() BDA 2 weeks fter Py () AAVs Cre or GFP t P1 BDA 2 weeks fter Py CSMN CST () Py t P7 or 2 months () Py t 2 months Supplementry Figure 1. Sheme of unilterl pyrmidotomy used for deteting ompenstory sprouting of intt CST xons. In the experimentl set indited with (), left pyrmidotomy (Py) ws onduted t the ge of postntl dy 7 (P7) or 2 months, BDA ws injeted to the right sensorimotor ortex t 2 weeks post-injury, nd the nimls were terminted for nlysis in nother 2 weeks. In the experimentl set (), AAV Cre or AAV GFP ws injeted to the right sensorimotor ortex of PTEN f/f t P1 nd the left prymidotomy onduted t the ge of 2 months. At 2 weeks post-injury, BDA ws injeted to the right ortex nd the nimls were terminted 2 weeks lter for histologil nlysis.
Forelim Hindlim Spinl Cord Supplementry Figure 2. AAV-Cre injetion results in effiient Credependent PLAP expression in reporter mouse. -, AAV-Cre ws injeted to the right side of t he sensorimotor ortex of reporter mouse (Ros26-lox-STOP-lox-PLAP) t the ge of P1. The mouse ws terminted for PLAP detetion t the ge of 6 weeks. Left nd right pnel represents the ortil region innervting the forelim () nd hindlim (). White str denotes the sensorimotor ortex., spinl ord trnsverse setion showing the PLAP+ CST xons nd their rnhes. Sle r in -: 1 mm.
C4 d C7 e T8 f g h CST i j.14 k C7.12 18.1 14.8 1.6.4 6.2 2.2 Numer of leled CST fiers Sprouting xon density index (ontrllterl / ipsilterl).7.6.5.4.3.2.1 Mid Z1 Z2.16 T8.7.6.5.12.4.8.3.2 AAV GFP.4.1 AAV Cre Mid Z1 Z2 Supplementry Figure 3. Postntl PTEN deletion does not lter the CST numers nd projetion ptterns.,, Trnsverse setions of the medullry pyrmid showing BDA leled CST xons in the PTEN f/f mie injeted with AAV GFP () or AAV Cre (). The quntifition results of the CST numers in these two groups re shown in (i). h, Trnsverse setions of the spinl ord t C4 (, f), C7 (d, g) or T8 (e, h) showing the distriution of BDA leled xons in the PTENf/f mie injeted with AAV GFP ( e) or AAV Cre (f h). Sle r: 5 μm. j, k, The quntifition results of the C7 nd T8 level of CST xon sprouting density index (ontrlterl/ipsilterl) (j) nd rossing xons (see Fig. 1f) (k). p >.5 for ll of omprisons. N = 6 for eh group. In these experiments, AAVs were injeted to the right side of the sensorimotor ortex of the PTEN f/f mie t P1. The mie were terminted for nlysis t the ge of 6 weeks. Sprouting xon numer index C7 T8
Intt spinl ord Dorsl hemisetion Spinl ord rush Supplementry Figure 4. Shemes of three different spinl ord injury models used in this study., Intt spinl ord: With BDA injetion to the right side of the sensorimotor ortex, CST on the left spinl ord re leled in trnsverse setion (Upper pnel). The pperne of leled fiers in sggitl setion is shown in the low pnel., T8 dorsl hemisetion: This injury trnsets the dorsl spinl ord (oth dorsl min CST trt nd dorsolterl trt) s shown in the upper pnel. In the sggitl setion (lower pnel), leled CST fiers re terminted t the proximl end of the lesion site., T8 spinl ord rush model. This injury trnsets ll pssing CST xons s shown in the upper pnel. In the sggitl setion (lower pnel), ll leled CST xons re terminted t the proximl end of the lesion site.
8 weeks fter T8 dorsl hemisetion, BDA/GFAP d e.35 Retrtion ul index.3.25.2.15.1.5 AAV GFP Supplementry Figure 5. Redued retrtion in CST xons in PTEN deleted mie fter T8 dorsl hemisetion., Representtive imges of sggitl setions from PTEN f/f mie with right ortil injetion of AAV GFP () or AAV Cre () t P1 nd T8 dorsl hemisetion t 6 weeks. BDA ws injeted to the right sensorimotor ortex t 6 weeks post-injury nd the nimls were terminted 2 weeks lter. The setions were o-stined with BDA (red) nd GFAP (lue). Sle r: 2 μm. d, High mgnifition imges showing the morphology of leled CST xons in the terminl res. Sle r: 5 μm. e, Quntifition of the retrtion uls per.1 mm 2 t the CST ends, normlized y the numer of leled CST xons in these two groups. : p <.1, Student s t-test. 9 nimls in AAV GFP group nd 11 in AAV-Cre groups were used. At lest three setions ontining the min CST undle were quntified per niml. AAV Cre
BDA/GFAP BDA d Supplementry Figure 6. CST retrtion ours in the ute phse of T8 dorsl hemisetion.,, Representtive imges showing sggitl setions with BDA (red) nd GFAP (lue) stining from PTEN f/f mie injeted with AAV GFP () or AAV Cre (). Note tht in oth ses, BDA leled CST xons retrt from the lesion site. Sle r: 1 μm., d, High mgnifition of the imges showing the retrtion uls in the terminls of BDA-leled xons in oth groups. White str denotes the lesion site. Sle r: 3 μm.
1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28
Supplementry Figure 7. Seril setions from PTENf/f mouse injeted with AAV Cre t P1 nd T8 dorsl hemisetion t 6 weeks showing numerous BDA-leled xons in the spinl ord distl to the lesion site. #25 mrks the midline setion through the entrl nl (#1 24 re from the ontrlterl nd #26 44 re from ipsilterl side of the spinl ord). While some xons in the distl ord ould e tred k to the ventrl spinl ord, the mjority of xons pper to pss through the lesion sites through disrete res in the dorsl side of the lesion sites. While BDA ws injeted only to the right side of the sensorimotor ortex, mny leled fiers oulde seen in oth left nd right sides of spinl ords oth proximl nd distl to the lesion site. The fiers in the ipsilterl ord proximl to the lesion (for exmple, in #27 29) re likely due to inresed sprouting ross the midline. In the distl ord, mny fiers lter the projetion trjetory towrds ventrl nd distl ord in oth sides of the spinl ord. The imges of #17 nd #19 re shown in Figs. 3 nd 3d. Sle r: 5 μm.
BDA/GFAP Fier numer index.14.12.1.8.6.4.2 AAV GFP AAV Cre 5 1 25 5 75 1 15 2 25 Distne from lesion (μm) Supplementry Figure 8. Sprouting nd regenertion of BDA leled CST xons fter T8 dorsl hemisetion in seond set of experiments., Representtive imges from sggitl setions from PTEN f/f mie with AAV GFP () or AAV Cre () t P1 nd T8 dorsl hemisetion t the ge of 6 weeks (, ). The setions were lso o-stined with nti GFAP ntiodies (lue). Sle r: 3 μm.,the numers of leled CST xons were ounted t different distnes distl to the lesion sites in PTEN f/f mie injeted with AAV GFP or AAV Cre. : p <.5, two wy ANOVA followed y Fisher s LSD.
BDA/GFAP d Supplementry Figure 9. Mny regenerting xons re ssoited with GFAP positive mtrix in the lesion sites.,, Sggitl setions showing BDA leled CST xons in the lesion sites of PTEN f/f mie injeted with AAV GFP () or AAV Cre (). Sle r: 2 μm. High mgnifition imges re shown in nd d. These setions were lso stined with nti-gfap ntiodies (lue). Sle r: 3 μm.
1 dys fter rush 4 weeks fter rush 12 weeks fter rush GFAP Supplementry Figure 1. GFAP positive mtrix develops fter spinl ord rush injury., Sggitl setions stined with nti GFAP ntiodies tken from dult mie t 1 dys (), 4 weeks () or 12 weeks () fter rush injury. GFAP negtive lesion sites re lerly seen in the ute phse () nd GFAP-positive mtrix grdully develop in the lesion sites in the hroni phses (, ). Sle r: 2 μm.
BDA/GFAP d Supplementry Figure 11. No CST regenertion deteted t 1 dys fter spinl ord rush injury.,, Sggitl setions with BDA (red) nd GFAP (lue) stining from PTENf/f mie injeted with AAV GFP () or AAV Cre () nd rush injury 1 dys efore termintion. White str denotes the lesion sites. Sle r: 2 μm., d, High mgnifition imges show the retrtion uls in the terminls of CST xons in PTENf/f mie with AAV GFP () or AAV Cre (d). Sle r: 5 μm.
BDA/GFAP d e f g
Supplementry Figure 12. Etopi ilterl projetion of regenerting CST xons fter rush in PTEN deleted mie. f, Sggitl setions with BDA (red) nd GFAP (lue) stining from ontrlterl (, ), midline (, d) or ipsilterl (e, f) side of the spinl ords from PTEN f/f mie injeted with AAV GFP (,, e) or AAV Cre (, d, f). In these experiments, AAVs were injeted to the right sensorimotor ortex t P1 nd the nimls were sujeted to T8 rush injury t the ge of 6 weeks. 1 weeks post-injury, BDA ws injeted to the right side of the sensorimotor ortex, thus resulting in leling of ontrlterl (, ) nd midline (min) CST xons (, d) in oth groups. However, different from ontrol group (e), mny BDA leled xons re lso seen in ipsilterl spinl ord (oth rostrl nd udl) in PTEN f/f with AAV Cre (f). Sle r: 2 µm. g, An extended imge of d showing tht regenerting xons extend up to 3 mm udl to the lesion sites. White str denotes the lesion sites. Sle r: 5 µm.
d Fier numer index e.2.18.15.12.1.7.5.2 25 5 1 15 AAV Cre AAV GFP 2 25 3 Distne from lesion (μm) Supplementry Figure 13. CST regenertion in PTEN f/f mie wi th neontl AAV injetion nd T8 spinl ord rush injury t 5 months. -d, Representtive imges from sggitl setions showing the min dorsl CST trt (, ) or lterl CST xons in the gry mtter (, d) in PTEN f/f mie with ortil injetion of AAV-GFP (, ) or AAV - Cre (, d) t P1 nd T8 rush injury t 5 months. BDA ws injeted to the sensorimotor ortex t 1 weeks fter injury nd the nimls were terminted 2 weeks lter. The setions were o-stined to detet BDA (red) nd GFAP (lue). Sle r: 2 μm. e, Quntifition of leled xons in the spinl ord udl to the lesion site in oth groups. : p <.5, two-wy ANOVA followed y Fisher s LSD. 5 nimls in eh group were used. 3 setions per nimls were quntified.