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BASP1 labels neu al s em
cells in he neu ogenic niches
o mammalian b ain
Louis N. Manganas1,7*, I ene Du á2,4, Si an Osenbe g5,6, Fai h Seme ci5,6, Mehme Tosun5,6,
Rachana Mish a5,6, Luke Pa ki ny5,6, Juan M. Encinas2,3,4 & Mi jana Male ic‑Sa a ic1,5,6,8*
The mechanisms esponsible o de e mining neu al s em cell a e a e nume ous and complex.
To begin o iden i y he speci ic componen s in ol ed in hese p ocesses, we gene a ed se e al
mouse neu al s em cell (NSC) an ibodies agains cul u ed mouse emb yonic neu osphe es. Ou
immunohis ochemical da a showed ha he NSC‑6 an ibody ecognized NSCs in he de eloping and
pos na al mu ine b ains as well as in human b ain o ganoids. Mass spec ome y e ealed he iden i y
o he NSC‑6 epi ope as b ain abundan , memb ane‑a ached signal p o ein 1 (BASP1), a signaling
p o ein ha plays a key ole in neu i e ou g ow h and plas ici y. Wes e n blo analysis using he NSC‑6
an ibody demons a ed mul iple BASP1 iso o ms wi h a ying deg ees o exp ession and co ela ing
wi h dis inc de elopmen al s ages. He ein, we desc ibe he exp ession o BASP1 in NSCs in he
de eloping and pos na al mammalian b ains and human b ain o ganoids, and demons a e ha he
NSC‑6 an ibody may be a use ul ma ke o hese cells.
To de e mine he po en ial o neu al s em and p ogeni o cells as he apeu ic agen s, he in ica e pa hways
ha media e hei p oli e a ion, su i al, and di e en ia ion mus be unde s ood1–3. To accomplish his goal,
we need selec i e ma ke s ha would allow speci ic s udies o he he e ogeneous popula ion o neu al s em and
p ogeni o cells (collec i ely labeled NPCs). Exis ing ma ke s such as nes in, Sox-2, b ain lipid-binding p o ein
(BLBP), glial ib illa y acidic p o ein (GFAP) and o he s, a e exp essed no only by NPCs bu also by o he cell
ypes, mainly o he as oglial lineage4–9. While se e al o he ma ke s appea o be selec i e o NSCs10–13, hey
a e all in acellula , limi ing cha ac e iza ion o hese cells o ex- i o s udies. To iden i y no el ma ke s o hese
cells and cap u e possible memb ane-bound an igens, we gene a ed se e al mouse an ibodies (Abs) agains
cul u ed mouse emb yonic neu osphe es ha con ain bo h neu al s em and p ogeni o cells. The NSC-6 Ab,
which p oduced he mos obus s aining o NPCs, co esponded o B ain-Associa ed Signal P o ein 1 (BASP1),
a p o ein no p e iously desc ibed in NPCs.
BASP1 (also known as NAP-22 and CAP-23) belongs o a amily o g ow h-associa ed p o eins, which include
g ow h-associa ed p o ein 43 (GAP-43) and my is oyla ed alanine- ich p o ein kinase C subs a e (MARCKS). I
is conside ed a signal p ocessing p o ein ha plays c i ical oles in synap ic plas ici y and neu i e ou g ow h14–19.
I is a hyd ophilic 23kDa p o ein wi h SDS-PAGE elec opho esis mobili y o 58 kDa20. BASP1 also con ains a
basic domain ha allows binding o phospha idylinosi ol-4, 5-bisphospha e (PIP2) and calmodulin, egula ed
by p o ein kinase C (PKC)-media ed phospho yla ion on Se -521. In u n, i s hyd ophobic p ope ies h ough
N-my is oyla ion a e c i ical o plasma memb ane20, PKC, and calmodulin binding21. Du ing de elopmen ,
BASP1 accoun s o almos 1% o he o al p o ein in b ain and almos hal o ha in he adul b ain22,23 hus
suppo ing a ole o synapse o ma ion du ing de elopmen and synap ic unc ion in adul hood. No su p is-
ingly, BASP1 mouse gene knockou s a e non- iable18. In con as , BASP1 o e -exp ession in adul neu ons24
and in PC12E2 cells and hippocampal neu ons25 s imula es neu i e ou g ow h. This e ec is belie ed o occu
independen ly o he neu al cell adhesion molecule (NCAM) pa hway26 and o ely on p ecise plasma memb ane
OPEN
1Depa men o Neu ology, S ony B ook Uni e si y Medical Cen e , S ony B ook, NY, USA. 2Achuca o
Basque Cen e o Neu oscience, Leioa, Spain. 3The Basque Founda ion o Science, IKERBASQUE, Bilbao,
Spain. 4Depa men o Neu oscience, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain. 5Depa men s
o Pedia ics, Neu ology and Neu oscience, Baylo College o Medicine, Hous on, TX, USA. 6Jan and Dan Duncan
Neu ological Resea ch Ins i u e a Texas Child en’s Hospi al, Hous on, TX, USA. 7Depa men o Neu ology,
S ony B ook Uni e si y Medical Cen e , Heal h Sciences Cen e T-12, oom 020, S ony B ook, NY 11794,
USA. 8Depa men s o Pedia ics, Neu ology, and Neu oscience, Baylo College o Medicine, Jan and Dan Duncan
Neu ological Resea ch Ins i u e a Texas Child en Hospi al, 1250 Mou sund S ., Rm 1250, Hous on, TX 77030,
USA. *email: [email p o ec ed]; [email p o ec ed]
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localiza ion and o ganiza ion25. In he adul b ain, BASP1 is exp essed in he ce eb al co ex, hippocampus,
ol ac o y bulb, basal ganglia, halamus and ce ebellum27. In addi ion o plasma memb ane, i has been epo ed,
simila ly o GAP-43, MARCKS and CAP-23, in he cy oplasm28–30 and nucleus31,32. I is mainly dis ibu ed o he
synap ic e minals, dend i ic spines, and synap ic esicles, sugges ing an impo an ole in synap ic unc ion33.
BASP1 has no been epo ed in NPCs and hus, ou inding ha an NPC-de i ed an ibody labels his p o ein
led us o pu sue de ailed cha ac e iza ion o BASP1 in he de eloping and adul mammalian neu ogenic egions.
Du ing de elopmen , BASP1 is exp essed h oughou he b ain, while in adul hood, i is es ic ed o neu ogenic
egions. In e es ingly, in he adul hippocampal niche, i is es ic ed o ype I, adial neu al s em cells (NSCs)
while in he sub en icula niche, i is limi ed o B and C cells and GFAP-exp essing cells in he os al mig a-
o y s eam (RMS)— hese likely ep esen as ocy ic ubes h ough which C cells mig a e. O e all, ou esul s
sugges ha BASP1 has po en ial alue as a ma ke o NSCs.
Resul s
NSC‑6 s ains mouse and human NPCs. In his s udy, we gene a ed se e al mouse Abs agains mouse
NPCs and cha ac e ized one Ab in pa icula , NSC-6. Ini ial sc eening o ail bleeds ob ained om each o he
h ee mice immunized wi h NPCs e ealed ha one mouse (numbe 3) p oduced he mos obus immunola-
beling agains cul u ed neu osphe es, while ail bleeds om mice 1 and 2 showed li le o no immunolabeling,
compa able o ha ob ained wi h wild- ype non-immunized mouse se um (Fig.1A). Immunoblo analysis using
he ail bleed om mouse numbe 3 agains p o ein ex ac s isola ed om NPCs e ealed mul iple bands a
49kDa, 46kDa and 22kDa (Fig.1B), while immunoblo s using ail bleeds om mice 1 o 2 did no display
any speci ic immuno eac i i y and we e simila o non-immunized mouse se um. Based on hese consis en
immunoblo and immunocy ochemical esul s, we used mouse numbe 3 as a sou ce o splenocy es o gene -
a e hyb idomas. As desc ibed in he “Ma e ials and me hods” sec ion, one Ab, NSC-6, yielded obus signal by
ELISAs pe o med agains neu osphe es and was selec ed o u he analyses. O no e, we could no isola e a
monoclonal clone despi e se e al ounds o NSC-6 hyb idoma subcloning. I is unclea whe he u he subclon-
ing is necessa y o usion wi h mo e han one lymphocy e occu ed du ing hyb idoma p oduc ion.
To alida e he speci ici y o NSC-6 immunolabeling agains NPCs, we cul u ed neu osphe es om Nes in-
GFP ansgenic mice in which he g een luo escen p o ein (GFP) exp ession is d i en by he nes in egula o y
elemen s5. Nes in is a speci ic ma ke o neu oepi helial s em cells34. Cul u ed neu osphe es, gene a ed om
Nes in-GFP emb yos, we e immunolabeled wi h NSC-6 (Fig.1C). All Nes in-GFP exp essing cells we e posi i e
o NSC-6 immunolabeling. NSC-6 also immunolabeled a mino i y o cells adhe ing o he pla e ha exhibi ed
low o unde ec able le els o Nes in-GFP. In addi ion, we de i ed human neu op ogeni o cells (hNPCs) om
inducible plu ipo en s em cells (iPSCs) ob ained om a heal hy adul and s ained hem wi h NSC-6. The NSC-6
labeled a po ion o hNPCs (Fig.1D), sugges ing di e en popula ions o hNPCs in ou cul u e. Indeed, so ing
o he hNPCs labeled wi h he NSC-6 con i med he exis ence o NSC-6 posi i e and nega i e popula ion in wo
independen expe imen s (Fig.1E).
NSC‑6 an ibody ecognizes BASP1. To de e mine he iden i y o he NSC-6 an igen, we employed Liq-
uid Ch oma og aphy–Mass Spec ome y (LC–MS) wi h pep ide mass inge p in ing o LC–MS/MS ( andem
MS) on he spo excised om he 2D gel con aining human hippocampal ex ac (Fig.1F). We obse ed ou
dis inc , s a is ically accep able pep ide sequences in six MS/MS spec a, accoun ing o 34.8% co e age o he
p o ein BASP1 (Fig.1G). As expec ed, he excised spo con ained an abundance o bo ine p o eins (casein and
albumin) esul ing om he immunoblo ing p o ocol. The only de ec able human p o eins we e a small a i-
e y o ke a ins (common con aminan s obse ed in LC/MS/MS s udies) and he b ain abundan , memb ane
Figu e1. Mouse-de i ed polyclonal an ibody agains mouse neu osphe es iden i ies BASP1 as i s an igen. (A)
Neu osphe es immunolabeled wi h ail bleeds om mice 1, 2, and 3, and no mal mouse se um (nms) (all a
1:1000). (B) Immunoblo o he neu osphe e lysa e wi h mouse 3 ail bleed and nms (bo h a 1:10). The a ows
indica e h ee dis inc bands o 49kDa, 46kDa and 22kDa. Full leng h s ips a e shown in a single c opped
image. (C) Epi luo escen images o Nes in-GFP neu osphe es (g een) labeled wi h NSC-6 an ibody ( ed) show
high immunolabeling o he neu osphe e (me ged, yellow). All he Nes in-GFP cells a e NSC-6 posi i e, bu
some NSC-6 immunoposi i e cells a e nega i e o Nes in-GFP (a ow). Scale ba is 100µm in A and 50µm
in C. (D) Human neu al p ogeni o cells (hNPCs) labelled wi h DAPI (blue) and NSC-6 an ibody (1:20, ed).
Scale ba is 20µm. (E) So ing o hNPCs s ained wi h unconjuga ed NSC-6 an ibody and Day Ligh 405 as he
seconda y an ibody. 250,000 li e cell e en s we e acqui ed. Single iable cell ga ing was ca ied ou as shown
in uppe panels. Exclusion o NSC-6neg and alse-posi i e signals was done by ga ing ou side uns ained cells
and cells s ained wi h he seconda y an ibody only (middle panels). Two independen ly gene a ed hNPC lines
(hNPC.1 and hNPC.2) we e examined, showing a subpopula ion o NSC-6 labeled cells. (F) Le A 2-DE gel
ob ained wi h 90µg o human hippocampus lysa e and s ained o o al p o eins wi h Syp o Ruby luo escen
s ain. Righ Immunoblo ob ained om he same p o ein load un on an iden ical gel in pa allel wi h ha o (B),
and immunolabeled wi h he NSC-6 Ab. The spo o in e es , co esponding o molecula weigh o 45kDa, is
indica ed in he cen e o he blo . The loca ion o a blank, con ol spo is also indica ed. Sepa a e blo s, shown
in hei en i e y, we e c opped and sepa a ed by whi e space. (G) LC/MS/MS analysis indica es ha he spo o
in e es is accession # IPI00299024 BASP1, b ain abundan , memb ane a ached signal p o ein 1. Fou pep ides
we e iden i ied in six spec a, wi h 34.9% co e age. aEach pep ide sequence was de e mined in dis inc MS/MS
spec a. All six spec a we e manually con i med. bp- alues < 0.025 we e conside ed s a is ically accep able. (H)
RT-PCR o BASP1 mRNA in cul u ed adul mouse NPCs and mouse li e cells (nega i e con ol).
▸
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a ached signal p o ein 1 (BASP1). As con ol, we excised a simila ly sized piece o blo om he ma gin no
exposed o he gel-p o ein ans e bu ea ed o he immunos aining p ocess. We obse ed all bo ine p o eins
and human ke a ins in he con ol spo diges , bu he e was no e idence o BASP1 in his sample (Fig.1F,
igh panel). To hen con i m he BASP1 exp ession in adul mouse neu osphe es, we pe o med RT-PCR and
obse ed signi ican di e ence be ween BASP-1 mRNA in mouse NPCs compa ed o mouse li e , used as a
nega i e con ol (Fig.1H).
The NSC‑6 an ibody localizes BASP1 o adial neu al s em cells (NSCs) in he emb yonic mouse
b ain. To de e mine whe he BASP1 is exp essed in NSCs o he de eloping mouse b ain, we immunolabeled
b ain sec ions om emb yonic day 12 (E12) Nes in-GFP mice wi h he NSC-6 Ab (Fig.2). Du ing emb yonic
de elopmen o he ce eb al co ex, adial glia no only se e as sca olds o mig a ing neu oblas s, bu also as
p ecu so cells ha gene a e neu ons and glia35. Radial glia exp essing Nes in-GFP we e localized h oughou
he de eloping emb yonic b ain (Fig.2), including co ex (Fig.2A) and he u u e hippocampal egion (Fig.2B).
All cells exp essing Nes in-GFP also exhibi ed NSC-6 immunolabeling, p esen on he GFP-posi i e adial glia
longi udinal p ocesses (Fig.2C). Fu he mo e, Sox2 s aining con i med ha NSC-6 did no iden i y ampli ying
neu op ogeni o s in he sub en icula zone a E12 (Supplemen a y Fig.S1). These esul s sugges ha BASP1,
ecognized by NSC-6 Ab, is exp essed in adial glia du ing emb yonic b ain de elopmen .
The NSC‑6 an ibody localizes BASP1 o known neu ogenic egions in he pos na al mouse
b ain. To cha ac e ize BASP1 exp ession in he pos na al mouse b ain, we ca ied ou diaminobenzidine
(DAB)-based immunolabeling in 4-week old mice (Fig.3). In gene al, NSC-6 Ab immunolabeled neu ogenic
Figu e2. NSC-6 an ibody immunolabels adial glia in he emb yonic mouse b ain. NSC-6 immunolabeling
( ed) is p esen along he p ocesses o adial glia in Nes in-GFP emb yonic (E12) co ex (A) and hippocampus
(B). (C) High magni ica ion image o he emb yonic co ex shows Nes in-GFP exp ession in he nucleus and
soma o adial glia (a ows). These compa men s we e no immunolabeled by he NSC-6 Ab. Scale ba s a e
50µm in (A,B); and 5µm in (C).
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a eas o he pos na al mouse b ain—p esumably NPCs—and cells in he whi e ma e such as he co pus cal-
losum (Fig.3A), he an e io commissu e, and he ce ebellum, whe e NSC-6 immunolabeling was p esen in he
Be gmann glia adial p ocesses in he molecula laye (Fig.3B). NSC-6-immunoposi i e cells we e also ound
in he hilus, he g anule cell laye , and he molecula laye o he den a e gy us (Fig.3C). Rema kably, obus
s aining was ound in p esump i e NSCs o he subg anula zone (SGZ), known o ha bo he neu ogenic s em
cells o he adul hippocampus36. In addi ion, NSC-6 immunolabeling was obse ed in he sub en icula zone
(SVZ) o he la e al en icle (Fig.3D), while bo h caudal (Fig.3E) and os al (Fig.3F) pa s o he RMS we e
conspicuously immunolabeled. NSC-6 immunolabeling was no obse ed in o he b ain egions, such as co ex
and s ia um (Fig.3A,B,E). Omission o he p ima y Ab p oduced no immunolabeling (Fig.3G).
The NSC‑6 an ibody localizes BASP1 o NSCs in neu ogenic niches o he pos na al mouse
b ain. To u he in es iga e he exp ession o BASP1, we double-immunolabeled b ain sec ions wi h NSC-6
Ab and a panel o diagnos ic cell ma ke s (Fig.4). In he SVZ and he den a e gy us, NSC-6 immunolabeling
colocalized wi h ma ke s o NSCs, such as imen in (Fig.4A,C) and GFAP (discussed below). In con as , i did
no colocalize wi h ma ke s o neu oblas s, imma u e, and ma u e neu ons, such as PSA-NCAM (Fig.4B,D),
P ox-1 (Fig.4E), and NeuN (Supplemen a y Fig.S2), espec i ely, as well as mic oglia (Iba-1), oligodend ocy e
p ogeni o s (NG2), o ma u e oligodend ocy es (myelin basic p o ein (MBP). These da a con i m ha BASP-
1exp ession, as de ined by NSC-6 Ab immunolabeling, is es ic ed o he NSCs in bo h pos na al neu ogenic
niches.
The NSC‑6 an ibody localizes BASP1 o NSCs and no ANPs in he mouse hippocampus. In
he adul hippocampus, neu ogenesis begins wi h p ima y adial NSCs ( ype I cells), which exp ess nes in,
imen in, GFAP, and BLBP in hei apical p ocesses35,37–39. These NSCs di ide asymme ically, gi ing ise o
ampli ying neu op ogeni o s (ANPs, ype II cells), which p oli e a e symme ically be o e exi ing cell cycle and
di e en ia ing slowly in o neu ons35,36,39,40. ANPs and NSCs di e in mo phology and exp ession ma ke s: ANPs
a e small, ound cells ha exp ess low le els o nes in and lack imen in and GFAP. In Nes in-GFP ansgenic
mice, NSC-6 Ab immunolabeling colocalized wi h GFAP, indica ing ha ype I NSCs, and no ype II ANPs,
Figu e3. NSC-6 an ibody immunolabels di e en adul mouse b ain egions. (A) NSC-6 immunoposi i e
glia-like cells in he co pus callosum (cc). No e he absence o NSC-6 immunolabeling in he co ex (cx) and
he s ia um (s ). (B) NSC-6 immunolabeling o Be gmann glia p ocesses in he molecula laye (mol) o he
ce ebellum. The g anula laye (g ) lacks NSC-6 immunolabeling. (C) NSC-6 labels spa se as ocy es-like
cells in he hilus (h), he molecula laye (mol), and he lacunosum molecula e laye (Lmol) o he den a e
gy us. Radial-as ocy e p ocesses—mos likely belonging o NSCs due o hei pe pendicula o ien a ion—
in he g anule cell laye (gcl) a e also immunolabeled. (D) NSC-6 immunolabeling o he SVZ adjacen o
he la e al en icle (l ) and su ounding pa enchyma. (E) NSC-6 immunolabeling o he RMS. No e he
absence o s aining in he s ia um (s ). (F) NSC-6 immunolabeling o he os al aspec o RMS, en e ing and
dissemina ing in he ol ac o y bulb (ob). The a ow poin s he os al di ec ion. (G) Omission o he p ima y
an ibody esul ed in absence o de ec able immunolabeling h oughou he en i e b ain. The co pus callosum
be ween he co ex (cx) and hippocampus (hc) is shown. Scale ba s a e 50µm in all images.
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exp ess BASP1 (Fig.5A,B). In addi ion, we obse ed andom and spa se NSC-6 and GFAP colocaliza ion in
he hilus and he hippocampal molecula laye , indica ing ha BASP1 migh be exp essed in den a e gy us
as ocy es. This is no su p ising, gi en ha adial NSCs gi e ise o as ocy es and ha GFAP, imen in, nes in
and BLBP a e all exp essed in as ocy es as well. To hen solidi y ou inding ha NSC-6 does no label ANPs,
we did a iple s ain wi h NSC-6, GFAP and Sox2 an ibodies. We con i med ha NSC-6 did no iden i y ANPs,
which we e only Sox2-posi i e (Fig.5C). In addi ion, we u ilized ano he ansgenic mouse line in which nes in
egula o y elemen s d i e he exp ession o he cyan luo escen p o ein (CFP) con aining a signal o nuclea
localiza ion35,36. Since only he nuclei o NPCs can be isualized in he Nes in-CFPnuc mouse s ain, NSCs and
ANPs canno be dis inguished, unless an addi ional ma ke such as imen in o GFAP is used. Ou da a employ-
ing NSC-6 immunolabeling in b ain sec ions om Nes in-CFPnuc mice con i med ha NSC-6-labeled BASP1
is exp essed only in NSCs wi hin he SGZ neu ogenic niche (Supplemen a y Fig.S3A). Immunos aining wi h
he comme cially a ailable BASP-1 polyclonal an ibodies did no yield any de ec able s aining (Supplemen a y
Fig.S3B,C).
The NSC‑6 an ibody localizes BASP1 o B and C cells in he pos na al SVZ and NSCs o he
spinal co d. In he SVZ, NSC-6 co-localized wi h GFP and GFAP in Nes in-GFP mice (Fig.6A,B), he e o e
labeling B cells, he equi alen o NSCs in he hippocampus40. Howe e , in he SVZ, NSC-6 also labeled C cells,
he ansien ampli ying p ecu so s (equi alen o hippocampal ANPs), which exp ess nes in bu no GFAP o
imen in. The NSC-6 Ab did no label neu oblas s, e med A cells41,42, in he SVZ, as he e was no co-localiza ion
wi h PSA-NCAM (Fig.5B). In he Nes in-CFPnuc ansgenic mice, NSC-6 immunolabeling was p esen in he
cy oplasm o all Nes in-CFPnuc exp essing cells (B and C cells, Fig.6C). In addi ion, he NSC-6 Ab labeled
ependymal cells, which also exp ess nes in, a e adjacen o he cells bo de ing he en icle, and a e labeled wi h
FoxJ1 (Supplemen a y Fig.S4).
In he RMS, A and C p ecu so s mig a e owa ds he ol ac o y bulb enshea hed by B cells42. NCS-6 immu-
nolabeled as ocy ic ubes ha enshea h C cells (immunoposi i e o Nes in-GFP and GFAP) as hey mig a e
owa d he ol ac o y bulb and as ocy es (immunoposi i e o GFAP bu no Nes in-GFP) loca ed in he icini y
(Fig.6D). Nes in-GFP-posi i e and GFAP-nega i e C cells we e no immunolabeled wi h NSC-6 (Fig.6D), which
is in con as o wha we obse ed in he SVZ. Because o he dissimila i ies be ween hei espec i e immunola-
beling pa e ns, we concluded ha he NSC-6 Ab does no ecognize nes in, GFAP, o imen in and we alida ed
his conclusion by biochemical analyses ha showed dis inc immunolabeling o he espec i e a ge p o eins
on immunoblo s o whole mouse b ain ex ac s (Fig.8A–D). Taken oge he , NSC-6-labeled BASP1 exp ession
is limi ed o B and C cells in he SVZ and only as ocy ic ubes ha enshea h C cells in he RMS.
To examine whe he NSC-6 also labels NSCs localized a ound he cen al canal o he spinal co d, we s ained
spinal co d sec ions om wild- ype (Fig.6E), Nes in-GFP (Fig.6F), and Nes in-CFPnuc ansgenic mice (Fig.6G).
NSC-6 co-localized wi h imen in, GFP in Nes in-GFP, and CFP in Nes in-CFPnuc mice. Howe e , while co-
localized wi h GFAP in Nes in-GFP cells, i also labeled some Nes in-GFP-posi i e bu GFAP-nega i e cells,
sugges ing ha in he spinal co d, i migh label some neu op ogeni o s and no only NSCs.
Figu e4. The NSC-6 an ibody does no label neu onal lineages in he adul mouse neu ogenic egions. NSC-6
immunolabeling colocalizes wi h imen in (A) bu no PSA-NCAM in he SVZ. (B) NSC-6 immunolabeling
colocalizes wi h imen in (C) bu no PSA-NCAM (D) o P ox-1 (E) in he den a e gy us (DG). Scale ba s a e
20µm in (A–C); 10µm in (D); and 40µm in (E).
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NSC‑6 an ibody labels NSCs in human b ain o ganoids. We used human iPSCs o gene a e b ain
o ganoids using modi ied Pasca p o ocol, a guided app oach based on he supplemen a ion o ex e nal ac o s o
induce iPSCs o di e en ia e owa ds do sal o eb ain-like issue43. We selec ed his me hod because i ecapi u-
Figu e5. NSC-6 an ibody immunolabels adul hippocampal NSCs bu no ANPs. (A) NSC-6 immunolabeling
co-localizes wi h GFAP in he hilus, he SGZ, he g anula cell laye , and he molecula laye o he den a e
gy us. (B) NSC-6 immunolabeling co-localizes wi h GFAP and Nes in-GFP in he adial p ocess o he NSCs
loca ed in he SGZ. (C) NSC-6 does no label Sox2+ GFAP− ANPs (a ows) adjacen o NSC-6+; GFAP+;
Sox2+ NSCs (a owhead). Scale ba s a e 25µm in A, 5µm in B, and 10µm in (C).
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la es wi h conside able accu acy he de elopmen o he human co ex in gene al and he o ganiza ion o NSC
zones in pa icula 44,45. The key s ages in o ganoid gene a ion a e shown (Fig.7A). By day 55 (55d), he o ga-
noid con ains en icula -like zones whe e neu al s em/p ogeni o s eside; neu ons (MAP2+), and as ocy es
(S100β +) (Fig.7B). A 79d, en icula -like zones s ill exis (PAX6 ) and neu ons a e now abundan (MAP2+)
(Fig.7C, uppe panel). By 104d, he o ganoid con ains ma u e neu ons wi h synap ic con ac s as e idenced by
SYN1 immunos aining (Fig.7C, lowe panel) and exhibi s spon aneous elec ical ac i i y and ac ion po en ials
(Supplemen a y Fig.S5). We examined he BASP1 exp ession in 55d o ganoids and obse ed ha NSC-6 Ab
s ongly immunolabeled PAX6+ NSC subpopula ion (Fig.7D). In con as , we did no de ec such s aining in
imma u e DCX+ neu ons in a 55d o ganoid (Fig.7E) o ma u e S100β+ as ocy es in a 104d o ganoid (Fig.7F).
These da a u he solidi y ou indings in he mouse models, demons a ing ha human NSCs a e pa icula ly
en iched wi h BASP1 compa ed o o he b ain cells.
NSC‑6‑labeled BASP1 is egula ed empo ally in he mammalian b ain. We hen examined em-
po al exp ession o he NSC-6-labeled BASP1. Biochemical analysis o he whole b ain ex ac s om E15, P1,
P30 and P60 mice by SDS-PAGE and immunoblo ing e ealed mul iple polypep ide iso o ms ecognized by he
NSC-6 Ab (Fig.8A). The majo iso o ms obse ed in E15 and P1 mice co espond o ela i e elec opho e ic
mobili ies (M ) o 35, 38, 47 and 51kDa. In P30 mice, he 35 and 38kDa iso o ms we e educed ela i e o he
47kDa iso o m (Fig.8A). Fu he mo e, in he P60 mouse b ain his ela i e di e ence in iso o m exp ession
is e en mo e appa en ; he e is a u he educ ion in he in ensi y o he 35 and 38kDa bands while hose co -
esponding o 47 and 51kDa pe sis . These da a sugges ha BASP1 is modi ied du ing b ain de elopmen such
ha iso o ms exhibi ing lowe M alues p edomina e ea ly in de elopmen , while iso o ms ha exhibi highe
M alues become p e alen in adul hood. Posi i e con ols included GFAP, imen in, and nes in espec i ely
(Fig.8B–D). As expec ed, exp ession o GFAP was ela i ely low du ing de elopmen and inc eases wi h ma u a-
ion (Fig.8B). On he con a y, imen in (Fig.8C) and e en mo e nes in (Fig.8D) displayed obus exp ession
du ing de elopmen wi h a apid decline du ing ma u a ion.
To hen cha ac e ize BASP1 exp ession in he human b ain, we compa ed e al and adul human b ain p o ein
ex ac s by SDS-PAGE and NSC-6 immunoblo analysis. NSC-6 labeled a majo band a 35kDa and a mino
band a 51kDa in he e al human b ain (Fig.8E). In he adul human b ain, he majo pep ide ecognized by
NSC-6 mig a ed a 45kDa (Fig.8E) and ela i ely smalle quan i ies o he 51, 35 and 38kDa iso o ms we e
also de ec ed. These esul s a e gene ally consis en wi h hose obse ed in he emb yonic and adul mouse
b ain. Finally, we compa ed NSC-6 immunolabeling in samples p epa ed om speci ic egions o he human
adul b ain (Fig.8F). Consis en wi h he da a ob ained in mice, ou esul s show high BASP1 exp ession in he
hippocampus, he b ains em, and he spinal co d (Fig.8F). As expec ed om he immunohis ochemis y esul s
epo ed abo e, ela i ely low le els o BASP1 exp ession we e obse ed in he co ex and ce ebellum.
Discussion
In his s udy we aimed o gene a e mouse an ibodies agains epi opes ound on NPCs. We isola ed one an i-
body (NSC-6) and cha ac e ized i in de ail. Mass spec ome y using human hippocampal issue e ealed he
iden i y o he ecognized an igen as BASP1, a signaling p o ein ha plays a key ole in neu i e ou g ow h and
plas ici y14–19, bu he e, we demons a e ha i migh be u ilized as a ma ke o NSCs in he adul b ain.
Simila app oaches o de eloping an ibodies agains mouse emb yonic s em cells ha e been a emp ed in he
pas u ilizing mice46,47 and abbi s48. Majo d awbacks in mice include immune ole ance o mouse emb yonic
s em cell su ace an igens leading o low an ibody p oduc ion, which could be o e come by immunizing abbi s
ins ead. Rega dless o he animal used as a hos , a signi ican numbe o an ibodies a e ypically gene a ed agains
in acellula epi opes when animals a e immunized wi h whole cells as was obse ed in ou s udy.
We ound ha NSC-6-labeled BASP1 localizes o all adial glia a he E12 s age o b ain de elopmen , while
pos na ally, i es ic s o he neu ogenic a eas o he mouse b ain bu no he co ex. This exp ession pa e n
con as s p e ious s udy using DAB-based immunolabeling o NAP-22 (BASP1 alias) in he adul a b ain,
which demons a ed obus labeling o ce eb al co ex27. While we do no know he basis o his di e ence in
immunolabeling o co ex, possibili ies include species a ia ions be ween a and mouse exp ession o BASP1,
o di e ences in epi ope ecogni ion be ween he wo an ibodies used ha could yield dis inc pa e ns o
immuno eac i i y. Indeed, he wo comme cial BASP1 polyclonal an ibodies did no immunolabel NSCs and
in gene al, exhibi ed poo s aining o he mouse b ain issue.
Figu e6. NSC-6 an ibody immunolabels cells in he adul SVZ and RMS as well as a ound he spinal co d
canal. (A) NSC-6 an ibody immunos ain co-localizes wi h GFAP and Nes in-GFP in he SVZ and he RMS
(i s o igin is shown in he uppe igh co ne o he images). (B) NSC-6 also immunolabels he cy oplasm o
some GFAP-nega i e Nes in-GFP cells in he SVZ. (C) NSC-6 immunolabels he cy oplasm o SVZ neu al s em
and p ogeni o cells in Nes in-CFPnuc ansgenic mice. (D) In he RMS, NSC-6 immunolabels cy oplasmic
p ocesses o GFAP-posi i e Nes in-GFP cells and co-localizes wi h GFAP in Nes in-GFP-nega i e cells. (E)
NSC-6 co-localizes wi h imen in in cells su ounding he cen al canal o he spinal co d. I also spa sely
s ains imen in-nega i e cell p ocesses in he neighbo ing pa enchyma. (F) NSC-6 labels Nes in-GFP-posi i e
GFAP-nega i e cells a ound he cen al canal, as well as Nes in-GFP-nega i e GFAP-posi i e cell p ocesses in he
su ounding pa enchyma. (G) NSC-6 an ibody labels he cy oplasm o he Nes in-CFPnuc cells su ounding he
cen al canal in he Nes in-CFPnuc ansgenic mice. Scale ba s a e 10µm in (A,C–E,G); and 5µm in (B), and
40µm in (F).
◂
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Acknowledgemen s
We a e g a e ul o G igo i Enikolopo o c i ically e iewing he manusc ip , Dwigh Ma in o expe ech-
nical assis ance, and Huda Zoghbi o he use o low cy ome e . This wo k was suppo ed by he NIGMS
(5R01GM120033), U.S. A my Medical Resea ch (DAMD170110754), Cyn hia and An ony Pe ello Endowmen ,
and Ma k A. Wallace Endowmen (M.M.S.); he Na ional Ins i u e o Diabe es and Diges i e and Kidney Diseases
(T32DK07521-16) (L.N.M.); MINECO SAF-2015-70866R (J.M.E), FPI MICINN p edoc o al Fellowship (I.D.);
he P o eomics Cen e a S ony B ook Uni e si y (NIH/NCRR 1S10 RR023680), and he BCM IDDRC G an
(P50HD10355) om he Eunice Kennedy Sh i e Na ional Ins i u e o Child Heal h and Human De elopmen
o use o he Mic oscopy Co e acili ies, he RNA InSi u Hyb idiza ion Co e acili y, and he Human Neu onal
Di e en ia ion Co e acili y.
Au ho con ibu ions
L.N.M. con ibu ed o he concep ion and expe imen al design, collec ion and assembly o da a, da a analysis
and in e p e a ion, and manusc ip w i ing. I.D. collec ed some o he ansgenic mouse da a. S.O. collec ed all
human model immunolabeling da a and p o ided hNPCs o low cy ome y expe imen s. F.S. collec ed da a
on comme cial BASP1 an ibodies and di e en ial NSC-6 immunos aining in NSCs and ANPs. R.M. gene -
a ed mouse neu osphe es and pe o med RT-PCR. L.P. pe o med low cy ome y. M.T. collec ed some o he
wild- ype and emb yonic immunos aining da a. J.M.E. collec ed ansgenic mouse da a and con ibu ed o da a
analysis and in e p e a ion, and manusc ip w i ing. M.M.S. con ibu ed o he concep ion and expe imen al
design, inancial suppo , da a analysis and in e p e a ion, and manusc ip w i ing. All au ho s app o ed he
inal e sion manusc ip .
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Addi ional in o ma ion
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o g/10.1038/s4159 8-021-85129 -1.
Co espondence and eques s o ma e ials should be add essed o L.N.M.o M.M.-S.
Rep in s and pe missions in o ma ion is a ailable a www.na u e.com/ ep in s.
Publishe ’s no e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
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Scien i ic Repo s | (2021) 11:5546 | h ps://doi.o g/10.1038/s41598-021-85129-1
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