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1 doi:0.08/nture0987 SUPPLEMENTARY FIGURE Structure of rbbit Xist gene. Exons re shown in boxes with romn numbers, introns in thin lines. Arrows indicte the locliztion of primers used for mplifiction.
2 RESEARCH b Xist / Hprt Jridc Xist Hprt Rbbit X chromosome Xist / Jridc c Merged Xist α-hk7me SUPPLEMENTARY FIGURE Xist nd X-linked genes expression nd HK7tri-methyltion in femle rbbit culture cell (Synoviocyte). () Rbbit X chromosome mp with the studied genes. (b) Representtive cells re shown for Xist (green), gene primry trnscripts (red) nd (blue). (c) Representtive cells re shown for Xist (green), HK7 tri-methyltion (red) nd (blue).
3 % of nuclei RESEARCH Giems Xist / α-hk7me / α-hk7me Femle 96hPC 00μm Mle μm b Xist / α-hk7me c 00 Xist ( n= ) α-hk7me 80 0hPC Femle Blstocyst (TE) (96hPC) Blstocyst (TE) (0hPC) SUPPLEMENTARY FIGURE Histone H K7 methyltion in rbbit blstocysts. () Immunolbelling (red) with ntibodies ginst HK7 tri-methyltion ws combined with Xist FISH (green) t 96hPC stge rbbit blstocyst. For ech sex, n intct embryo (Giems) together with severl enlrged representtive nucleus re shown. XX cell (,,). XY cell (,5). (b) Representtive isolted trophectoderm from 0hPC XX blstocyst by immunolbelling with ntibodies ginst HK7 tri-methyltion (red) combined with Xist FISH (green). Representtive cells (boxed nuclei -). (c) Proportion of cells showing enrichment of HK7me on the Xist coted X chromosome t 96hPC nd 0hPC blstocyst stge embryos. TE, trophectoderm.
4 RESEARCH SUPPLEMENTARY FIGURE TUNEL stining on rbbit femle nd mle 0hPC blstocysts. () TUNEL stining FITC, (green) on region of control blstocyst following tretment with DNAse for 0 minutes. (b) TUNEL stining (green) on region of femle blstocyst (sexed by PCR). (c) TUNEL stining (green) on region of mle blstocyst (sexed by PCR). A totl of six control embryos, seven femle embryos nd nine mle embryos were exmined nd ll gve
5 % of nuclei % of nuclei RESEARCH similr results to those shown in pnels (), (b) nd (c) respectively. Note tht exposure times were incresed - fold for the FITC chnnel in the mle nd femle blstocyts compred to the control, to ensure tht ny fint TUNEL stining would be detected. Giems Xist / Hprt X / Y chromosome / Xist / Hprt X / Y chromosome / 96 hpc 50μm b Giems Xist / Hprt Xist / Hprt c c Femle ICM Xist μm hPC Blstocyst (ICM) 8 (5) % 0hPC Blstocyst (EPI+PE) Embryonic Stge 8 () No. of cells exmined (No. embryos) 0 hpc d α-oct / Xist α-oct / Xist α-oct / Xist f HK7me Oct- 0 e 00μm α-oct / α-hk7me α-oct /α-hk7me α-oct / α-k7me % (5) 8 (5) No. of cells exmined (No. embryos) 00μm SUPPLEMENTARY FIGURE 5 Xist nd Hprt expression nd HK7me profiles in the inner cell mss of rbbit blstocysts. Exmples of ICM cells (Giems) isolted from blstocysts nlysed by FISH for Xist (green) nd Hprt (red) expression; followed by DNA FISH using X (red) nd Y (green) chromosome probes. () An intct femle ICM (Giems-stined) is shown, together with severl enlrged representtive nuclei. Two representtive cells (boxed nuclei -) with no Xist ccumultion but two punctte Xist nd Hprt signls re shown. (b) Dorsl view of one representtive isolted embryonic disc (pre-epiblst ICM cells) from lte (0hPC) blstocysts nlysed by FISH for Xist (green) nd Hprt (red) expression is shown, together with severl enlrged representtive nuclei. Four representtive blstomeres (boxed nuclei -). (c) Percentge cells showing different Xist expression 5
6 % of nuclei RESEARCH ptterns in ICM nd epiblst cells in erly (96hPC) nd lte (0hPC) blstocyst stge embryos rbbit embryos. (d) Immunolbelling (red) with ntibodies ginst Oct- ws combined with Xist FISH (green) in embryonic disc from lte (0hPC) stge rbbit blstocyst. Four representtive blstomeres (boxed nuclei -). (e) Dorsl view of one representtive isolted embryonic discs from lte (0hPC) blstocysts nlysed by immunofluorescence for Oct- (green) nd HK7me (red) is shown, together with severl enlrged representtive nuclei. Four representtive blstomeres (boxed nuclei -). (f) Proportion of cells showing enrichment of HK7me in Oct- positive (green) or Oct- negtive (white nuclei) cells of (0hPC) blstocyst stge embryos. Xist / Hprt X / Y chromosome / Xist / Hprt X / Y chromosome / Prthenogenetic embryos b Xist Xist X-linked gene primry trnscripts % % Blstocyst Embryonic Stge Hprt X-linked Gene 0 No. embryos 0 No. of embryos SUPPLEMENTARY FIGURE 6 Xist nd Hprt expression in rbbit prthenogenotes () A region of rbbit prthenogenetic blstocyst nlysed by FISH to detect Xist (green) nd Hprt (red) followed by DNA FISH using X (red) nd Y (green) chromosome probes, together with enlrged representtive nuclei re shown. (b) Percentge of cells showing different Xist expression ptterns (left) nd different Xist/Hprt expression profiles (right), in rbbit prthenogenetic blstocysts. 6
7 RESEARCH A B XIST / FGD FGD XIST ATRX Humn X chromosome XIST / ATRX C Merged Xist α-hk7me SUPPLEMENTARY FIGURE 7 X-inctivtion sttus nd HK7me enrichment on the humn X in cultured femle primry cells (WI8). () Humn X chromosome mp. (b) Representtive cells showing Xist (green), gene primry trnscripts (red) nd (blue). (c) Representtive cell showing Xist (green), HK7me (red) nd (blue). 7
8 RESEARCH XIST / FGD X / Y chromosome / XIST / FGD FGD X chromosome / Y cen / Femle 50μm 50μm Mle 50μm 50μm XIST / ATRX SUPPLEMENTARY FIGURE 8 XIST, X-linked gene expression nd heterochromtin detection in humn blstocysts cultured for 7 dys. One femle nd one mle humn embryo cultured for n extr dy (up to dy 7) re shown. FISH ws performed to detect XIST (green) nd primry trnscription from the X-linked FGD gene (red). This ws followed by DNA FISH for the X chromosome (red) nd Y centromere (green). Representtive XX blstomeres (boxed nuclei -), nd XY blstomeres (boxed nuclei -) re shown. Zones of intense green signl outside nuclei re bckground. stining revels the bsence of heterochromtic Brr body in femle blstomeres (boxed nuclei nd ), but the presence of dense Y-chromosome body in mle blstomeres (boxed nuclei nd ). 8
9 RESEARCH b Dynmics of XCI during femle pre-implnttion development ICM Epiblst or Rodents Imprinted XCI Pternl X chromosome () Mternl X chromosome () Xist X-linked gene trnscript HK7me enrichment Zygote -cell -cell 8-cell Morul Erly Blstocyst or Lte Blstocyst Humn Rbbit Mouse Pternl Xist On Mternl Xist Off (Imprint ) Acivtors (Both XX+XY) Xist Xist Off Rndom XCI Pternl Xist On Pre-Xist up-regultion Choice Xist Activtors (XX only) or Tsix Xist Mternl piring Xist On CI XCI inctive ctive or inctive EGA EGA Pternl Xist On Mouse Mternl Xist On or Rbbits Rndom XCI? -) re shown. Zones of intense green signl outside nuclei CI Xist Activtors Post-XCI Choice? (XX only) or inctive Xist CI Humns Rndom XCI? Rbbit Pternl re bckground. stining revels the bsence of heterochromtic Xist On Xist Brr CI? or Activtors Pre-XCI Choice? (Both XX+XY) body in femle blstomeres (boxed nuclei nd ), but the Mternl presence of Xist CI? Xist On dense Y-chromosome body TE in mle blstomeres TE Humn (boxed nuclei nd ). Post-XCI Choice? or inctive SUPPLEMENTARY FIGURE 9 () Summry of the Xist expression nd X-inctivtion kinetics during preimplnttion rbbit nd humn development compred to mice. (b) A working hypothesis to explin some of the differences in Xist/XIST regultion nd XCI initition observed in mouse, rbbit nd humn embryos. ICM, inner cell mss ; TE, trophectoderm. 9
10 RESEARCH SUPPLEMENTARY DISCUSSION In recent study on humn pre-implnttion embryos vn der Berg et l concluded tht XCI initites by the blstoyst stge nd tht XIST is lrgely monolleliclly up-regulted even in moruls. There study ws bsed on FISH for XIST nd single gene (CHIC) on the one hnd, nd IF using HK7me or mcroha on the other hnd, lthough the IF ws not combined with XIST FISH nd thus they could not specificlly identify the inctive X. One possible difference could be the culture conditions used, which were probbly not identicl to ours. However, both their study nd ours used tmospheric oxygen so this cnnot be n explntion. Another possible difference could be t the level of the FISH. Although monollelic expression of XIST/CHIC ws reported, this could be due to less efficient FISH thn in our hnds, leding to reduced detection of signls nd therefore pprent monollelic, or no expression. In our study we find billelic XIST expression nd billelic primry trnscript detection of three genes in the vst mjority of blstomeres, with high efficiency (Fig. ). Furthermore, we combine XIST FISH with nti-hk7me nd show tht lthough there re some enriched foci of HK7me in the nuclei of humn blstocysts these do not correspond to the XIST coted chromosoml domin (Fig. ). 0
supplementary information
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