Pglyrp1-Cre Marks Distinct Epithelial and Immune Lineages Across Mucosal Sites

14 Muc os a-a ss oc iat ed l y mph oid tis su e ( MAL T) init iat es im mune r e spo nses a t 15 muc os al ent ry si t es . Wi th in MAL T , m ic r ofold (M ) c ells sa mple lu min al antig ens and 16 d e l i ver th em to u n de rlyi n g i mm un e c e l ls . D e spit e th ei r funct i onal i mpo rtan ce , f ew 17 t ool s enabl e sele ct i v e manipul at ion of M c ells in vivo . H er e w e r ep or t the gen er ati on 18 and ch ar ac t er iza ti on of a p eptid o gl y c an r ec ogni ti on pr ot e in 1 ( Pglyrp1 )-Cr e kno ck-in 19 mou se de sign ed t o all ow c ondi ti ona l gene ti c a cc ess t o M c ells. Using R o sa26-t d T o mat o 20 re p or ter mi ce , we fo und s tr ong Pglyrp1 p r o mo t e r ac tivi ty i n g u t epit heli al c ells, 21 including go ble t and M c ells, w he r ea s a c ti vi ty in na sal-a ss oc iat ed l y mpho id tis sue 22 (N AL T ) w a s mo r e het er ogen eou s and sk ew ed t ow ar d s i mm une cell s, pa rt ic ula rl y 23 neutr oph ils. T o func ti onall y int e rr o g at e Pglyrp1 -e xpr ess ing c ells, w e p erfo rm ed C r e-24 med iat ed abl ati on us ing thr ee D T A- bas ed m odel s. Th e R os a26 GFP- DT A line c aus ed 25 ma rk ed pe rina tal l et hali ty in d ou ble-po si ti v e pups , s ugges ting es se ntial r ol es for 26 P gl yrp1-p os it i v e cell s e ar l y in l ife . In c on tras t , R o sa 2 6 DT A and Rosa 2 6 iD TR cr osse s 27 pr odu ced m ini mal depl eti on of m uco sal popul at ions , includ ing M c e lls, e v en a t th e 28 high es t n on-leth al d ipht her ia toxi n d o se . T he se fi ndings de mons tr at e ti ssu e-spe c ific 29 Pglyrp1 p r o mo ter ac tivi ty and h ighl igh t ch allenge s in ach ie v ing M cell-sp ec ifi c 30 ta rge ting. Alt hou gh n ot M c ell-r e st ri ct ed, th e P gl yrp1- Cr e m ous e pr o v id es a useful t o ol 31 for man ipula ting P gl yrp1-e xp r es si ng line age s and p r o bing the ir r ol es in mu c osal 32 hom eo st as is and i mm unity . 33 34 K e yw or d s: P gl y rp1- Cre; M c e l l s; M uc os a l i m muni ty 35 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint

36 Muc os a-a ss oc iat ed l ymph oid t iss u e ( M AL T ) p lays a c e nt r a l r o l e i n i m mune 37 defense at m aj or s it e s of pa thog e n ent ry , in cluding t he g as tr oint es t inal (G I) t r ac t , 38 r esp ir at o ry sy st em , skin, and g e nit ou rina ry trac t [1]. M AL T con si st of o rg an ized 39 l ymph oid foll icl es c o vere d b y s pe ci a l i ze d e p i thel ial l a y er s, wi th P e y er’ s pat che s 40 ser ving as th e be st-c h arac terized e x ampl e [2]. F r om s tudi es of P eyer ’ s p a t c he s , 41 mi cr ofold ( M ) c ells w er e id entified as r ar e ep ith eli al c ells t hat sa mple l uminal ant igens 42 and del i v e r t he m t o s ub epi thel ial i mmun e cells th r o ugh t r ans cyt os is [3]. Th is proce s s 43 is e xpl oit ed b y pa th ogens su ch as Salmonella , Y ersinia , Shigella , and Mycobacterium 44 tuberculosis t o ent er the h ost [4-7]. M c ells the r ef ore p l a y c r it ic a l r o le s i n mi crob ia l 45 path ogen esi s and mu c osal i mmun e su rvei l l a n ce [8]. 46 Despit e the ir i mpo rt ance , t ool s th at sel ec ti v el y t a rge t M cells in vivo r em ain 47 limit ed. Appr oa che s h a v e in clude d disrup ting RA NK-R A NKL s ignaling t o m odulat e M 48 cell ab undanc e [9], o r u sing mu c os al ad ju v an ts su ch as c hol er a t o xin [ 10]. Gen et ic all y 49 engine er e d m odels su ch a s Spib - or Sox8 -defi ci ent m i ce [11, 12] and Villin -C r e-dr i v en 50 cond iti onal kno ck o uts t ar get ing T nfrsf11a ( RA NK ) , Gp2, or Atoh8 [13-16] ha v e 51 a d va nc e d t he fi e l d, yet ma ny l ac k ce l l -typ e spe cific ity and af fec t br oad er ep ith eli al 52 co mpa rt ment s. 53 P ept ido gl y c an r ec ogni ti on pr ot e in 1 (PGL Y RP1 ), al so kn o wn as P GR P , PGRP-S , 54 T A G7, or TNF AF3L, is an innat e i mmun e r ec ept o r e xpr ess ed in se v er al c ell typ es . 55 PGL YRP1 h as b een det ec t ed in M c ells of P e y e r’s pat ch es, w h er e i t col oc alize s wi th M 56 cell m ark er s su ch as Ulex europaeus ag glut inin 1 (UE A-1) and GP2 [ 10, 17], and in 57 RA NKL-indu ced M cell s d er i v ed fr o m int es tin al o rg ano ids [18]. P GL YRP1 i s als o 58 e xpr es sed in neu tr ophil s, epit hel i al c ells, and s tr om al popul at ions [19-21]. The se 59 obs er v at ions sugg est ed Pglyrp1 a s a p ot en ti al pr om ot e r fo r M c ell-spe cif i c Cr e 60 e xpr es si on. 61 H e r e we de sc ri be t he g e n erat io n a nd c h arac te riza ti on of a Pglyrp1 -Cr e knock-in 62 mou se . Alth ou gh ini ti all y int ended fo r M c ell ta rg et ing, Pglyrp1 -Cr e d r o v e 63 r e co mb inat ion in a br oa der s et of epi th elial and i mmun e p opula ti o ns in a ti ssu e-64 spec ifi c manner . Us ing flu or es cen t r epo rt e r and d iphth er ia toxi n-bas ed abl at ion 65 mod els, w e m apped Pglyrp1 -e xpr ess ing line age s and unc o v er ed t hei r func ti onal 66 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint signifi c ance . Whil e n ot M cell-sp ec ific , thi s mod el pr o v ide s a v er sat ile t o ol f o r 67 manipul at ing Pglyrp1 -pos it i v e c ell s in vivo and for pr ob ing the ir c ontr ibu ti ons t o 68 muc os al bar ri er int eg ri ty and innat e i mm unity . 69 70

71 Gener ation of a Pglyrp1 -Cr e mouse model and founder scr eening 72 T o gen et ic all y ta rg et Pglyrp1 -e xpr e ssing cell s, w e g ene r at ed a kno ck-i n mou se 73 in w hi ch C r e r ec o mb inas e w as in s ert ed d o wnst r e am of e x on 3 of th e P gl y rp1 lo cus 74 using t he Ea si- CR ISPR me th od [22 ]. Thi s appr o ach pres er v e s endog e nous pr o mot er 75 r egul ati on w hil e m ini mizing di sru ption t o Pglyrp1 gen e funct ion (F igur e 1A ). T he 76 s i n g le - s tra n de d DNA (s s DNA) do n or u se d fo r r e co mb i na t io n i n c l u de d h o mo l ogy ar ms , 77 an int ern al ri bo so me entr y sit e ( IR ES), and th e Cr e c oding s eq uenc e ( Figur e 1B ). T he 78 full r e co mb ined e x on 3 se quen ce and don or t e mplat e s ar e sh o wn i n Supplem ental 79 Figur es 1A and B . 80 F ounder an im als w ere bo r n at ex pec te d M endel ian r a ti os , w er e p hen ot ypic all y 81 norm al, and r e main ed fe rt ile. F o r g enot yping w e us ed pr im er pai rs sp anning th e 5@iLH 82 and 3@iLH junct i ons of th e ins er ti on ( Figur e 1C ) th at g ener at ed d is tinc t PCR pa tt e rns f o r 83 wild-t ype and kn ock- in all ele s. R e str ic ti on enzym e d ige sti on wi th E co RI and Nh eI 84 furth er c onfir med c or r e ct ca ss ett e i nt egr a ti on (Supple ment al F igur es 1 C-1E ). 85 The se anal y se s v e rifi ed suc ce ssful g ener at ion of the Pglyrp1 - Cre k n oc k -i n line . 86 87 Reporter expr ession in newborn Pglyrp1 -Cre mice r ev eals tissue-specific 88 patterns 89 T o e x am ine P glyrp1 - d ri ve n C r e ac tivi t y , w e c r o ss e d Pglyrp1 - C re mi ce w i th 90 B6.Cg-G t( R OS A)26S or tm14(CA G-td T om ato )Hze /J (als o kn own as R o sa26-t d T o mat o) r epo rt e r 91 mi ce [23]. T o e x am ine w h ole- bo d y r ep ort er e xpr es si on dur ing e ar l y de v elop men t , 92 one-d a y-old pups w er e fi x ed, s ec ti oned, and st ain ed f o r RFP and th e M c ell ma rk er 93 GP2 [17]. 94 T h e G I trac t ex hi bi te d st r ong T om a t o e xp r es si on in Pgly r p1 Cr e/wt R osa2 6 tdT om/wt 95 pups, w ith cle ar c olo cal izat ion be t w een T om at o and GP2 (F igur e 2A ; Supplem ent al 96 Figur e 2 A). C ontr ol mi ce l ack ing C r e sh ow ed onl y GP2 s ta ining. A clos er exami n a ti o n 97 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint of the G I tr ac t sh ow ed th at T o mat o -onl y c ont r ols la ck ed o v e rlap bet w een T o mat o and 98 GP2 signal s, w ith onl y GP2-p os it i v e c ells vi si ble ( Fig ure 2A , t o p r o w , open 99 arr ow h ead s) . In c ontr as t , c olo cal iz ati on of T o mat o and GP2 ( ast er isk s ) w a s o bs erve d 100 exc l us i v e ly i n t he pres e nc e o f Cre r e co mb i na s e (Figure 2A , bo t t o m r o w) . Not a bly , 101 To m a t o + GP2 − c ells ( clo sed ar r ow h e ads) w er e m or e abund ant th an T o mat o − GP2 + cell s 102 (op en ar r ow he ads ), indi ca ting P gl y rp1 e xpr ess ion in ep ith eli al li neage s be y ond 103 clas si cal M c ells. 104 In th e N AL T , T o mat o e xpr es si on w a s det ect ed onl y in Cr e-po si ti v e mi ce ( Figur e 105 2B, bo tt o m r ow , clos ed ar r ow h ea ds). GP2 s ta ining w as r a rely ob ser v e d i n e it h er 106 cont r ol mi ce or Pglyrp1 -Cr e m ic e, s ugges ting e ith er v e ry l ow M c ell a b undance at t hi s 107 stag e (F igu r e 2B , Supplem ent al Fi gur e 2B ) o r th at G P2 is n ot a r el i able ma rk er fo r 108 N AL T M cells , con sist ent wi th r e cen t finding s in hum an a ir w a y ti ssu e [2 4]. 109 The se obs er v at ions indi cat e t issu e-spe cific Pglyrp1 p rom o t e r a ctiv it y , wi th 110 r o bus t epi thel ial e xpr ess i on in the GI tr a ct bu t li mit ed or und et e ct abl e e xpr es si on of 111 canon ic al M cell ma rk ers su ch as GP 2 in t he upper a ir w a y mu co sa. 112 113 Quantitati v e anal ysis of P gl yrp1-Cre–dri v en T omato expression in adult mice 114 highlights tissue-specific patterns 115 T o ch ar act er ize P gl yrp1-dr i v en r ep ort er e xpr ess ion at s ingle- cell r es ol uti on, w e 116 perfo rm ed f l ow cyt om et ry on G I t r ac t and N AL T t is sue s fr o m adul t mi ce ( 8-12 w e eks 117 of ag e) c arr ying Cr e, the T o mat o r e port er , or bo th allele s. Th e ant ib od y panel includ ed 118 ma rk e rs fo r ep ith eli al line age s, im mune s ubs et s, and M c el l populat ion s 119 (Supplem ent al F igur e 3 A, o r ang e boxes ). Imm une p opul ati ons w ere i nc l uded b as ed on 120 pr e vi ou s r epo rts i mplic at ing PGL Y RP1 in b oth innat e and adapt i v e i mmun ity , as w ell 121 as its in v ol v e ment in inflam mat or y r esp onse s [19-21, 25, 26]. 122 Init ial anal y se s confi rm ed tha t t h e pr es ence of Cr e r e co mb ina se al lele, th e 123 T o ma to r e p or ter a l l e l e, o r bo th did n o t a l t e r th e o vera l l com p os i tion o f m aj or c e l l 124 populat i ons in ei the r the G I trac t (Suppl em ental Fig ur e 3B ) o r the N AL T 125 (Supplem ent al F igur e 3C ). W e de t ect ed T o mat o flu or es cen ce onl y i n P gl y rp1 Cr e/wt 126 R o sa26t d To m / w t animal s, w ith st r on g e xpr es si on ac r o ss G I ep ith eli al s ubs ets (F igur e 127 3A). Go ble t c ells (Cd45 - Ep ca m + Gp2 + T nfaip2 - ) , i mm atu r e M cells (Cd 45 - Ep ca m + Gp2 -128 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint T nfaip2 + ), and m atu r e M cells (C d45 - Ep ca m + Gp2 + T nfaip2 + ) a l l ex hib i t e d v e ry h ig h 129 T om at o po si ti vi ty , indi ca ting b r o ad Pglyrp1 p r o mo ter a ctivi t y i n g u t e pi thel ial l ine ages 130 (Fig ur e 3A ). 131 In c ont r as t , T om at o e xpr ess i on in t he N AL T of P gl ryp1 Cr e/wt Ro sa 2 6 tdT o m/wt adult 132 m ic e was mo r e he tero ge n eo u s ( Fi gu re 3B ) . Ne u tro ph i l s ( Cd45 + Epc am - L y6g + ) sh ow ed 133 the h igh es t r epor t er e xpr e ssi on, w hile ep it heli al su bs ets d ispl a y ed v ari abl e T o mat o 134 le v el s. T uft cell s (Cd45 + Ep ca m + ) sh ow ed the s tr onges t epi thel ial e xpr ess ion ( ~70 %), 135 w her eas g oble t cell s (Cd45- Epc am +Gp2+T nfa ip2-) and M cell su bs ets , b oth i mm at ure 136 (Cd45- Epc am+ Gp2-T nfa ip2+) and m at ure (Cd45- Epc am+ Gp2+T nfa i p2+), di spla y ed 137 int er med iat e e xpr es si on ( ~50 %) . 138 T oge ther , th es e da ta r e v e al di st inct tis sue- spe cifi c p att erns of Pglyrp1 pr o mot er 139 ac ti vi ty: pr edo min antl y ep ith eli al in the gu t , and m or e im mune- enri ch e d in t he N AL T . 140 141 Pglyrp1 -Cre–Rosa26 DTA mice show no det ectable changes in epithelial or 142 immune composition 143 T o t es t w h eth er Pglyrp1 - ex p ress i n g popul at ions c ould b e d eplet ed b y 144 cons ti tut i v e diph the ri a t o xin e xpr es sion , w e c r o sse d Pglyrp1 -C r e mi ce wit h B6.1 29P2-145 Gt( R OS A)26S or tm1(D T A)Lky / J ( h erea ft e r R o sa 2 6 DT A ) mi ce , e xp ect ed t o d ri v e c ell-int rins ic 146 diphth er ia t o xin (D T A ) e xpr e ssi on in C r e-p os it i v e line age s [27]. W e ana l yzed the 147 impa ct of thi s c ons ti tut i v e abl at ion appr o a ch on mu co sal cell p opula t ions in th e gu t 148 and N AL T b y flo w cyt o met ry , us i ng a r ef ined ant ib od y pan el d es ig ned t o ident ify 149 epit heli al su bse ts , includ ing GP2 + a nd GP2 – popul at ions , and m aj or i m mune line age s 150 (Supplem ent al F igur e 4A , or ange boxes ). A na lysi s by fl ow cy tome t ry de m onst r at ed no 151 signifi c ant d iff er enc es in epi thel ial or i mmun e p opula ti ons in GI trac t o r NA L T a cros s 152 genot ype s ( Fig ure 4 A, B) . N ei th e r GP 2 – nor GP2 + ep ith eli al pop ulat ion s w er e r edu ced. 153 Thus , cons ti tut i v e D T A e xpr es si on did no t d eplet e Pglyrp1 -e xpr ess ing cell s in 154 muc os al t is sue s, sugge st ing li m it ed t o xin effi ca cy or c omp ens at o ry sur vi v al 155 me chan ism s. 156 157 Pglyrp1 -Cre–dri v en expr ession of GFP -D T A r esults in perinatal lethality and 158 mosaic expr ession in survi v ors 159 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint F ollo wing th e l ack of d et e ct able c ell lo ss in Pglyrp1 - Cre– R o sa26 DT A mi ce, w e 160 ne xt t est ed a se c ond D T A-e xp ress i n g mo us e l ine t ha t als o inc or por at es a Cr e-161 independ ent GFP r epo rt e r ups tream o f t he D T A c a ss et te at t he R o s a26 l oc us. Thi s 162 mod el, B6.129S6 ( Cg)-G t( R OS A)26 Sor tm1(D T A)Jpm b / J ( h erea fte r Ros a 26 GFP-DT A ) [28], 163 enabl es b ot h funct ion al abl at ion of Cr e- e xpr es sing cells and fluor es cen t lab eling of all 164 cells ca rry ing th e ta rg et ed allele , i ndependent of r e co m bina ti on. A nalys is o f cros s es 165 r e v eal ed ma rk edl y r edu ced su rv i v a l of P gl yrp1 Cr e/wt R o sa26 GFP-D T A/wt p ups at w e aning 166 (Supplem ent al Fig ur e 5 A ), w ith c lose r exam i n at io n re v e a l i n g s i g n i fi c ant pe rina tal 167 leth alit y a mong P gl y rp1 Cr e/wt R osa2 6 GFP-DT A/wt pups. M ost P gl y rp1 Cr e/wt R o sa26 GFP-D T A/wt 168 pups d ied wi th in t he f i rst 24-48 h o urs of life and de ce as ed pup s e xh ib it ed a bdo min al 169 dist en si on wi th fluid ac cu mula ti on sugg est i v e of int e st inal d y sfu ncti on. Ma rk ed 170 inflam mat i on and t is sue deg r ad at i on o bs er v ed in t he abd om inal r eg i on of d ec ea sed 171 pups pr e v ent ed the ir c olle ct ion for hist olog ic al ana lysi s. 172 Despit e r ep eat ed br eed ing att emp ts, onl y a s mall num be r of d ou ble -pos itiv e 173 anim als su rv i v ed t o w e aning. Th es e r ar e su rviv o rs were u se d to ex a min e th e e ff ec ts o f 174 cons ti tut i v e D T A e xp r es si on on m uc osal c ell p opulat i ons in th e g ut (F ig ur es 5A , B ) and 175 N AL T (F igur es 5 C, D) using th e sa me fl ow c yt o me tr y p anel emplo y e d in t he T o mat o 176 re p or ter e xper i ment s (Supple men t al F ig ure 3 A) . Be ca u se M c e l l s a re p re se n t at low 177 abundan ce , th e y w er e an al yzed sepa r at el y fr om ot her epi th elia l and im mun e 178 populat i ons in bo th t he gu t (Fig ur e 5B ) and N AL T (F igur e 5D) . S urpri sin gl y , the 179 a n a lysi s r ev e a le d no sign ifi cant d iff er en ce s in o vera l l ce l l p opula ti ons ( Figur e 5A ) o r M 180 cell sub se ts (F igur e 5 B) in the GI t r ac t , n or in co rr esp onding p opula ti o ns in the N AL T 181 (Fig ur e s 5C, D) , w hen c omp ar ing Pglyrp1 -Cr e–p os it i v e GFP-D T A m i c e wit h c ontr ol 182 litt e rm at e s t ha t on l y con ta ined the Cr e- inse rt ion . 183 T o unde rst and w h y s om e do ubl e-pos itiv e a ni ma l s su r vi ve d wh i le ot h ers d ied , 184 w e e x a mined G FP e xpr ess ion a s a surr og at e for D T A e xpr ess i on in GI t ract and N AL T 185 tis sue s of su rv i v ing m ic e. De spit e b eing dr i v en b y a C r e- independ ent prom o t e r at t he 186 R o sa26 l oc us, s urv i ving do ubl e-pos it i v e ani mal s sh ow ed m os ai c GFP e xpr ess ion , wi th 187 onl y 32 % of gu t c ells and 55 % of N AL T c ells e xpr es sing GF P , sugge st ing inc ompl et e 188 tr an sgene a ct i vit y in su r vivi n g m ic e (Supple men tal F igu r e 5 B) . 189 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint The se findings sugg est th at wid esp r ead D T A e xpr es si on in Pglyrp1 + li neage s i s 190 inc ompa ti ble w ith ne ona tal su rviv a l , w h e r e as m os ai c ex p r e s sio n i n surv i v ors li mi ts 191 det e ct able depl et ion. 192 193 Assessment of mucosal cell depletion using Rosa26 iDTR and diphtheria toxin 194 T o a v oid per ina tal l eth alit y and all o w t e mpo r al c ontr ol of a blat i on, w e used the 195 induc ible C57 BL/6-G t( R OS A)26S o r tm1(HBE GF)A wai /J str ain (h erea fte r re f e rre d t o a s 196 R o sa26 iD TR ), w h ic h e xpr e sse s th e d i phthe ri a t o xin r ec ep tor ( D T R) i n a C re - d epend ent 197 manne r [29]. In t his sy st em , Pglyrp1 -e xpr ess ing cells r em ain vi abl e un less sel e ctiv e l y 198 deplet ed b y adm inis tr at ion of e x ogen ous d iphth er ia t o xin (D T). T o det er mine an 199 appr op ri at e D T d os e, w e fi rst p erf o rmed a t i trat io n s tu dy u s i n g i n tra pe r i t o n ea l ( 1 00 , 200 200, 3 00, and 500 ng) and int r ana sal (200 , 300, 500, and 1 ,000 ng) deli v e ry r out es 201 (dat a no t sh own ), foll ow ing tr e atm ent r egi men s p revi ou s l y re po r t e d fo r th e 202 R o sa26 iD TR str a in [29-32]. A t d os es ab o v e 200 ng, ani mals e xhi bit ed s igns of t o xi ci ty 203 including pe ri anal blee ding, r e duc e d m ob ility , and a bdo min al sw ell ing, in s o me c ase s 204 as ea rl y a s 24 hou rs aft e r t he f irs t adm inis tr at ion . B ased on th ese o bs ervat i ons, w e 205 sele ct ed 2 00 ng as the h ig hes t d os e th at pr odu ced n o let hali ty and m inim al dis tr es s 206 for bo th intr ape rit one al and int r ana sal a dm inis tr at ion. 207 In th e gu t , int r ap erit one al D T in cr eas ed B c ells (Cd4 5 + Epc am - Ly 6 g - F4/80 -208 B220 + ) re gard l es s o f geno typ e and c aus ed mod es t , n on-sign ifi cant d ecre as es i n so me 209 epit heli al sub se ts ( Fig ures 6A , B ). M ce l l p opula ti ons r em ained un c hanged. In th e 210 N AL T , D T r edu ced B cells and ne utr ophil s in P gl yrp1 Cr e/wt Ro sa 2 6 iD TR/wt mice and 211 i n c r e as e d u n ch arac ter i z e d e pi t he l ia l c e l l s ( Cd45 - Ep ca m + Gp2 - T n fai p 2 - ), bu t ag ain M 212 cell fr eq uen ci es w er e un aff ect ed (Fi gur e s 6C , D ). 213 Intr anas al D T p r odu c ed s im ila rl y m odes t chang es in b ot h gut and N AL T , and did 214 not signific antl y alt er M c ell a bunda nce (Fi gur es 6 E –H ). 215 Thus , Pglyrp1 -C r e –dr i v en D T R e xp r es si on all ow s c ont r oll ed abl at ion of so me 216 populat i ons but fail s t o d eplet e M cells or o the r Pglyrp1 -e xpr e ssi ng linea ges a t 217 to l era t e d DT d os e s. 218 219

220 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint W e g ene r at ed a Pglyrp1 -Cr e kno ck-i n mou se mod el d esign ed t o pr o v ide gene tic 221 ac ce ss t o Pglyrp1 -e xpr e ssing line ages , includ ing M c ells. T he knock- in s tr at eg y 222 pr es er v ed endog enou s pr om ot er r egula ti on and r esult ed in h eal th y , fertil e an im als, 223 indic at ing m ini mal dis rupt ion t o P gl yrp1 fun ct ion. 224 Our findings de m onst r at e tha t P glyrp1 p r o mo ter a ctiv it y is hi gh l y ti ss u e 225 spec ifi c. In th e gu t , Pglyrp1 - Cre m ar ks M c e l ls , g ob l et c e l l s, and oth er ep ith eli al 226 subs et s, w h ile in the N A L T i t m ar ks neutr ophil s and tuft c ells m or e pr om inentl y . Th is 227 unders c or e s the i mpo rtan ce of ti ss ue con text i n i n ter p r e t i n g Cre driv e r a ctiv it y a n d 228 highl ig hts t he c halleng e of a chie vin g M cell-sp ec if i c g ene ti c ta rg et ing acr os s mu co sal 229 s i tes . 230 F unct ion al s tudi es fur the r und ers co r ed th es e li mi ta ti ons. C ons tit ut i v e D T A 231 e xpr es si on cau sed p er inat al l eth alit y , sugg es ting es sent ial r oles for Pglyrp1 -e xpr essing 232 cells ea rl y in l ife, po ss ibl y r el at ed t o gut ba rr ier ma tur at ion du ring ne o natal feed ing. In 233 cont r ast, indu ci ble iD TR- med iat ed abla ti on r e sult ed in lim it ed d eple ti on at t ole r at ed 234 D T d os es, s ugge sting tha t inc omp let e upt ak e or r es tr ict ed t o xin a c ces s c ons trai n s 235 eff ect i v e line age a bla ti on. 236 Despit e th ese ch allenge s, Pglyrp1 -Cr e pr o v ide s a us eful t o ol f or pr ob ing 237 Pglyrp1 -e xpr es sing pop ulat i ons in vivo . I ts ac tivi ty i n e pi t he l ia l and im mune 238 co mpa rt ment s ena ble s st udi es of m uco sal h o me ost as is, innat e im mune signal ing, and 239 bar ri er func ti on. T he le thal it y ob s er v ed w ith G FP-D T A hi ghli gh ts th e physi o lo g ic a l 240 imp ort anc e of t hes e l ineag es dur ing the ne onat al pe ri od. 241 M ore b r o a dly , t h is w o r k i l l u stra t e s th at M ce l l - a ss oc ia te d gene e x pr es si on 242 p a t ter n s v a ry a cros s ti s su e s a n d o ver l a p wi t h o t he r e pi t he l ia l and i mmun e sub se ts. 243 Insi ght s fr om t his m od el w ill gu id e fut ur e eff ort s t o d e v elop ne xt-ge ner a ti on M cell-244 spec ifi c Cr e dr i v e rs wi th imp r o v ed t issu e spe cifi ci ty . 245 246 247

248 Animals 249 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint The P gl y rp1-Cr e m ou se w as g ene r at ed us ing th e E as i-C RISP R t e chni que [22], 250 as de tail ed b elo w , in c olla b orat ion w it h t he C hi l d r e n ´ s Me d ic a l Ce n te r Res ea r c h 251 Insti tut e G eno me Engin ee ring C ore a t UT S ou t hw e s t ern. 252 The R os a26 DT A str a in ( B6.129P 2-G t(RO SA )26S or tm1(D T A)Lky /J) [27], R osa 26 GFP-D T A 253 str ain (B6.129S6 ( Cg)-G t( R OS A)26 Sor tm1(D T A)Jpm b /J) [28], and R os a26 iD TR s t rai n 254 (C57B L/6-G t( R OS A)26S or tm1(HBEGF)Aw a i /J) [29] w er e o bt ain ed dir ec tl y fr om Th e 255 Ja ck son L a bora t o r y . T he T om at o r e p or ter st r a i n (B6. Cg- Gt(ROSA)26Sor tm14(CAG-256 tdTomato)Hze /J) [23] w a s gen erou s l y prov ided b y Deni se Mar c iano at UT Sou th w est ern. 257 All m ic e w er e main ta ined on a C57 Bl/6J gene ti c ba ck grou n d . 258 Geno typing of mi c e and em br y os w as p erf o rm ed a cc or d ing t o t he p r ot oc ols 259 pr o v ided for ea ch s t r ain b y Th e J a ck son L ab or at ory . Th e gen otyp ing str at eg y fo r th e 260 P gl yrp1- Cr e mo use i s d es cr ib ed in th is m anus cr ipt . Br iefl y , g eno mi c DNA w a s 261 e xtr act ed fr om t ail bi ops ies of n e w bo rn pups or e ar pun ch es of w ean ed m ic e u sing a 262 pr e vi ou sl y des cr ib ed p r ot oc ol [33]. 263 All e xper im ent al pr oc edu r es w ere appr o v ed b y t he Inst it ut ional Ani mal C are 264 and Use Co mm itt ee ( IA CUC) of UT South w e st ern . M i ce w er e h ous ed under sp ec ifi c 265 path ogen-fr ee c ondit ion s in a cc or d a nce wi th inst itu ti onal g uid eline s. 266 Gener ation of Pglyrp1 -Cre mice 267 Suit able pr ot o spa ce r adja cen t mo ti f (P A M) s it es w er e i dent ifi ed w it hi n e x on 3 268 of t he Pglyrp1 lo cus t o gu ide C RIS PR-as so ci at ed pr ot ein 9 (C as9 )-me diat e d d oubl e-269 str and br ea ks . Th e DN A r epai r t e m plat e c ons ist ed of a 2,0 15-ba se p ai r ssD N A d ono r 270 cont ain ing h o mol ogy a rm s flank in g th e ta rg et ed cut s it e , an I R ES, and t he c oding 271 seq uenc e of C r e r e co m bina se (F igu r e 1B ; Supple men tal F igu r e 1 A) . T h e I RES ele men t , 272 pr o v ided b y the Child r en´s Me dica l Cent er R e se ar ch Ins ti tut e G eno me Engine er ing 273 Co r e at U T S outh w est ern , ens ur ed independ ent t r anslat i on of Cr e reco mb i n as e from 274 the endog enou s Pglyrp1 tr ans cr ipt . The Cre s equ enc e (1 ,026 bp, e x cluding th e st ar t 275 and st op cod ons ) w as ob tai ned fr o m th e A ddgen e Seq u ence Anal yzer 276 (ht tps: //w ww . addgen e. o rg/b r ow s e /se quen ce /199783/ ), and th e ssD N A d ono r 277 cons tru c t w as s ynth es ized vi a G ene wiz s ingle- st r anded D N A s ynth esi s ser vi ce . 278 T o fac ili tat e g enot yping, Ec o RI and Nh eI r e str ic ti on enzym e r ec ogni ti on s i tes 279 w er e ins ert ed a t th e 5@iLH and 3@iLH e nds of th e c ass ett e. The endog eno us Pglyrp1 s to p 280 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint cod on w as p r es er v ed, w h ile the Cr e se qu enc e w as flank ed b y ind epe ndent st ar t and 281 st op c odons t o ens ur e ind ependen t e xpr es si on of b oth pr ot e ins. Fin all y , w e int r odu ce d 282 a sil ent p oint mut at ion wi thin the P AM s eq uenc e ( hi ghli ght ed in r ed , Supplem ental 283 Figur e 1B ) t o pr e v ent r ep eat ed C a s9 r ec ogni ti on and r e- cut ting. Th e r eq ui r ed g uid e 284 RN A w a s or d er e d fr om ID T . 285 Z ygo te i n jec t io n s w e r e pe r for me d by t he C hi l d r e n ’ s Me d ic a l C e n ter Res ea r c h 286 Insti tut e Gen om e Eng ine ering C ore a t U T S outh w es t ern u sing s tand ar d Eas i-C RISP R 287 pr ot oc ols. 288 Whole-mount immunofluor escence on newborn pups 289 The eu than asi a and fix a ti on of w hole ne wb orn p ups w er e p erf o rmed in 290 ac co r dan ce wi th th e g uid eline s of I A CUC of UT Sou th w est ern. Br iefl y , 1-da y- old pup s 291 w er e eu than ized, d ec api tat ed, and skinned t o fac ili tat e th e f ix at i on. Th e ent ir e b odi es 292 and h eads w er e fix ed wit h 4 % p arafo r ma l d ehyde ( P F A, T he rm o S ci e nt if i c ), e mb e dded 293 sagi tt all y in p ar affin, se ct ioned a t 5 μm, and m ount ed on glas s sl ide s. 294 F or im muno st aining , sl ides w ere depa r affin ized u sing xy l ene and eth ano l 295 w as hes , foll ow ed b y he at-m edi at ed antig en r e tr ie v al in 10m M s odiu m c itr at e (pH 6 .0) . 296 Tis sue se ct ions w er e th en p er me ab ilized and bl o ck ed fo r 1 hou r a t room t e m pe r a t ure 297 in 0.4 % T r it on X-100 and 5 % bovin e s er u m a lb u mi n (B SA ) i n PBS (bl ock ing s olut ion 298 wit h T r it on ). Aft e r w a shing wi th PBS, slid es w ere i nc u ba te d o vern i g h t at roo m 299 te m p erat ure wi t h a 1: 1 0 0 d i l ut io n of r a bb it anti- RFP (R o ckland #D600-401-379S) and 300 r at ant i-Gp2 ( MBL #D278-3 ), in blo cking solu ti on w it h T r it on. Th e foll ow ing d a y , 301 slide s w er e w ash ed wi th PBS and incu bat ed fo r 1 h ou r a t r o om t e m per a tur e wi th a 302 1:400 dilu ti on of go at anti -rab bi t IgG-Ale x a Flu or 48 8 (In vi tr ogen #A11008) and 303 d o n key a n ti - r a t I g G A l exa F l uo r 594 ( I n v it r ogen #A21209 ), in blo ckin g solu ti on wi th 304 T r it on. Aft e r a final se ri es of PBS w as hes , sl ide s w er e in cub at ed wi t h D API , w as hed 305 ag ain, mount ed in Pr ol ong Gold a ntifade r e agent, and i mag ed us ing an Axio sc an.Z1 306 slide s canne r ( Ze iss ). 307 Flow cytometry 308 Single- cell s uspen si ons fr om g ut and N AL T ti ssu es w er e pr epa r ed using a 309 m o di fi e d v e rs io n o f a previ ou s l y d es c ri be d pro t o co l [34]. Bri efl y , a se ct ion of t he ileu m 310 and the N AL T w er e c olle ct ed s epa r at el y , min ced wi th s cis so rs , and di gest ed in RP MI 311 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint 1640 m ediu m (G ib co ) con tain ing 1 0mM H EPES , 5 % h ea t-ina ct i v at ed FBS, 30 μg/ml 312 Dnase I ( R oc he ), and 1mg /ml Coll agena se II (W or thingt on) at 37 ºC for 45 m inut es . 313 The r e sult ing c ell su spens ion w a s pass ed th r o ugh a 70 μ m n y l on c ell str ain er (F alc on 314 #352350), cen trifu ged, and w ash e d in A CK (A m moni um- Chlo rid e-P ot ass ium ) l y si s 315 buff er ( Gi bc o #A10492-01 ). Cell s w er e th en r es uspend ed in 5 % BS A in PBS 316 (F A CSbuff e r) . 317 F or i mm unos ta ining, cells w e r e in c ubat ed w ith the anti b od y p anels sp ec ified in 318 ea ch s ec ti on of th e R e sults , f oll o w ed b y w ash ing and in cub at ion wit h se c ondar y 319 anti bod ie s in F A CS buff er , if n eed ed . Aft er st aining , cells w ere w a s he d and fix ed in 4 % 320 PF A in PBS for 3 h ou rs ( or 1 % PF A in PBS o v e rnig ht ) follo w ed b y ana l y sis on an LS RII 321 flow cyt o met er (BD Bi os ci enc es) an d anal yzed u sing Fl owJ o sof tw ar e . 322 The foll ow ing ant ib od ies w er e used: r a t Ep ca m ( Cd326 )-B V421 (1:200 , 323 Bio Leg end #118225) , r at Cd45- AP C-C y7 (1 :200, Bi oleg end #103115 ), r a t L y6g-P E-324 eFluo r610 (1 :200, In vit r o gen #6 1-9668-82), r at L y6G-A lexa F lu or 4 8 8 ( 1: 200, 325 Bio Leg end #127625 ), r a t F4 /80-P e rCP-C y5.5 (1 :200, Bi oleg end #1231 28), r a t F4 /80-326 PE (1 :200, Bi oleg end #123110), rat B220-PE-C y7 ( 1:200 , Bi oleg end #103221), r a bb it 327 T nfaip2-C F647 (1:100 , B io rb yt # or b101923- CF647 ), r a t Cd3 -Per CP - Cy 5 . 5 ( 1: 200, 328 Bio Leg end #155615) , rab bi t Gp2-FI T C (1:100 , B i orby t # or b 37776) , r ab bit Gp2 (1:10 0, 329 B i orby t # or b623866 ), g oa t ant i-r a bb it IgG-A lexa F lu or 6 4 7 ( 1: 20 0 , I n v it r ogen 330 #A21245) . 331 Diphtheria toxin inoculations 332 R o sa26 iD TR mic e, wi th o r w ith o ut P g l yrp 1-Cr e inse rt ion, w ere i n j ec te d 333 intr ape rit oneal l y fo r t hree co n se c ut i ve day s w ith 100 μl of ei the r PBS or 200ng of D T 334 (Sig ma ) pe r inje ct ion using a 1 ml tu ber cul in syr inge (BD #309626) . 335 F or int r ana sal in oc ulat ion s, th e sa me r egi men w as foll ow ed , wi th ea ch m ous e 336 r e ce i v ing e it her 10 μl of PBS or 200 ng of D T (S igm a) vi a a 2-20 μl pipett e. 337 All m ic e w er e 8-11 w e ek s of ag e, a nd all an im als w er e se x- and age- mat ched . 338 Ea ch e xper im ent w a s r epe at ed a t le ast tw ic e. 339 Statistical anal ysis 340 Stat is ti cal anal y ses w ere perfo rm ed us ing G r aph P ad Pri sm 341 (R RID:S CR _002798 ). F or fl ow cyt o met ry st udi es, tw o- w a y AN O V A w it h c o rrec ti o n fo r 342 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint mult iple co mpa ri sons w as used w h en t hree ex p er im e nt a l gro u ps w e r e p re s ent. W hen 343 onl y tw o gr oups w e r e co mpa r ed , mult iple Mann-W hi tne y t est s w i th H olm-Sid ak 344 co rr ec ti on w e r e p erfo rm ed. Su rv i v al s tud ies w er e an al yze d us ing Kaplan-M ei er 345 a n a lysi s. P - v a l u es a r e indi c at ed i n th e figu r es ; o the rw is e, c omp ar i sons w er e not 346 signifi c ant . Sta ti st ic al signif ic anc e w as d efin ed as f ollow s: *p<0 . 05, **p<0 .005, 347 ** *p<0.0005 , ** **p<0 .0001. 348 349

350 The aut ho rs thank th e c or e fa cil it i es a t UT Sou th w est ern Med ic al C e nt er fo r 351 the ir v alua ble cont ri but i ons t o th i s w o rk, includ ing th e Child r en´s Med ic al Cent e r 352 R e se ar ch Ins ti tut e Gen om e Eng inee ring c or e, p ar ti cula rl y t o the c or e d irec tor H ao Z hu , 353 and Lin L i, Y u Zhang , and T r ipt i Sh arm a; th e H ist opat hol ogy C ore, p art ic u l ar ly to t he 354 co r e d ir ect or Br et E v e rs, and J oh n Shelt on; th e Wh ole Br ain Mi cr os cop y F ac ilit y 355 (R RID:S CR _017949 ), pa rt ic ula rl y t o D enis e R a mi r ez; the Fl o w C yt o met ry F a cil ity , 356 part ic ula rl y t o Da vid F a rrar ; a n d th e Mo ody F o undat ion Fl o w C yt om etr y F ac ilit y for 357 pr o v iding a cc ess t o G r aph P ad Pri sm softw ar e. 358 359 Competing inter ests 360 All au th ors de cla r e tha t the y h a v e n o co mpe ting int er es ts. 361 362 Funding 363 Thi s w ork w as suppo rt ed b y t he Na ti onal In st itut es of H eal th U01 AI12593 9 364 and R01 A I184584 t o M.U .S. 365 366 Author contributions 367 Con cept ualiz ati on, S.A .A ., M.U .S. ; F or mal anal y si s, S.A .A. , M.U .S. ; In v e stig a ti on, 368 S.A. A.; F unding a cq uis it i on, M.U .S. ; Pr oj e ct ad mini s trat io n , S. A .A . , M . U .S . ; S u pe rv i sio n , 369 M.U .S.; W r it ing – ori ginal dr aft , revie w and ed it ing, S. A. A., M .U .S. 370 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint

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J., et al. , Mycobacterium tuberculosis infects dendritic c ells with high 454 frequency and impairs their function in vivo. J Im mun ol, 2007. 179(4) : p. 2509-455 19. 456 457 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figur e legends 458 459 Figur e 1. Generation of a Pglyrp1 -Cre mouse model and founder screening. (A ) 460 Geno mi c se quen ce of m ou se c hromo so me 7 s ho wing th e Pglyrp1 g ene, as vi sual ized in 461 the U CSC Gen om e br ow ser . (B ) Sc h ema ti c r epr es enta ti on of t he 2 ,015 bp ssD N A d ono r 462 cons tru c t , illus trat i n g a l l e le m ents includ ed fo r th e t a rget ed kn ock- in. ( C) Pr im er 463 loc at ions u sed t o ident ify th e Cr e r ec om bin ase inse rt ion a t the 5@iLH and 3@iLH ends of t he 464 mod ified gen om ic s equ enc e. IR ES = Int ernal r ib os om e en tr y s it e . 465 466 Figur e 2. R eporter expr ession in newborn Pglyrp1 -Cr e mice r ev eals tissue-467 specific patterns. ( A) C onfoc al i m ages of r epr es ent ativ e Pglyr p 1 wt/wt R osa26 tdT om/w t 468 (t op ), and P gl yrp1 Cr e/wt Ro sa 2 6 tdT om /wt (b ott om ) on e-da y-old p ups fr o m th e G I t is sue 469 (A ), and N AL T (B ) s ec ti ons st ain ed wi th anti- r ed fluo r es c ent p r ot ein ( RFP ), and an ti-470 GP2 anti bo die s. T he w hit e b o x h ig hligh ts th e ar ea s h own at h igh er magnific at ion in 471 the r igh t p anels . Clo sed a rr ow h ead point s t o T o mat o + GP2 - cells , open arr ow h ead t o a 472 To m a t o - GP 2 + c ells, and ast e ri sk t o T om at o + GP2 + cell s. Sc ale b ar s i ncluded in t he 473 pict ur es. 474 475 Figur e 3. Quantitati v e anal y sis of Pgl yrp1-Cr e dri v en T omato expr ession in adult 476 mice highlights tissue-specific patterns. F lo w c y t o me tr y a n alysi s o f P g lyr p 1 Cr e/w t 477 (bla ck s qua r es ), R o sa26 tdT om/w t ( gr a y t ri angle s), and P g l yrp1 Cr e/wt Rosa 2 6 tdT om/wt (r ed 478 cir cle s) gut ( A) and N AL T ( B) t issu es in ad ult mi ce . Ma rk ers us ed t o define ea ch c ell 479 populat i on are s pe ci fie d i n t h e x - a xis. Ind i vid ual da ta ar e plott ed wi t h m eans ± SD 480 anal yz ed wit h tw o-w a y ANOV A wi t h Ge i ss e r-Gr eenh ous e c o rrec ti o n ( no sphe ri ci ty 481 assu med ). *p<0.05 , **p<0 .005, * **p<0. 0005, * * **p<0. 0001. Whe r e n ot s ho wn, 482 co mpa ris ons w er e no t s ignifi can t. N = 7 ( Pglyr p 1 Cr e/wt ) and 10 ( R o sa26 tdT om/wt , 483 P gl yrp1 Cr e/wt R o sa26 tdT om/wt ). Ma r k ers used t o ident ify ea ch c ell populat i on ar e 484 desc ri bed in Suppl em ental T abl e 1 A . 485 486 Figur e 4. P gl yrp1-Cr e–R osa26 DTA mice show no detectable changes in epithelial 487 or immune cell composition. P e r cent age of t o tal cells in gut (A ), a nd N AL T (B ) in 488 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint R o sa26 D T A/DT A (black squ ar es ), P gl yrp1 Cr e/wt R o sa26 DT A / w t ( g ray t ri angl es ), or 489 P gl yrp1 Cr e/wt R osa 26 D T A/DT A mic e (bl ack c ir cles ). Ind i v idual da ta are p l o tte d wi th m ea n s 490 ± SD anal yzed wi th t w o- w a y A NO V A w it h Ge iss er- Gr e enh ous e co rr ec ti on (no 491 spher i cit y as sum ed) . Wh ere n ot sh ow n , c om p ar i sons w er e no t sig nific ant . N = 9 492 (R os a26 DT A / D T A ), 11 (P gl y rp1 Cr e/wt R o sa26 DT A / w t ), and 7 (P gl yrp1 Cr e/wt R o sa26 DT A / DT A ). 493 Ma rk ers us ed t o d efine e ach c ell p o pulati on thr ou gho ut all the gr aphs ar e sp ec ified in 494 the x-axis . Ma rk ers us ed t o id entify e ac h cell popul ati on ar e des cr ib ed in 495 Supplemen tal T a ble 1B. 496 497 Figur e 5. P gl yrp1-Cr e–dri v en expres sion of GFP -D T A r esults in perinatal lethality 498 and mosaic expr ession in survi v ors. (A ) Fl o w cyt om et ry anal y sis of cell p opula ti on 499 le v el s, and (B ) M c ell popul at ions i n gut t iss ue of P gl yrp1 Cr e/wt Ro sa 2 6 wt/wt ( gr a y b ar s) , 500 and P g l yrp1 Cr e/wt R o sa26 GFP-D T A/wt ( gr een b ar s) m i ce. (C ) Flo w cyt o m etr y anal y si s of 501 cell p opula ti on le v els , and (D) d et ailed le v els of M c ell pop ulat ion s of sa me m ic e in 502 N AL T . Ind i v idual da ta ar e plott ed wit h me ans ± SD anal yzed w ith mult iple M ann-503 Whi tne y t e st wi th H olm-Sid ak c o rrec t ion. ns = n ot s ignifi cant. N = 9 504 (P gl yrp1 Cr e/wt Ro sa 2 6 wt/wt ) , 9 ( P g lyrp 1 Cr e/ wt Ro sa 2 6 GFP-D T A/ wt ). M ar k er s u sed t o ident ify 505 ea ch cell p opula ti on are d es cr i bed i n Supplem ental T able 1A . 506 507 Figur e 6. Assessment of mucosal cell depletion using Rosa26 iDTR and Pgl yrp1-Cr e 508 under intr aperitoneal and intranasal D T tr eatment. (A ) Fl o w c yt o m etr y anal y sis of 509 cell p opula ti on le v el s, and ( B) M cell popul at ions in g ut tis sue of 510 P gl yrp1 wt/wt R o sa26 iD TR/wt (squar es ) and P g l yrp1 Cr e/wt R o sa26 iD TR/wt (trian gles ) mi ce 511 aft er in tr aper it oneal in oc ulat ion o f ei th er PBS o r 2 00 ng D T . ( C) F low c yt om etr y 512 a n a lysi s o f ce l l popula ti on le v els, a nd (D) M cell p opulat i ons of s am e mi ce in N A L T . 513 Indi v idu al da ta ar e plo tt ed wi th me ans ± SD anal yzed w it h tw o- w a y A NO V A and Sid ak 514 co rr ec ti on for mul tiple c omp ari s ons. Wh er e not sh own , c omp ar i sons w er e no t 515 signifi c ant . N = 6 (PBS ), 4 (200 ng D T). (E ) Fl ow c yt o me try anal y si s of cell p opulat i on 516 le v el s, and (F) M cell p opula ti ons i n gut ti ssu e of P gl y rp1 wt/wt R osa2 6 iD T R/wt (squa r es ) 517 and P gl yrp1 Cr e/wt Ro sa 2 6 iD TR/wt (tria ngles ) mi ce aft e r in tr anas al ino cul ati on of e it he r 518 PBS or 200 ng D T . (G ) Fl ow c yt o met ry an a lysi s o f ce l l p opula ti ons, and (H ) M c ell 519 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint populat i ons of s am e mi c e in N AL T . Ind i vidu al d at a ar e plo tt ed w it h me ans ± SD 520 anal yz ed wi th tw o-w a y AN O V A and Sid ak c orr ec ti on for m ultipl e c omp ari son s. Wh er e 521 not sh own, c omp ar is ons w er e n ot sign ific ant . N = 6 (P BS), 3 (200 n g D T) . Ma rk e rs 522 used t o iden tify e ach c ell pop ula tion a re d es cr ib e d i n Suppl em ental T a ble 1 A. 523 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figure 1 B C A Pglyrp1 (Exon 3) CreIRES EcoRI NheI Pglyrp1 (Exon 3) CreIRES EcoRI NheI 5´ Fw 3´ Fw 5´ Rv 3´ Rv Pglyrp1/ NM_009402.2 Pglyrp1 Scale chr7: .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint A B Pglyrp1Cre/wt Tomatofl/wt Pglyrp1wt/wt Tomatofl/wt Merge Merge Tomato GP2 50 µm 200 µm * * * * * * * * * * * * * * * 200 µm Merge Tomato GP2 50 µm Merge Pglyrp1Cre/wt Tomatofl/wt Pglyrp1wt/wt Tomatofl/wt 200 µm Merge TomatoMerge GP2 200 µm Merge Merge 50 µm Tomato GP2 50 µm Figure 2 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figure 3 A B .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figure 4 A B .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figure 5 A B C D .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint Figure 6 BA DC FE HG .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 24, 2025. ; https://doi.org/10.64898/2025.12.22.696025doi: bioRxiv preprint