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1 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 inducer of ne~hro~enesis, is an and is also reauired for dedo~ment eve 1 and &tterning skeletal ~ronckers,'~~ Melanie ~ohocki,' Clementine LUO,' Guangbin ~ofrnann,~?~ J. Antonius J. L. ~radley? Allan ~arsent~l~ Gerard and Genetics, 77030 USA; The 'Department of of Texas M.D. Anderson Cancer Center, Houston, Texas, Molecular University Baylor Genetics, 2Department of Biochemistry, 3~oward Hughes Medical Human Molecular and of Institute-Department Institut fur 4GS~, Forschungszentrum fur Umwelt und Gesundheit, College of Medicine, USA; 77030 Texas, Houston, D-85758 Saeugetiergenetik, Oberschleissheim, Germany by factors originally ability to Bone morphogenetic proteins (BMPs) are multifunctional growth identified their of generated BMPs, BMP-7, we have one induce ectopic bone formation. To the investigate the function of after birth die shortly mice technology. stem cell using embryonic mice BMP-7-deficient BMP-7-deficient at several stages of poor kidney because of of mutant embryos development. Histological analysis resulting a virtual absence development revealed that metanephric mesenchymal cells fail to differentiate, in hybridization analysis showed In of BMP-7 affects kidneys. newborn in glomerulus of that situ the absence days and Pax-2 Wnt-4 between 12.5 and 14.5 such nephrogenesis, of markers molecular of expression the as In postcoitum (dpc). This identifies mice BMP-7 as an inducer of nephrogenesis. addition, BMP-7-deficient BMP-'/-deficient mice also have during Finally, have eye defects that appear to lens induction. originate and the hindlimbs. skull, rib restricted to the defects patterning skeletal cage, the Words: BMP-7; kidney; eye; limb] [Key 29, Received August 18, 19%; revised version accepted September 1995. is vertebrate the permanent development kidney During tion the of epithelialization and metanephric mesen- tissue components: generated by the interaction of two Grobstein 1957; chyme (Gruenwald 1937; al. et Saxen the metanephric mesen- the and bud, epithelial ureteric 1 1.0 dpc mice cul- 1976). Metanephric mesenchyme of At 11.0 days Saxen 1987). 1955; chyme (Grobstein 1953, presence of the ureteric bud, or other induc- tured in the branches out bud from the (dpc) postcoitum the ureteric proliferates tive tissues such as embryonic spinal cord, Wolffian metanephric mesen- the invades duct and epithelial structures. In contrast, into differentiates and chyme. the nephrogenesis is in event key the Thereafter, cultured cells metanephric mesenchymal in the absence reciprocal inductive interaction between these two tis- and died. of proliferate not did other inductive tissues sues. The metanephric mesenchyme induces the ure- Transfilter to the induction experiments have led postu- teric collecting the form to and bifurcate grow to bud late by caused be could these inductive phenomena that ducts. At the same time, signals from the bud ureteric (Saxen diffusible and/or interactions cell-cell molecules into metanephric mesenchyme of conversion the induce molecules Wnt-4 has 1987). been identified as one the of met- epithelial structure. The epithelialization of an the for metanephric mes- of epithelialization necessary the involves At 11.5 steps. several anephric mesenchyme enchyme. de- Wnt-4-deficient mice, kidney in However, dpc metanephric mesenchyme condenses around the 1994), et al. velopment is normal until 14.5 dpc (Stark ureteric bud, and condensed mesenchyme segregates other inducing molecules are impor- suggests which that These into small pretubular aggregates. aggregates will development. tant earlier during comma- process epithelialization an undergo to become that is expressed One molecule the at kidney in the the eventually and shaped bodies, S-shaped bodies, epi- the bone morphoge- time is earliest induction event of (Saxen 1987). of the nephron thelial component protein-7 netic also called osteogenic (BMP-7)) protein-1 have indicated organ the culture experiments Classic the of trans- (OP-1) (Ozkaynak et al. 1990), a member condensa- the to that lead factors inducing of presence (TGF-P) superfamily. Origi- factor-p forming growth the from bone on purified were BMPs nally, of basis when their ability to induce ectopic bone formation Academic Center for address: 5Present Department of Oral Cell Biology under Sam- 1965; implanted the skin of rodents (Urist Vrijie Boechorstraat, van Universteit der Am- Dentistry in Amsterdam, Netherlands. The sterdam, author. 6Corresponding 198 1 Wozney 1988). The skeletal al. ; et Reddi path and 2808 Laboratory Press & Cold Spring Harbor 9~2808-2820 ISSN 0890-9369/95 $5.00 GENES DEVELOPMENT O 1995 by

2 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 nephrogenesis and two members this of familyl of function morphogenetic necessary for rnetanephnc mesenchymal condensation 5 differentiation factor BMP-5 and and growth the be (GDF-51, factor for a survival to appears and dpc 12.5 after genetically (Kingsley et al. 1992; has documented been metanephric mesenchyme. absence of In the BMP-7, et Storm func- skeletal patterning this Besides al. 1994). between and metanephric mesenchymal 12.5 cells die tion of are BMPs evidence indicate lines several that the 4.5 ceases dpc and, consequently, glomerulus formation f dur- of tissues range morphogenetic molecules for a wide the first at BMP-7 for requirement The after 12.5 dpc. ing development. First, BMP homologs have been iden- indicates that this mole- stage of glomerulus formation no that has Drosophila, an organism in tified skeleton cule is early glomerulus inducer. BMP-7-deficient an Second, Anderson 1992). (Ferguson mediates and BMP-4 developmental defects mice also exhibit other eye de- in early the in embryo Xenopus dorsallventral patterning velopment and skeletal patterning with variable pene- al. 1992). Third, mouse (Dale et trance and expressivity. For BMP- 7-deficient instance, Bmp-6, Bmp-4, Bmp-2, originate that mice have eye defects lens of the time at and Bmp-7 are expressed early during development, long of BMP-7 absence the in Finally, formation. develop- the that will the onset of skeletogenesis, and in areas before of ment et al. 1989,1990, (Lyons skeleton of the parts become not digits are affected; the the ribs, the skull, and the 19951. it has been shown that inactivation of Finally, in polydactyly absence leads BMP-7 of hindlimbs. to mice in or Bmp-4 Bmp-2 leads to early embryonic lethal- been any skeletal formation has ity, before initiated (Winnier et al. i985; Zhang H. and A, Bradley, in prep.]. Results that Thus, these studies demonstrate the biological func- mice BMP-7-d&cient of Generation of tion the BMPs is not restricted to skeletal patterning during development. a targeting vector was allele null generate a To of BMP-7, that would generate an the ma- missing allele designed Like many other Bmps, Bmp-7 is expressed in a wide polypeptide processed of the domain ture deleting ex- by 19951. variety of tissues during development (Lyons et al. system developing urogenital Bmp-7 is expressed in the ons has and 6 pBMP7T replacement-type vector The 7. of development, and at every stage after birth it is mainly sequences of are kb 6.1 that with the homologous mouse kidney, suggesting in expressed that it may involved be Bmp-7 locus, a phosphoribosyl transferase hypoxanthine of in kidney development. To investigate the function sim- jHprt) for minigene positive selection and a herpes we have generated BMP--/-deficient mice BMP-7, em- by virus plex nega- for cassette tkJ (HSV thymidine lunase bryonic stem tive selection 1A). Recombinant clones were iden- (Fig. We cell technology, (ES) report here that tified by using both Southern blot hybridization required several discrete events involving is BMP-7 in 5'- and most impor- The different organs during development. 12% in the hypo- 3'-flanking probes, at a frequency of of these, tant of penetrance terms on the and the effect in xanthine immunopterin and thymidine (HAT\ and is viability the mouse, is kidney development. BMP-7 of FIAU-resistant clones (Fig. IBj. were con- Chimeras Bmp-7 Gene targeting 1. Figure lo- at the [A) Schematic representation of the cus. the mouse expected gene at replacement [E7), 6 and (E6) cod- Bmp-7 locus. Exons 7 of the six for ing of cysteine residues seven BMP-7, are represented as the mature dark, stippled boxes. The 5' and 3' external I I \ / I TK PGK-hprt probes used for Southern blot analysis are targeting vector shown as solid and the diagnostic bars, (X) HindEI. (H) XbnI; lines. fragments as puta- the of Southern blot analysis (B) I 5' ex- 3' and Both 5A1. tive targeted clone targeted allele detected restric- predicted probes ternal 5' {GI Southern analysis blot fragments. tion Xba kb I 13.0 of neonates from intercrosses between kb I11 Hind 9.2 wild-type heterozygous ) + / + ( mutant BMP-7 mice. kb I Xba 10.9 kb 5.6 Hind 111 mutant j + / - ) heterozygoous Wild-type mutants; - [ ) homozygous mutants. / - mutants; blot of total RNA Northern (D) analysis from a heterozy- from 12.5-dpc embryos the probe gous intercross, using as a 3'- of mouse Bmp-7 region untranslated the two Bmp-7 transcripts, 4.0 cDNA. The kb, 2.0 glyceraldehyde and are indicated. A probe cDNA was (GADPH] phosphatase - for as an used internal control amount the probe 3' lane. of in RNA each loaded DEVELOPMENT & GENES 2809

3 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press Luo al. et ES into cells C57BLl6 blas- injecting targeted by structed tion in the cortical region (Fig. 2H), whereas in the wild- fe- tocysts. Chimeric males were crossed with C57BLl6 glomerulogenesis evident (Fig. is type control, active the mutant (bmp-7"') males to transmit allele through BMP-7 that indicate observations These 2~). crit- plays a 1C). Chimeras generated from one (Fig. line germ the any detect not did We ical role during nephrogenesis. mutation targeted clone transmitted the through the in or system urogenital the abnormalities parts other of with Northern blot analysis performed RNA germ line. in any other internal organs of the bmp-7m1/bmp-7m' samples from any detect to failed embryos 12.5-dpc mice. mRNA (Fig. homozygotes 7"' bmp- 7"'l in bmp- BMP-7 determine the embryological basis of this To abnor- Likewise, ID). immunohistochemistry performed on the embryos mality, mutant and wild-type of kidneys kidney sections of newborn mice using a monoclonal were examined during development. In times different at antibody against the mature domain of BMP-7 distal the embryos, 12.0-dpc the normal ure- the of part not 1994) could pro- al. et (Vukicevic detect any BMP-7 teric and the buds are several branched has bud times tein kidneys of mutant mice (data not shown). in the mesenchymal condensed by surrounded that At cells. stage, the kidney rudiments of mutant embryos are than those but smaller wild-type embryos of are other- BMP--/-deficient mice phenotypes of Morphological Just half 2A,B). wise histologically (Fig. indistinguishable (bmp-7"'/+) mice were phenotypically Heterozygous a day metanephric later, the recognizably is rudiment heterozygotes between these Crosses normal. produced 7"' and wild-type ' bmp- / between the bmp- different 7" size, although about litters one-quarter of of the normal noninduced In the 12.5-dpc embryos. wild-type kidney, had morphological features. neonates distinctive metanephric mesenchyme periphery the at present is they 85% size (body weight, in smaller were all Grossly, cells sur- (cortical region), and condensed mesenchymal of in the exhibited polydactyly 82% normal), hindlimbs, condensed every ureteric bud. Part of the round mesen- and 71 microphtalmia or either anophtalmia had % (Ta- chymal cells have fractionated already to form pretubu- ble died abnormal mice morphologically 1). the All of the (n kidneys 7m'/bmp-7m' lar aggregates. In bmp- 8), = within 48 hr after birth. Southern blot hybridization mesenchymal noninduced in the present cells are also analysis that all of these abnormal mice were revealed region, cortical cells mesenchymal condensed the but they were 7"' 7"'lbmp- homozygotes and that bmp- surround that should the ureteric buds are either par- expected Mendelian ratio at the recovered (0.005

with section compared ments. is expressed in the 9.5 Bmp-7 dpc, Wolffian At bmp-7"'l bmp- 7"' kid- dysgenic The wild-type kidneys. rise al. et buds (Lyons ureteric the to give ducts, which do have an identifiable neys not metanephric mesen- expressed 12.5 dpc, At shown). is not Bmp-7 1995; data chyme, was no evidence of glomerulus forma- and there and, a to predominantly in the ureteric bud epithelium mutant mice l. neonatal of analysis Morphological Table - digit Extra Anophtalmia and/or Phenotype forelimbs microphtalmia in Dysgenic kidneys hindlimbs in 12/17 14/17 2/17 abnormal/number examined 17/17 Number of DEVELOPMENT & GENES 2810

4 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 nephrogenesis and devel- kidney of Histological analysis 2. Figure in sag- are mice. Shown BMP-7-deficient opment sections of 12.0-dpc (A,B), kidney 12.5- of ittal (EJ) 14.5-dpc (C,D), embryos, and newborn dpc (G,HJ. At 12.0 dpc, the mutant mice is kidney ureteric the both (A,B); control than the smaller bud (u) and the condensed mesenchymal cells At 12.5 (cm) present in the mutant kidney (B). are dpc, there is a severe reduction mesenchymal in condensation kidney (arrowhead in the mutant CJ; D) compared with the control (cm in pre- in in are present (pa] aggregates tubular the both the mutant and wild type (C,DJ. At 14.5 dpc, the clearly smaller mutant kidney (F) is than the wild (El. is no mesenchymal condensation There type a reduction in the mutant kidney. There is also in the number of noninduced mesenchymal cells (arrowhead in comma- FJ. (g) Glomerulus; (cb) body; shaped the neonatal S-shaped body. At (sb) stage, kidney is much smaller than the mutant the wild type. In addition, there is an absence of mesenchymal cells and glomerogenesis in the (G,H) (G,H). (A-FJ Bar, 100 pm; mutant kidney (G,H). pm bar, 500 WTI lesser kidneys, was surrounding condensed mesenchy- in the extent, expressed in the mesenchymal cells WTI transcripts in the devel- Bmp-7 transcripts are (Fig. presence At 14.5 dpc, 3A). of ma1 The 3B). cells (Fig. pretubular in same structures, in the present and also oping kidney of a 12.5-dpc bmp-7m1/bmp-7m1 is embryo aggregates, in the but are absent comma-shaped and the consistent with the histological analysis showing S-shaped bodies. In glomeruli, the dpc, stage. At 14.5 at this of mesenchymal cells presence transcripts Bmp-7 in the kidneys, bmp-7m1/bmp-7m' were WT1 transcripts 3B). Bmp-7 is also in the (Fig. podocytes were detected expressed in the cord spinal (data not shown), a tissue were present in the podocytes of the few glomeruli but metanephric mes- bud to induce ureteric that can replace the nonin- region where cortical in the greatly reduced 1987). WT1 tran- differentiation enchyme in vitro (Saxen duced metanephric mesenchyme should be. scripts were also completely absent around the ureteric in kidney phenotype the To characterize more detail, condensed mesenchymal cells should be buds where the patterns several well- the examined we expression of of absence The 3F). (Fig. present of the WT1 expression in either metanephric of characterized molecular markers bmp-7m1/bmp-7m1 embryo, mesenchyme kidney epithelial cells at 12.5 and or 14.5 cortical 14.5-dpc of region mesenchyme con- where both the the and noninduced The dpc. et tumor gene WT1 (Call al. 1990) en- Wilms' present, be densed mesenchymal cells should is consis- codes a transcription factor essential for kidney develop- histological observations the tent with in the few very that WTl expressed is al. 1993). ment (Kreidberg et cells at metanephric mesenchymal noncondensed mesenchymal cells are present in the mu- and 12.5 in dpc kidney the tant addition, at this stage of development. In the mature glomeruli of podocytes starting dpc 14.5 at fact that few glomeruli WT1 expression is detected in the bmp-7m'/bmp-7m1 et al. 1992). In 12.5-dpc (Armstrong GENES & DEVELOPMENT 2811

5 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press Luo al. et in kidney marker genes and mutant kidneys. (A,B) Bmp-7 expression during 12.5-dpc and of Expression 3. Figure 14.5-dpc wild-type in Bmp-7 and the condensed mesenchymal cells (cm) around the is expressed ureteric buds normal dpc, 12.5 kidney development. At glomeruli at re tubular is but aggregates and the high levels, further them to the expression is extended At 14.5 its dpc, (A). expression in wild-type ( + 1 + J and mutant ( - I - ) kidneys. At 12.5 (BJ. (C-FJ WTI down-regulated in the comma- and S-shaped bodies expressed both the wild-type (C) and the mutant (D). At 14.5 dpc, WT1 dpc, the in kidney noninduced mesenchyme of WTI is and the (cm] in the cortical region of condensed mesenchymal cells (m) noninduced mesenchyme in the that transcripts are detected (EJ but are no longer present the wild-type kidney in of (G-I) Pax-2 expression in the same region the mutant kidney (arrowhead F). Pax-2 condensed mesenchymal cells and pretubular aggregates in is expressed in the and kidneys. wild-type in At 12.5 dpc, mutant kidney Note level of expression in the condensed mesenchymal cells of the mutant (HI. the mutant and (G) that the wild type the both wild-type ludney. At 14.5 dpc, is much lower compared with that in the expressed condensed mesenchymal cells and in is Pax-2 detected in the mutant Pax-2 (I). be kidney, in same stage the At expression can only in agreggates the wild-type kidney pretubular areas adjacent to the and pretubular aggregates ureteric buds where condensed mesenchymal cells in the not buds but the ureteric is I). (K-N) Wnt-4 expression in wild-type and mutant kidneys. At 12.5 dpc, Wnt-4 in kidney (arrowhead should be present in normal pretubular aggregates in expressed expression is barely in areas adjacent to only some of Wnt-4 kidney, the mutant In (K). detectable the mutant Wnt-4 pretubular aggregates and the collecting duct system (MI. In in kidney, 14.5 dpc, At (L). is expressed buds ureteric the adjacent to the ureteric buds in the cortical region (arrowhead in N). (0-R) Laminin A Wnt-4 expression is absent from the areas 12.5 dpc, A is expressed in the ureteric buds of both the wild-type (0) and expression in wild-type and mutant kidneys. At Laminin both Laminin dpc, in the ureteric buds can be detected in A the wild type and the mutant, 14.5 At kidneys. (P) expression the mutant mutant the (Q), from is absent kidney type detectable the in bodies, epithelialized comma-shaped the whereas its expression in wild (R). Bars, (*m. 100 indicates are formed that the absence of that ex- WT1 transcrip- of family Pax the of a member encodes Pax2 in the mutant kidney pression a direct conse- not is tion vitro, In regulates factors. epithelial con- the Pax-2 quence the absence of of et (Dressler mesenchyme the of version metanephric BMP-7 simply the but al. reflects (i.e., appropriate the metanephric absence of cell type the in the developing kidney of normal 1993). At 12.5 dpc, mesenchyme). in ureteric bud, the sur- is the Pax-2 expressed embryo, DEVELOPMENT GENES &

6 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 nephrogenesis and the our histological analysis and raises with agreement at a higher rounding condensed mesenchymal cells, and, BMP-7 of absence that hypothesis stage, aggregates. in the programmed At pretubular in the level, to leads this (PCD) cell death cells metanephric mesenchymal of kidneys, is expressed in the bmp-7m1/bmp-7m1 Pax-2 early during nephrogenesis. in the wild-type control but at a re- same structures as During normal kidney development, PCD is normally 3G,H). in At 14.5 dpc, duced level (Fig. wild-type em- stromal cells (Koseki et al. 1992). Analysis to restricted bryos, be continues Pax-2 at expressed to moderate lev- the by kidneys bmp-7m1/bmp-7m' of of sections the in cells, mesenchymal buds and condensed ureteric els TUNEL showed PCD in stromal cells, but also assay in and at a higher level the and aggregates pretubular in metanephric noninduced was de- PCD the mesenchyme. bmp-7m1/bmp-7m' comma-shaped bodies (Fig. 31). In the tectable stages three the in mesenchyme metanephric in kidneys, cells surrounding in the expression Pax-2 the (n=4) examined: 12.0 dpc data not shown], 13.5 dpc buds is ureteric in absent, but virtually its expression unaffected [Fig. ureteric buds appears to re- The 3J). be Fig. =4; (n 4; data not shown). These 4), and 14.5 dpc (n = duction of in the 12.5-dpc bmp-7"'/ Pax2 expression suggest BMP-7 may act as a survival factor results that histo- kidney result with the consistent is of for metanephric mesenchymal cells and that the absence bmp-7"' were less mesenchy- analysis showing that there logical of of expression molecular markers of mesenchymal condensations complete absence The stage. this ma1 at death cells may be a consequence of the of mesenchymal of expression cortical region of the 14.5-dpc in the Pax-2 cells. with bmp-7m'/bmp-7m' kidney is also consistent the of reduced number noninduced mesenchymal cells at development BMP-7 is involved in eye this stage. the family of of a member Wnt-4, growth factors Wnt with unilateral mice are born or bi- bmp-7m11bmp-7m1 [Parr and McMahon is in the ureteric expressed 19941, lateral eye defects (Table 1). These are variable in expres- al. bud and in aggregates pretubular (Fig. 31,K; Stark et sivity, ranging from an absence of eye to eyes structures of the embryo, in normal kidney 12.5 dpc 1994). At detected Wnt-4 the expression pretubular aggre- was in the bud (Fig. 3K). At ureteric same gates adjacent to every stage in the mutant embryo, was Wnt-4 expression aggregates (Fig. pretubular in detectable barely At 3L). embryo, normal of kidney the in 14.5 dpc was Wnt-4 3M]. expressed in pretubular aggregates (Fig. At this Wnt-4 expression is com- stage in the mutant kidney is consistent with the pletely absent (Fig. 3N), which absence pretubular aggregates distinguishable histo- of The clear reduction of logically. Wnt-4 expression ob- served in the kidney of 12.5-dpc bmp-7"'/ bmp-7"' em- bryos suggests either that the pretubular aggregates are or intrinsically abnormal in the mutant that kidney is Wnt-4 the pretubular aggregates in regu- expression by BMP-7. lated bmp-7"'lbmp- study ureteric bud development in To mice, we examined the expression of Laminin A, a 7"' buds and marker of the epithelial cells of ureteric the expres- Laminin A ducts [Klein et al. 1990). collecting kidneys bmp-7m1/bmp-7m1 in the buds of ureteric sion wild-type kid- was with that in the unchanged compared ney at both 12.5- and 14.5-dpc embryos (Fig. 30,P,Q,R). ureteric Thus, the absence of BMP-7 did not affect epi- thelium consistent with the re- development, which is sult of histological analysis. At 14.5 dpc, Laminin A is that also highly expressed in the comma-shaped bodies transition. In epithelial to mesenchymal are undergoing Laminin kidneys, bmp-7m'/bmp-7m1 A expression in comma-shaped bodies was observed (Fig. 3R), consis- not tent with 14.5-dpc lack of comma-shape bodies in the Figure In 4. situ detection of apoptosis in developing wild-type kidneys. bmp-7m1/bmp-7m' and mutant kidneys. Section of wild-type 13.5-dpc embryos (A) indi- summary, In in hybridization analysis the situ or TUNEL the by analyzed bmp-7m1/bmp-7m1 embryos (B] was absence the that cates of expression the affects of BMP-7 in dark-field Materials and methods, and by assay, as described molecular markers specific the metanephric mesen- for apo- are shown. Note the microscopy. Representative sections 12.5 dpc, but not the expression of the chyme as early as ptosis region in the cortical the of the mutant kidney where ketanephric Bars, mesenchymal cells are located. pm. 100 in marker of molecular result This cells. is epithelial DEVELOPMENT & GENES 2813

7 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press Luo al. et Four mice lacking of normal size. bmp-7m11bmp-7m' had developed normally whereas the other eye remained identifiable eyes were examined histologically. In these no optic cup formation with rudimentary and no lens 5B). In mice, (Fig. absent lens, retina, and cornea were the not did We 5C-G). (Fig. observe any vesicle invagination cornea, lens, bmp-7m11bmp-7m' the other mice, mutant bmp- younger in abnormalities phenotypic consistent Eye not shown). (data retina were well and developed 7m11bmp-7m1 embryos not shown). This phenotype (data development was examined during embryogenesis to es- expression of coincident is with the eye during Bmp-7 dpc, 11.0 cause the tablish eye phenotype. At the of development. transcripts Bmp-7 the in optic are present cup has of and invagination when the optic developed surrounding head ectoderm vesicle and the as early as 9.5 wild-type embryos (Pei and the lens vesicle is visible in 1995; data not shown). To gain insight al. dpc (Lyons et optic cups and Kaufman lens 1992), the 1970; Rhodin in examined was Paxd of eye defect, expression into this ) or bilat- % 1 (2 unilaterally vesicles have not developed Pax-6 bmp-7m'/bmp-7m1 embryos. the eye of developing erally in bmp-7m1/bmp-7m1 embryos [n=20). In (14%) eye development the for is essential earliest events of in embryos remaining mutant the (65%], optic cups were be expressed to continues both flies and vertebrates and lens of the 12.5-dpc mouse embryo in the retina and the however, lens lens formation occurred; present and had vesicles were smaller than those of wild-type embryos at (Hogan et al. Halder et al. 1995; al. et Grindley 1988; We also examined eye stage (data not shown). the same 1995). In a subset of bmp-7m11bmp-7m1 embryos that brnp-7"'/bmp-7"' embryos 10.5-dpc development in 12.5 dpc, at eyes had expression Pax-6 indistinguish- was of in that embryos, one eye and found the mutant one 5E,F]. able from that in (Fig. wild-type embryos 5. Analysis the eye phenotype of BMP-7-deficient Figure of Histological analysis of eye development. mice. (A-GI [A) Fron- sections of the head of a newborn wild-type mouse with tal well-developed cornea retina (r), lens (le), and lacrimal glands (c), the (B) of the (lg). the vicinity of Frontal section head through eye of a BMP-7-deficient newborn mouse showing the absence [C- structure glands. of any identifiable eye lacrimal the except G) Eyes sections of the head of 10.5 dpc embryos. (C) of Frontal with optic cup and lens ves- well-developed a wild-type embryo (D) Frontal section of the head of a mutant littermate. Note icle. that the optic cup has developed and the lens ves- on one side, is present, whereas on the other side icle optic cup is rudi- the mental the lens vesicle is absent (arrowhead). (E-G) Higher and the magnification of one eye of the wild-type embryo (El, right the mutant of left eye (F), and the embryo eye of the mutant which has embryo, identifiable lens vesicle (G). no (H.1) Pax-6 mutant expression the eyes of 12.5-dpc wild-type (HI and in (I) embryos. In both the wild type and the mutant, ex- is Pax-6 vesicle. lens the and cup optic the in eye the that Note pressed the of the mutant is much smaller than that of structure wild type. In addition, the eye development process was delayed, lens vesicle connected leaving the surface ectoderm. with the Bar, (A,B) 200 ym; (C,D) bar, pm; 500 bar, (E-I) bar, 100 pm. 8r GENES DEVELOPMENT

8 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 nephrogenesis and skeletal patterning Bmp-7 is expressed in normal is required for 7 BMP- buds In developing mouse limb dpc, limb mes- the including 9.5 the entire limb field at the 1995; al. et overlying ectoderm (Lyons enchyme and 7"' 7m1/bmp- mice, skeletal bmp- abnormalities In the data not shown). At 10.5 dpc, when the apical ectoder- cage, rib the discrete areas: in the were identified skull, distinct ridge (AER) becomes in the developing ma1 the rib cage, the phenotype varied; In the hindlimbs. and mouse limb, in the mostly are found transcripts Bmp-7 the all of had fol- or mutants the of however, all some they although AER, in a lower level at are also present asymmetric pairing of of fusion lowing features: ribs, the anterior and posterior mesenchyme underlying the seven pairs than reduction ribs, attached of of less ribs, Bmp-7 expression is shown). 11.0 dpc At AER (data not or pair most the of both posterior one of ribs, mal- and in the prominent AER but is also expressed weakly in formation xiphoid process (Fig. GA,B). of the the In skull, the underlying mesenchyme ex- (Fig. At 11.5 dpc, 7A). (1) observed: consistently abnormalities were three the AER pression is down-regulated in epithelial cells of the center; in a cavity with smaller basosphenoid bones appears in underlying mesenchymal cells of the and (2) occipital bone basosphenoid with the the of fusion progress zone (data not shown). At 12.5 and 14.5 dpc tissue; and through of cartilaginous a piece marked (3) cells in was expressed Bmp-7 interdigital mesenchymal reduction of the pterygoid bones (Fig. 6C,D). Despite the and of subdermic layer the the bud, outlining in limb high level of expression of skeleton axial the in BMP-7 digits future (Fig. 7B,C). al. 1995), et (Lyons patterning the a consistent in defect To identify changes in gene expression patterns that axial observed. of the was skeleton not two polydactyly, of determinants represent early might mice Most of the be could (82%) BMP-7-deficient iden- molecular markers, Sonic HedgeHog (Riddle (Shh) al. et an extra tified phenotypically because of the presence of 1991)) were examined 1993) and Hoxd-13 (Dolle et al. in animals few A digit hindlimbs in the (polydactyly). the of and wild-type 11.0-dpc buds limb developing bmp- (12%) the forelimbs. The in also exhibited polydactyly embryos. The partial mirror image dupli- 7m1/bmp-7m1 extra digit was always a preaxial duplication and had the observed in cation bmp-7m1/bmp- 7"' limbs might be of digit GF). V (Fig. or IV, 111, 11, morphology of rib cages (A) and mutant of BMP-7-deficient Skeletal phenotype 6. Figure mice. (A,BJ Skeletal preparations of newborn wild-type rib rib are indicated: misalignment of the rib pairs on the sternum (open arrows); mutant fusion (B] mice. Several defects cage in the of the (xp); size reduction of rib xiphoid process shape of modification (rf); the (C,D) Ventral view of 13 (short, solid arrow in B). bones in frontal bones were removed. The three defects parietal the mutant skull are skeleton of skulls of newborn mice after the and and the fusion between right alisphenoid and inner ear (arrowhead in D); (ba]; in inhcated: presence of a cavity basosphenoid bone pterygoid bones (pt). reduction in the sizes of the right hindlimb of (El and mutant (F) (E,F) Skeletal preparation of the wild-type present in the mutant newborn mice. Digits are numbered from anterior to posterior; an extra is indicated (ED]. digit DEVELOPMENT & GENES 2815

9 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press Luo al. et and Shh, BMP-7-deficient mice. (A] Whole-mount Figure Hoxd-13 during limb development 7. Expression of Bmp-7, of wild-type and AER. (B,C] development. expression of Bmp-7 is predominant in the limb the At during Bmp-7 of hybridization situ in 11.0 dpc, is expressed and (C] hindlimbs. At both 12.5 and 14.5 dpc Bmp-7 dpc in the [B] dpc 12.5 in 14.5 of Bmp-7 hybridization in situ Section that outlines the future digits. (D,E) interdigital mesenchyme (D) and Shh expression in the developing limb buds of 11.0-dpc wild-type (F,G] and mutant in both wild type forelimb and the hindlimb. the Hoxd-13 the between difference is no embryos. There (El the mutant limb, [F] and mutant (G) embryos. In the bmp-7m1/bmp-7m1 Hoxd-13 11.0-dpc wild-type expression in the developing limb buds of posterior area limb bud compared with the wild-type control. The lower level smaller part of the a of the expression is restricted to in the wild-type and the mutant embryos is similar. of Hoxd-13 expression was forelimb between the consistently. Hoxd-13 observed pm. Bars, 100 Hoxd-13. Mutant Shh or of related to altered expression chyme undergoes an epithelial transformation to form and wild-type embryos were cut the midsagitally, and This the of tubules the the glomerulus and nephrons. embryo halves were probed for separately Shh and Hoxd- process begins mesenchy- at nephrogenic 11.5 dpc with 13 expression. Shh expression was identical in wild-type ma1 and is initiated by signals coming condensation limbs and mutant (Fig. Hoxd-13, 6D,EJ; in contrast, Classic coculture 19871. bud (Saxen ureteric from the posterior the in is expressed which the of distal part and inductive ex~eriments have established that signals to a smaller region hindlimbs, was restricted wild-type coking from the ureteric bud could be short-range dif- in the the mutant hindlimbs. Hoxd-13 part of posterior as factors, fusible such growth factors (Gruenwald 1937; expression was weaker hindlimbs. mutant the in overall 1957; Saxen al. 1976). et Recently, specific Grobstein Hoxd-13 embryos mutant of forelimb expression In the as Wnt-4 factors required for glomerulus formation, such was slightly weaker. genetic begun al. 1994), have (Stark by et identified be to In Wnt-4-deficient mice, mesenchymal cells con- means. around dense express the ureteric buds and molecular Discussion such markers, as and Pax2 dpc, least 14.5 at until WTI, the suggesting that the absence of Wnt-4 does not affect have generated BMP-7-deficient mice by deleting the We Therefore, different early stage of glomerulus formation. mature protein. the of domain coding for the sequence of signals are involved the early steps in nephrogenesis. This allele is recessive because heterozygotes carrying observations reported The suggest that paper this in the bmp- are normal, whereas bmp- 7"' / bmp- allele 7"' inducer early nephrogenesis. an is BMP-7 of Bmp-7 is nephrogene- in of a multitude exhibit mice 7"' defects time appropriate the at bud ureteric the of expressed in and ocular development, sis, skeletogenesis. in development. Moreover, the absence of BMP-7 only incomplete mesenchymal condensations are identified kidney and BMP-7 development in the kidnev at 12.5 d~c. In the absence of BMP-7 the mesen- the vertebrate development metanephric During a metanephric mesenchymal cells died before 14.5 dpc, DEVELOPMENT GENES &

10 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press BMP-7 nephrogenesis and observed the when meta- also that is phenomenon most hindlimb development. Polydactyly is one of the absence mesenchyme is cultured in nephric the of proper observed abnormalities frequent humans in (Mellin inductive tissues (Saxen 1987). The few mesenchymal 1978) and has McKusick and Tentamy 1963; docu- been condensations observed in bmp- 7"'/ bmp- 7"' mice at in repeatedly mented et al. 1994; (Mendelsohn mice probably are the handful of 12.5 dpc responsible for et Wurst two 1994; 1995). At least al. Fawcett hy- et al. in the kidneys of neonatal mutant glomeruli identified an presence the potheses could explain of in digit extra mice. passive by explained be might These placental that mice. One possibility would be BMP-7-deficient been has that a phenomenon BMP-7, of maternal transfer the BMP-7 prevents excessive proliferation cells in of another member the of documented for TGF-P superfam- progress in later or AER the below zone the interdigital TGF-P1 (Letterio et al. 1994). Because nephrogenesis ily, digit another BMP-7, absence the In mesenchyme. of 7"' bmp- 7"'l proceeds bmp- in 12.0 dpc until normally would form the anterior part of the limb because in of mice, to before acting be must signals other inductive This the condensation of excessive mesenchyme. would that regulates tar- BMP-7 this time. Our findings imply of mesenchymal survival controls BMP-7 that imply are genes in metanephric mesenchymal cells that get any limb; however, we did sig- not observe in the cells in- genes These critical for nephrogenesis. might be number in the difference cells undergoing of nificant ap- volved in the directly differentiationlproliferation of mesenchyme between wild-type and limb in the optosis alternatively, or, metanephric mesenchyme whose genes not shown). (data embryos mutant second hypothesis A tar- products are necessary for cell survival might be the the would be that the absence of BMP-7 affects pattern of BMP-7. of gets the expression of genes the anteroposterior controlling axis in digit formation. We analyzed the expression of mice and normal in Shh and Hoxd-13 BMP--/-deficient BMP-7 development eye and and could Hoxd-13 at littermates show that 11.0 dpc of the formation is result inductive in- reciprocal Lens poste- more expression was weaker and restricted to its surface vesicle the teraction between the optic and ecto- in the that rior mutants. These results part suggest has 1901 derm (Spemann Genetic dem- 12). 19 evidence ex- abnormal hindlimb growth and that BMP-7 inhibits onstrated for- lens for required is vesicle optic that the of BMP-7-deficient mice pression could lead Hoxd-13 in this mice, In 1984). al. et (Webster mice in mation expres- the formation of an extra (preaxial) digit. The to of formation the to leads interaction reciprocal lens the be shown also been has Bmp-2, Bmp, another of sion to and (Pei dpc vesicle from the surface ectoderm -10.5 at during bud limb expression Hoxd-13 to linked closely Rhodin 1992). In about 35% of bmp-7"'/ 1970; Kaufman has development, and it been suggested that BMP-2 bmp-7"' the embryos, interaction between optic vesicle could development (Fran- limb Hoxd-13 during activate surface ectoderm result- properly, and the occur did not Interestingly, et 1994). cis BMP-2 inhibits limb al. the uni- or in ing bilateral absence of lens formation in Martin 1993). and chick (Niswander growth in The these animals. expression of Bmp-7 during eye de- of BMP-7-deficient mice In summary, the generation BMP-7 that in involved directly is velopment suggests provided family has that this additional evidence of one factors be of it is, That lens formation. could the many processes morphogenetic growth factors regulate optic the by lens induction. released vesicle during The skeletal development. In including, but not limited to, also development importance has of BMP-7 during eye BMP-7 particular, our experiments demonstrate that acts been suggested in by vitro culture experiments showing formation and that it is glomeruli of inducer early an as that anti-BMP-7 antibodies can block lens formation in required for skeletal patterning and lens formation. Our Sampath pers. Solursh, M. and (K. embryos cultured rat not only that involved BMP-7 is demonstrate in results em- bmp-7m'/bmp-7m1 comm.). However, 86% of the several organs during development the differentiation of lens genes (or lenses), indicating that bryos did develop the in the mutations that hypothesis also raise but segregating in the of F, can suppress animals this aspect could genetic pathway in the itself or Bmp-7 gene be BMP-7-deficient phenotype. the responsible for several genetic diseases in which human glomerulus formation is impaired. and BMP-7 skeletal development mice have develop- skeletal BMP-7-deficient defects in and methods Materials ment that skeletal are restricted to a limited subset of of generation and targeting vector chimeras BMP-7 were elements. in absent skeletal abnormalities These To generate the targeting vector for positive-negative selection, the all of and were never studied animals heterozygous PCR- genomic library a screened, using was 1291SvEv a mouse a group observed mice strains as any of the mutant in amplified the re- fragment from cDNA 294-bp 3'-untranslated strongly suggests ES cell generated this This line. with (OP-1) Bmp-7 1397 to gion of (from nucleotide mouse the 1690) for that the mutation is responsible in the Bmp-7 locus (Ozkaynak et al. 199 1 cDNA phages the ). covering Overlapping defects. these replacement- A exons were isolated and mapped. 3' three most bmp-7"'/ in The most penetrant skeletal phenotype type targeting vector, pBMP7T, was constructed. The vector bmp-7"' a sixth of presence mice was digit preaxial the 5' to exon 6 and a 2.9-kb homology 3.2-kb homology contains a to The restriction of polydactyly both hindlimbs. in the 3' to 1). The PGK-hprt (where PGK is the exon phospho- 7 (Fig. hindlimbs illustrates nonequivalence the of forelimb and glycerate hnase expression was cloned 1 cassette promoter) DEVELOPMENT & GENES 2817

11 Downloaded from genesdev.cshlp.org on May 8, 2019 - Published by Cold Spring Harbor Laboratory Press Luo al. et the and expression arms, a homologous two MC1-tk between WTI, Wnt-4, and Pax-2 were described previously (Dressler et of the cassette was cloned into the polylinker site back- plasmid 1991; al. 1990; Klein et al. 1990; Ozkaynak et al. Armstrong et vector was The homologous regions. the which is outside bone, Pax-2, and WT1, for et al. 1994). cDNA probes al. 1992; Stark and 7 would be deleted after exons 6 both that so designed Laminin A (McGill Pelletier were Drs. from obtained Univer- J. EcoRI- targeting. The 5'-flanking 800-bp an was probe correct sity, Montreal, Canada), G. Dressler (University of Michigan, arm, and homologous 5' the of upstream 3.0-kb fragment BglII MD), respectively. Ann Arbor), and Y. Yamada (NIH, Bethesda, fragment just EcoRI-Hind111 3'-flanking the probe was a 700-bp probes for Bmp-7 and Wnt-4 were amplified by cDNA RT-PCR downstream correct targeting The homologous region. the 3' of cloned according to published information, into pBSKS ( - ) a XbaI event converts a 13.0-kb into fragment, wild-type 10.9- (Stratagene, probe, each For both and CA), Jolla, La sequenced. fragment, and kb XbaI a 9.2-kb into fragment HindIII wild-type antisense riboprobes were prepared either and sense using or T3 can revealed by Southern which fragment, HindIII a 5.6-kb be set of each in were used probes both polymerase, and RNA T7 blot hybridization using the 5'- and 3'-flanking probes, respec- situ were as Section in experiments. hybridization procedures 1). tively (Fig. (Sundin et al. 1990)) with the following modification. described and transfection cells was performed ex- ES of The culture polylysine-treated slides. 8 of Sections pm were mounted onto Briefly, (Ramirez-Solis et al. 1993). actly as described previously and hybridization The per- posthybridization washes were kg SalI-linearized of cells ES 25 AB2.1 were transfected with (Wilkinson formed as described 1992). were sections the Briefly, cells lo7 per gene pulser and grown using a Bio-Rad pBMP7T at were stringency washes The 50°C. at hybridized overnight targeted clones were The described. under double selection as were times 64°C. Exposure days. Autoradiography, 1&16 Ho- genomic DNA with Southern blot hybridization identified by echst 33258 staining, and photography were performed as de- ES targeted individual clone. from each then cells were The (Sundin et al. 1990), as well as whole-mount in situ scribed injected into (Bradley which C57BL16 1987), blastocysts were al. hybridization procedures (Takata et 1994). of uterus into the transferred then pseudopregnant females. Re- C57BLl6 females, and sulting male chimeras were mated to death analysis cell Programmed hybridiza- Southern blot by identified heterozygous offspring tion. heterozygotes The were (F,) generate then interbred to out published the to according was carried analysis The TUNEL mice (or homozygous BMP-7-deficient embryos) (F,). of (Gavrieli procedure 1992). Sections al. et mouse embryos were prepared as described for in situ hybridization. Southern blot analysis preparation Skeleton and tails mouse DNA isolation from double-resistant ES cells or blot performed described previously as analysis were Southern were prepared Skeletons as described (Kochhar Neonates 1973). digested 1992). DNA was al. (Matzuk et or XbaI either with were sacrificed, eviscerated, and fixed in 95% ethanol. skinned, a 0.7% agarose gel. DNA was in electrophoresed and HindIII 2% with were digested hydrox- potassium Nonskeletal tissues + (Amer- nylon membranes then transferred onto Hybond-N and 8GX, stained were ide. alcian blue Cartilaginous tissues by Inc., sham International, Amersham, UK), and standard South- S. by alizarin stained were tissues ossified red ern hybridization procedures were followed. Acknowledgments Northern blot hybridization RNA Total guanidinium the using prepared samples were thio- Drs. D. Duboule, M. Finegold, (G.E.), A. G. Eichele We thank cyanate-CsC1 method gradient blot and Northern as described, Pelletier, G. McMahon, K. Opperman, H. Sam- Overbeek, P. hybridization et al. 1989) was performed as described (Sambrook cDNA path, Y. Striker, R. dfSouza, L. Yamada, and H. Zhang for Briefly, with minor modification. total RNA samples were frac- help and advice during critical for probes and the course of this formaldehyde gel, and the acids were nucleic tionated on a her invalu- for Ducy to study. particularly G.L. is grateful P. Dr. a onto transferred The nylon membrane. Hybond-N+ hybrid- analysis the generating able help in the initial in of the mice, the blot Southern hy- for that as ization procedure was same her critical reading of manuscript. the also for figures, and We described above. bridization This by Guo X. thank for work was technical support. supported Basic Re- grant AR-41059, Health National Institutes of (NIH) Dimes of search Award IFY March the from Founda- 92-0871 Histological embryos analysis of Institute to Roussel Scientific the from a grant and tion, G.K. of development stages of were dissected free of various Embryos to NIH grant for G.E. awarded HD20209, by C.H. was supported yolk sacs were saved for the and decidua, and uterine muscle the the early part Medical Hughes Howard is a A.B. work. this of as described were Histology procedures analysis. DNA Institute Investigator; research also sup- in is laboratory his with embryos were The modifications. 1992)) minor (Kaufman the Health. of Institutes National by ported overnight 4% paraformaldehyde solution and trans- in fixed of publication costs The this article were defrayed in part by embryos ferred to 70% ethanol. The fixed were dehydrated This article payment therefore be hereby of page must charges. of through ethanol. Specimens were embedded in graded series section in 18 USC with marked "advertisement" accordance for sectioning. Sections stained plastic paraffin or either were 1734 solely to indicate this fact. al. et Culling by as described eosin and hematoxylin with kidney histology, which (1985), were stained by except those for Spicer periodic acid Schiff (PAS) staining according to the et al. References (1967). K. Armstrong, J.F., Pritchard- Jones, W.A. Bickmore, N.D. of 1992. Bard. expression The Wilms' the Hastie, and J.B.L. hybridization and in whole-mount Section in situ situ mammalian gene, tumor in the developing WT embryo. 1, hybridization 40: 85-97. Mech. Dev. the Bmp-7, of The probes used to survey expression Laminin A, analysis of chimeric mice. In Production 1987. and Bradley, A.. DEVELOPMENT & GENES 2818

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14 Downloaded from Cold Spring Harbor Laboratory Press on May 8, 2019 - Published by genesdev.cshlp.org BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. G Luo, C Hofmann, A L Bronckers, et al. Genes Dev. 1995, 9: Access the most recent version at doi: 10.1101/gad.9.22.2808 This article cites 50 articles, 20 of which can be accessed free at: References http://genesdev.cshlp.org/content/9/22/2808.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting click here. right corner of the article or Service Copyright © Cold Spring Harbor Laboratory Press

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