotypingPCRusing
genomicDNAobtainedfromalitterofembryos(embryonicday10.5)resultingfromcrossingtwoheterozygousparents.Primersusedare
describedinmethodsandtheirproductsdepictedin(A).Thelittercontainedtwoknockouts(-/-),twowildtypes(+/+)andfourheterozygotes
(+/-),asindicatedinthefigure.(D)Insituhybridization(ISH)withanantisenseprobeagainstthe3’UTRofInsm1onwildtypeandknockout
littermateembryos(E14.5,coronalsections)revealthepresenceofInsm1mRNAinthebrain,retinaandolfactoryepithelium(arrowheads)ofwild
typebutnotofknockoutembryos,functionallyconfirmingtheknockoutofInsm1.Scalebars:500μm.
The apical layer consists of densely packed, elongated terminally and produce neurons [1,25,27]. Eventually,
nuclei that correspond to proliferative progenitors. progenitors that remain in the apical layer generate the
These apical progenitors undergo mitosis at the luminal apically located sustentacular (glial-like) support cells
side of the epithelium and initially generate additional [28]. The neuro-basal layer consists of rounder, more
progenitors, some of which migrate basally to divide densely spaced nuclei that correspond to basal
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Figure2Increaseinthenumberofapicalcellsandconcomitantdecreaseinthenumberofneuro-basalcellsintheOEofInsm1-/-
embryos.RepresentativeDAPIstainedimagesfromcoronalsectionsof(A,D,H)Insm1+/+embryosand(B,E,I)anatomicallyalignedsections
frompairedInsm1-/-littermates.Solidlinesindicatetheapical(top)andbasal(bottom)marginsoftheOE,anddashedlinesindicatetheinterior
borderoftheapicalcompartment.InInsm1-/-miceat(D-G)E14.5and(H-K)E18.5,thenumberofapicalcellsper(100μm)2oftotalOEsurfaceis
57%greater(n=4embryopairs,totalof12alignedsectionpairs,P<0.05)and44%greater(n=3embryopairs,totalof9alignedsection
pairs,P<0.05),respectively,thaninpairedInsm1+/+littermates.Correspondingly,thenumberofneuro-basalcellsdecreasesby37%atE14.5(P<
0.001)andby24%atE18.5(P<0.01).Asapercentageoftotalcells,atE14.5apicalcellsmakeup30%oftheOEinInsm1+/+embryosand52%
inInsm1-/-embryos(F).AtE18.5,apicalcellsmakeup27%inInsm1+/+embryosand41%inInsm1-/-embryos(J).AtE12.5,theapicalandbasal
layersarenotyetapparent.However,thereisnodetectabledifferencebetweentheoverallthicknessoftheOEatanystage(P=0.98atE12.5,
P=0.07atE14.5andP=0.17atE18.5;n=3embryopairs,totalof9alignedsectionpairsatE12.5).Datain(C,G,K)arepresentedasmean
values±standarderrorofthemean(SEM).*P≤0.05,**P≤0.01,***P≤0.001.Scalebars:50μm.KO,knockout;WT,wildtype.
progenitors and to their progeny, the intermediately Deletion of Insm1 results in fewer nascent and mature
locatedneurons.Witha nuclearDAPIstainwe detected neurons in the embryonic OE
no change in the thickness of the entire OE at E12.5, The reduction in neuro-basal cells observed in the OE
E14.5 and E18.5 (Additional file 4). However, the apical of Insm1-/- embryos could reflect a reduction in basal
layer of proliferative progenitors was thicker in Insm1-/- progenitors, in neurons, or in both. We labeled nascent
than in Insm1+/+ embryos at E14.5 (+47%) and E18.5 neurons with antibodies against the bIII-tubulin isoform
(+59%),andtheneuro-basallayerwasconverselythinner (TuJ1). At most stages examined, the Insm1-/- OEs had
(Additional file 4). Nuclear counts confirm that, com- significantly fewer nascent (TuJ1+) neurons than the
paredtoInsm1+/+littermateOEs,Insm1-/-OEscontained OEs of their Insm1+/+ and Insm1+/- littermates (-43% at
moreapicalcells(+57%atE14.5and+44%atE18.5,mea- E11.5, -47% at E12.5, -39% at E14.5, and -37% at E18.5;
sured per unit area of the whole OE) and fewer neuro- Figure 3). At E10.5, the small reduction measured is not
basal ones (-37% at E14.5 and -24% at E18.5, measured statistically significant. Hence, we conclude that as early
perunitareaofthewholeOE)(Figure2). as E11.5 there are fewer neurons in the OEs of Insm1-/-
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Figure3Fewercellsexpressmarkersofyoung(bIII-tubulin)andmature(olfactorymarkerprotein)olfactoryreceptorneuronsinthe
OEofInsm1-/-embryos.(A-D)ImmunohistochemistryforbIII-tubulin(red)plus(C-D)olfactorymarkerprotein(OMP;green)inrepresentative,
anatomicallyalignedimagesfromtheOEof(A,B)E14.5and(C,D)E18.5Insm1-/-andanInsm1+/+littermateembryo.Fewercellsexpresseither
markerintheknockout(KO).Theapicalborderistowardthecenterofeachpanel,andthebasallaminaistowardtheperiphery.(E)Densityof
youngneurons(numberofcellsexpressingbIII-tubulinper(100μm)2)inOEatvariousembryonicstages.InInsm1-/-miceatE11.5,E12.5,E14.5,
andE18.5thenumberofyoungneurons(red)isreducedbyanaverageof43%(n=3embryopairs,totalof9alignedsectionpairs,P<0.05),
46%(n=3embryopairs,totalof9alignedsectionpairs,P<0.05),39%(n=4embryopairs,totalof15alignedsectionpairs,P<0.01),and37%
(n=3embryopairs,totalof9alignedsectionpairs,P<0.05),respectively.AtE10.5,themeasuredreduction(29%,n=3embryopairs,totalof9
alignedsectionpairs)doesnotachievestatisticalsignificance.(F)Densityofmatureneurons(numberofcellsexpressingOMPper(100μm)2)in
OEatvariousembryonicstages.AtE14.5andE18.5thenumberofmatureolfactoryneuronsisreducedby92%(n=3embryopairs,totalof9
alignedsectionpairs,P<0.01)and94%(n=3embryopairs,totalof9alignedsectionpairs,P<0.001),respectively.Datain(E,F)arepresented
asmeanvalues±standarderrorofthemean(SEM).*P≤0.05,**P≤0.01,***P≤0.001.Scalebars:50μm.WT,wildtype.
embryos than of Insm1+/+ and Insm1+/- littermates. We results in fewer nascent and mature neurons in the
also labeled with antibodies to the olfactory marker pro- embryonic OE.
tein (OMP), a marker of mature olfactory receptor neu-
rons, which are detected as early as about E14 [29-31]. Deletion of Insm1 only causes a minimal increase in
We also found a reduction (-92 to -94%) of OMP+ cells apoptosis at late embryonic stages
at E14.5 and E18.5 (at earlier stages there is little or no A decrease in neurons could be caused by an increase in
OMP expression) (Figure 3). Hence, deletion of Insm1 cell death. In the embryonic OE of the mouse, there are
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two waves of apoptosis. The first peaks at E11.5 and is displayed increased apoptosis in their OEs at E14.5 (P <
thought to correspond to morphogenetic changes. The 0.05) and perhaps also at E18.5 (P = 0.11), but not at
second ranges from E13.5 to E17.5 (peaking at E15.5) E12.5 (P = 0.62) or E11.5 (P = 0.6). Therefore, the
and corresponds to innervations of olfactory receptor reduction of neurons (detected at E11.5) precedes by at
neurons with their targets in the olfactory bulb. Both least 2 days any increase in apoptosis. Furthermore, the
nascent and mature olfactory receptor neurons undergo number of additional apoptotic cells at E14.5 in the
apoptosis during development [32]. We labeled apopto- Insm1-/- OE is approximately 100 times lower than the
tic cells with antibodies against activated cleaved caspase number of missing neurons (compare Figures 4G and
3 (ACC3) and confirmed the low levels of apoptosis at 3E). Hence, although apoptosis might contribute slightly
E12.5andthehigherlevelsatE11.5andE14.5(Figure4). to the reduction of neurons, it is not the primary cause
Compared to Insm1+/+ littermates, Insm1-/- embryos of this reduction.
Figure 4 Increased apoptosis in the OE of Insm1-/- embryos does not precede the decrease in nascent neurons. (A-F)
Immunohistochemistryforactivatedcleavedcaspase3(ACC3),amarkerofapoptoticcells,atselectedstages.ACC3+stainingisindicatedby
arrowheads.(G)AtE11.5(andcompare(A)and(B))andE12.5,thereisnosignificantdifferenceinACC3+cells(n=3embryopairs,P=0.63).By
E14.5(compare(C)and(D)),moreACC3+cellsaredetectedinInsm1-/-micethanintheirpairedInsm1+/+littermates(n=3embryopairs,P<
0.05),anincreasethatpersistsatE18.5(compare(E)and(F);n=3embryopairs,P=0.11).Datain(G)arepresentedasmeanvalues±standard
errorofthemean(SEM).*P≤0.05.Scalebars:100μm.
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Deletion of Insm1 does not alter cell cycle length, but deletion of Insm1 does not cause an overall lengthening
results in an increase in apical progenitors and a of the cell cycle among OE progenitors.
concomitant decrease in basal ones in the embryonic OE The above measurements did not distinguish between
A reduction in neurons might also result from a change apical and basal progenitors, and would not have
in progenitor proliferation. A decrease in progenitors or detected a cell cycle lengthening in basal progenitors
an increase in their cell cycle length would result in a (which could explain the reduction in neurons) if this
decrease in nascent neurons. Paradoxically, an increase was compensated by a cell cycle shortening in apical
in progenitors might also result in a temporary reduc- progenitors. However, we also detected no difference in
tion of neurons produced if, as in the egl-46 mutant BrdU+/Ki67+ ratios when considering only basal pro-
nematodes, progenitors continue dividing instead of pro- genitors (Figure 5). We therefore conclude that the
ducing two neurons [2-4]. We labeled OE progenitors at reduction of olfactory receptor neurons in Insm1-/-
E12.5 with Ki67 antibodies (which marks cells at all embryos is not caused by a lengthening of the cell cycle
phases of the cell cycle) [33-35] and by 30 minutes of 5- in basal OE progenitors.
bromo-2-deoxyuridine (BrdU) incorporation (which We also found no difference between Insm1-/- and
labels all cells in S-phase) and found no difference in Insm1+/+ embryos in the overall number of progenitors
the overall number of progenitors between Insm1-/- and in mitosis, which we labeled with antibodies against
Insm1+/+ littermates (Figure 5). phosphohistone H3 (pH3) [37] at E10.5, E12.5 and
Because the length of the S-phase is largely invariant E14.5 (Figure 6). This label, however, permits us to dis-
and changes in cell cycle length reflect changes in G1, tinguish between progenitors undergoing mitosis api-
we could estimate changes in overall cell cycle length by cally, which generate more progenitors, and progenitors
calculating the fraction of all dividing cells (Ki67+) that undergoing mitosis basally, which after one or more
are in S-phase (BrdU+) [36]. A lengthening of the cell divisions produce neurons [38]. In egl-46 mutant nema-
cycle, which would result in a delay in the production of todes, there is a transient increase in proliferative (P/P)
neurons, would be reflected in a lower BrdU+/Ki67+ divisions at the expense of neuronogenic (N/N) divisions
ratio. However, we detected no difference in this ratio [2-4]. In the mouse embryonic OE, this might translate
when comparing OEs of Insm1-/- and of Insm1+/+ litter- into increased apical and decreased basal progenitors.
mate embryos (Figure 5). Hence, we conclude that the Indeed, we found an increase of apical mitoses (+42% at
E12.5) and a decrease of basal mitoses (-45% at E12.5
and -35% at E14.5) in the OEs of Insm1-/- compared to
Insm1+/+ littermates (Figure 6D-I). The decrease in basal
mitoses could mathematically account for the decrease
in neurons (Figure 3) observed in the embryonic OEs of
Insm1-/- mice. Additionally, the increase in apical divi-
sions may explain the increase in thickness (Additional
file 4) and cells (Figure 2) of the apical layer of the
Insm1-/- mice.
The thickening of the apical layer and thinning of the
neuro-basal layer roughly compensate for each other
because, as indicated above, the overall thickness of the
OE does not change in Insm1-/- embryos. We also mea-
Figure5ThenumberofOEprogenitorsinthecellcycleorS- sured the entire surface areas of the OE sections used
phase,aswellascellcyclelength,isunalteredinInsm1-/- for estimating number of neurons (Figure 3) and con-
embryosatE12.5.(A)AtE12.5thereisnodifferenceinthe firmed no difference in the OE size of Insm1-/- com-
numberofOEcellsinthecellcycle(thoseexpressingKi67;P=0.91) pared to that of Insm1+/+ littermates (Additional file 5).
orinS-phase(thosehavingincorporatedbromodeoxyuridine(BrdU)
30minutesafterinjection;P=0.29)betweenInsm1-/-(greybars)
andInsm1+/+littermates(whitebars;n=3embryopairs,totalof9 Deletion of Insm1 results in more ASCL1-expressing and
alignedsectionpairs).(B)Thefractionofdividingcells(Ki67+)that fewer NEUROD1-expressing progenitors in the embryonic
areinS-phase(BrdU+),whichisindicativeofcellcyclelength,does OE
notdifferbetweenInsm1-/-miceandtheirInsm1+/+littermates(P=
In the OE the immediate neuronal precursors (INPs),
0.11).Cellcyclelengthdoesnotdifferamonggenotypeswhen
progenitors that divide terminally to produce two neu-
consideringallprogenitors(apical+basal;P=0.11)oronlythe
basalones(P=0.19).(C)ForBrdUincorporation,alargerdataset rons (N/N divisions), are located basally and express the
(n=6embryopairs,totalof18alignedsectionpairs)alsoshowsno basic-helix-loop-helix transcription factor NEUROD1
significantdifferenceofcellsinS-phaseatE12.5(P=0.08).Dataare [38-44]. These terminal progenitors are generated from
presentedasmeanvalues±standarderrorofthemean(SEM).
the transit amplifying progenitors (TAPs), which in the
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Figure 6 Mitotically active (pH3+) progenitors decrease basally and increase apically in the OE of Insm1-/- embryos. (A,D,G)
RepresentativeimagesfromcoronalsectionsofInsm1+/+mice.(B,E,H)AnatomicallyalignedsectionpairsfrompairedInsm1-/-littermates.Dorsal
istowardtheright,ventraltowardtheleft.(A-C)AtE10.5,thereisnostatisticallysignificantdifferenceinpH3+cellsoverall,orateitherthe
apicalorbasalborderoftheOE(n=4embryopairs,totalof12alignedsectionpairs,P=0.36overall,P=0.25apically,P=0.57basally).(D-F)
AtE12.5,Insm1-/-miceexhibitnodifferenceinoverallnumbersofpH3+(green)cellswhencomparedwithInsm1+/+littermates,butanaverage
42%increaseinpH3+cellsintheapicalsubpopulation(P<0.05),andanaverage45%decreaseinthebasalsubpopulation(P<0.01;n=4
embryopairs,totalof13alignedsectionpairs).(G-I)ByE14.5,thereisstillnodifferenceinoverallpH3+cellsbetweenInsm1-/-miceandtheir
pairedInsm1+/+orInsm1+/-littermates,andthereisalsonosignificantdifferencebetweenpH3+cellsintheapicalsubpopulation.However,
thereremainsa35%decreaseofmitoticallyactivecellsinthebasalsubpopulation(P<0.05;n=4embryopairs,totalof22alignedsection
pairs).Solidwhitelinesdelineatethebasallaminaanddashedlinesdelineatetheapicaledge(nasalairway).Datain(C,F,I)arepresentedas
meanvalues±standarderrorofthemean(SEM).*P≤0.05,**P≤0.01.Scalebars:100μm.
medial and dorso-lateral OE express another basic-helix- of Insm1-/- embryos at E12.5 (compared to Insm1+/+ and
loop-helix protein, ASCL1 (also known as MASH1) Insm1+/- littermates) there are fewer (-45%) NEUROD1-
(reviewed in [1,25,41,43-45] (Figure 7J). In the embryo- expressing cells (presumably the terminally dividing
nic OE, ASCL1-expressing TAPs appear to translocate neuronogenic progenitors, or INPs) and more (+24%)
from the apical compartment to the basal lamina and ASCL1-expressing cells (presumably the TAPs transi-
are present throughout the thickness of the OE. By tioning from apical to basal) (Figure 7K-O).
immunohistochemistry (IHC) on sections of E12.5
embryos, we confirmed that antibodies raised against Deletion of Insm1 results in fewer terminal divisions in
ASCL1 label cells located across the OE that undergo the embryonic (E12.5) OE
mitosis at apical and intermediate positions (Figure 7A, Although the reduction of NEUROD1-expressing cells is
D-F, J). In addition, antibodies to NEUROD1 label basal consistent with a decrease in terminally dividing (N/N)
cells, most of which (79%) express Ki67 and are there- neuronogenic progenitors, this reduction could also
fore dividing (Figure 7G-J). With double IHC we con- occur if Insm1 is an activator of NeuroD1. In fact,
firm that ASCL1 and NEUROD1 antibodies label Insm1 protein can bind to the NeuroD1 promoter,
different populations of progenitors at this stage (Figure although the presumed effect is to repress it [46]. In
7A-C). Using these antibodies, we find that in the OEs addition, although the reduction in basal mitoses is
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Figure7IncreaseinthenumberofprogenitorsexpressingASCL1(MASH1)anddecreaseinthoseexpressingNEUROD1inInsm1-/-
embryonicOE.(A-I)CoronalsectionsofE12.5wildtypeOE.Solidlinesdelineatebasallaminaanddashedlinesapicaledges.(A-C)Antibodies
toASCL1andNEUROD1labellargelydifferentsubsetsofprogenitors.(D-F)ASCL1isexpressedincellslocatedacrosstheepithelium(seealso
(A)),asexpectedforTAPstransitioningthroughintermediatecelllayers.SomeASCL1-expressingcellsatapicalandintermediatepositions
expresspH3(arrowheads),confirmingtheyareprogenitors.(G-I)NEUROD1-expressingcellsareatthebasaledgeand79%ofthemalsoexpress
thecell-cyclemarkerKi67(arrows;n=3sectionsofOEfrom2embryos),indicatingthatNEUROD1isexpressedinbasalprogenitors.(J)
RepresentationofaTAP,expressingASCL1andmigratingfromtheapicalairwaystothebasallamina,whereAscl1isdownregulated,NeuroD1is
upregulated,andNEUROD1+cells(INPs)divideterminallytoproduceneurons(’N’;‘M’representsmitoticcells,G0postmitoticcellsandSu
sustentacularcells).(K,L)IncreaseinASCL1-expressingcells(P<0.05;n=4embryopairs,totalof12anatomicallyalignedsections;compare(L)
and(N))anddecreaseinNEUROD1-expressingcells(P<0.05;n=4embryopairs,totalof12anatomicallyalignedsections;compare(N)and
(O))intheOEofInsm1-/-embryos(comparedtoInsm1+/+andInsm1+/-littermates).(K)Quantification.(L-O)CoronalsectionsofE12.5OEfrom
Insm1-/-andInsm1+/+labeledwithmarkersofnuclei(DAPI),presumedTAPs(ASCL1)andpresumedterminallydividingneuronogenicprogenitors
(NEUROD1).Dorsalistotherightandseptaltowardthebottom.Datain(K)arepresentedasmeanofalldata(columns)±standarderrorofthe
mean(SEM)(verticalbars).*P≤0.05.Smallsquaresdepicttheaveragedensityofeachembryo,andvaluesfromeachanatomicallypaired
sectionareconnectedbyobliquelines.Scalebars:50μm(A-I);100μm(L-O).KO,knockout;WT,wildtype.