Placental Defects and Embryonic Lethality in Mice Lacking Suppressor of Cytokine Signaling 3

Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3

Andrew W. Roberts*, Lorraine Robb*, Steven Rakar†, Lynne Hartley*, Leonie Cluse*, Nicos A. Nicola*, Donald Metcalf*, Douglas J. Hilton*, and Warren S. Alexander*‡

*The Walter and Eliza Hall Institute of Medical Research and Cooperative Research Centre for Cellular Growth Factors, Post Ofﬁce, Royal Melbourne Hospital, Victoria 3050, Australia; and †AMRAD Operations Pty. Ltd., Richmond, Victoria 3121, Australia

Contributed by Donald Metcalf, May 31, 2001 Mice lacking suppressor of cytokine signaling 3 (SOCS3) exhibited activity of which it ultimately inhibits. SOCS proteins may also embryonic lethality with death occurring between days 11 and 13 target associated signaling molecules for degradation. The SOCS ,of gestation. At this stage, SOCS3؊/؊ embryos were slightly smaller box domain has been shown to interact with elongins B and C than wild type but appeared otherwise normal, and histological proteins involved in the cellular ubiquitination machinery that analysis failed to detect any anatomical abnormalities responsible targets proteins for destruction via the proteasome (9, 10). Thus, for the lethal phenotype. Rather, in all SOCS3؊/؊ embryos exam- SOCS proteins may regulate cytokine responses through control ined, defects were evident in placental development that would of the turnover of signaling proteins as well as directly inhibiting account for their developmental arrest and death. The placental their catalytic activity or recruitment to the signaling complex. spongiotrophoblast layer was signiﬁcantly reduced and accompa- The physiological significance of SOCS-mediated control of nied by increased numbers of giant trophoblast cells. Delayed cytokine signaling has emerged from studies in which these branching of the chorioallantois was evident, and, although em- regulators have been functionally deleted in mice. SOCS1 is a key bryonic blood vessels were present in the labyrinthine layer of modulator of IFN-␥ signaling. Mice lacking SOCS1 display ؊ ؊ SOCS3 / placentas, the network of embryonic vessels and ma- deregulated responses to IFN-␥ that result in a lethal combina- ternal sinuses was poorly developed. Yolk sac erythropoiesis was tion of fatty degeneration and necrosis of the liver, excessive T ؊ ؊ normal, and, although the SOCS3 / fetal liver was small at day cell activation, lymphopenia, and hematopoietic infiltration of 12.5 of gestation (E12.5), normal frequencies of erythroblasts and multiple organs (11–14). In contrast, mice lacking SOCS2 are hematopoietic progenitor cells, including blast forming unit-eryth- healthy and fertile, but display excessive growth consistent with roid (BFU-E) and, colony forming unit-erythroid (CFU-E) were a negative regulatory role for SOCS2 in GH and͞or insulin-like present at both E11.5 and E12.5. Colony formation for both BFU-E growth factor (IGF)-I signaling (15). SOCS3 has been implicated ؊ ؊ and CFU-E from SOCS3 / mice displayed wild-type quantitative in control of cytokine signaling in several biological systems. For responsiveness to erythropoietin (EPO), in the presence or absence example, in addition to the potential roles for SOCS3 implied by of IL-3 or stem cell factor (SCF). These data suggest that SOCS3 is its capacity to interact with and inhibit signals from the GHR, required for placental development but dispensable for normal IL-2R␤, EPOR, and gp130 receptors (see above), SOCS3 has hematopoiesis in the mouse embryo. also been implicated in processes as disparate as leptin control of energy homeostasis (16) and modulation of intestinal inflam- he eight members of the suppressor of cytokine signaling mation (17). One report on mice lacking SOCS3 described Tfamily of proteins (SOCS1 to -7 and CIS) are characterized embryonic lethality with marked erythrocytosis (18). The au- by the presence of a centrally located Src homology 2 (SH2) thors concluded that SOCS3 is an essential physiological regu- domain, an N-terminal domain of variable length and divergent lator of EPO signaling, a model supported by the occurrence of sequence, and a conserved C-terminal SOCS box domain (1). lethal anemia in transgenic mice constitutively expressing SOCS1 to -3 and CIS have been shown to participate in a SOCS3 (18) and by data showing that SOCS3 binds to and negative feedback loop to regulate cytokine signaling, particu- inhibits signaling from the EPO receptor (6). larly from the hematopoietin class of cytokine receptors (2). In this study, we have independently generated mice lacking Signaling is initiated on cytokine-dependent receptor aggrega- a functional SOCS3 gene. We show that the death of SOCS3Ϫ/Ϫ tion, which activates Janus kinases (JAKs), resulting in tyrosine mice at mid-gestation is not linked to defects in the embryo but phosphorylation of the receptor as well as other signaling is associated with abnormalities in specific regions of the pla- proteins. Additional signaling molecules, such as the signal centa. The nature of these defects and our demonstration that transducers and activators of transcription (STATs) are subse- SOCS3 is expressed at these placental sites suggests that lethality quently activated via recruitment to specific receptor phospho- in SOCS3Ϫ/Ϫ mice results from placental insufficiency. In con- tyrosines (3). trast to the previous study, we found no evidence of defective The SOCS proteins modulate signal transduction by inhibiting erythropoiesis in SOCS3Ϫ/Ϫ mice. components of the JAK͞STAT pathway. Whereas SOCS1 is thought to directly bind the JAK kinases and inhibit their Materials and Methods catalytic activity, CIS appears to act by binding to the receptor, Generation of Targeted Embryonic Stem Cells and Mutant Mice. A preventing recruitment and activation of STATs (reviewed in 3-kb PCR product extending 5Ј from the SOCS3 ATG initiation ref. 2). Recent evidence suggests that SOCS3 action shares codon and a 4.3-kb XbaI-XhoI3Ј SOCS3 fragment (Fig. 1) were aspects of both these mechanisms. Studies of signaling from the GH receptor (GHR; ref. 4), IL-2 receptor beta chain (IL2R␤; ref. 5), erythropoietin receptor (EPOR; ref. 6), and gp130 (7, 8) Abbreviations: SOCS3, suppressor of cytokine signaling 3; BFU-E, blast forming unit- suggest that, whereas SOCS3 expression leads to inhibition of erythroid; CFU-E, colony forming unit-erythroid; STAT, signal transducer and activator of transcription; JAK, Janus kinase; EPO, erythropoietin; SCF, stem cell factor; E, day of JAK kinase activity, SOCS3 does not bind to JAKs with high gestation; CFC, colony-forming units. affinity and depends on the presence of cytokine receptor for ‡To whom reprint requests should be addressed. E-mail: alexander_w@wehi.edu.au. this action. The emerging model suggests that SOCS3 relies on The publication costs of this article were defrayed in part by page charge payment. This interaction with the activated receptor for recruitment to the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. signaling complex, providing access to the JAK kinases, the §1734 solely to indicate this fact.

9324–9329 ͉ PNAS ͉ July 31, 2001 ͉ vol. 98 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.161271798 Downloaded by guest on October 1, 2021 PstI chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment.

Yolk Sac and Fetal Liver Cultures. We suspended 5 ϫ 103 or 104 yolk sac or fetal liver cells in 1.5% methylcellulose (Fluka) in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% FCS and EPO (2 units͞ml at maximal concentration) with or without IL-3 (10 ng͞ml) and stem cell factor (SCF; 100 ng͞ml). After incubation at 37°C for 7 days [blast forming unit-erythroid (BFU-E) and mixed or myeloid colonies] or 2–3 days [colony forming unit-erythroid (CFU-E)] in a humidified atmosphere of 5% CO2 in air, colonies were scored at 35-fold magnification.

Histological Analysis and lacZ Staining. Embryos and placentas from timed matings of SOCS3ϩ/Ϫ mice (day of plug ϭ day 0) were fixed in a solution of 0.2% glutaraldehyde and 1% form- aldehyde, and lacZ activity was detected as described (21). Paraffin-embedded lacZ-stained embryos and placentas were serially sectioned, and alternate sections were stained with nuclear fast red or hematoxylin and eosin. Embryos were genotyped by extraction of yolk sac DNA (22) for PCR with an oligonucleotide set that allowed distinction between wild-type Fig. 1. Disruption of the SOCS3 locus by homologous recombination. (A) The and targeted SOCS3 alleles: 5Ј-CTT CAT GCC ATG ACG TCT structure of the murine SOCS3 locus is illustrated with exons as raised boxes GTG ATG C-3Ј,5Ј-GCC TTC GAG GCT GTC TGA AGA and the coding region shaded (Xb, XbaI; No, NotI; X, XhoI; RV, EcoRV; H3, TGC-3Ј, and 5Ј-GAA GCT GAC TCT AGA CGT TG-3Ј. PCR HindIII). In the targeted allele, the coding region was deleted and a cassette was performed for 30 cycles of 96°C for 30 s, 62°C for 30 s, and fusing the lacZ gene to the SOCS3 ATG and including the selectable PGKneo 72°C for 1 min before visualization of the amplification products gene was included. (B) Southern blot of genomic DNA from E11.5 embryos collected from a cross between SOCS3ϩ/Ϫ mice, including wild-type (ϩ͞ϩ), on 2% agarose gels with ethidium bromide staining. SOCS3ϩ/Ϫ, and SOCS3Ϫ/Ϫ samples. The DNA was digested with HindIII and hybridized with the 3Ј probe, which distinguishes between the targeted (9-kb) Derivation of Primary Embryonic Fibroblasts. Embryos from timed ϩ Ϫ and endogenous (20-kb) SOCS3 loci. (C) Northern blot of RNA extracted from matings of SOCS3 / mice were collected at day 11.5 of gesta- wild-type (ϩ͞ϩ), SOCS3ϩ/Ϫ, and SOCS3Ϫ/Ϫ primary embryo ﬁbroblasts after tion (E11.5) and dissected, and the trunks were individually stimulation with IFN-␥ (ϩ) or saline (Ϫ). The blot was probed with a SOCS3 treated with trypsin before disaggregation by pipetting. The cells coding region probe followed by a GAPDH probe to conﬁrm RNA integrity. were plated in DMEM containing 10% FCS and passaged as necessary when confluent. Yolk sacs were collected in parallel ␤ for genotyping. Near confluent cultures of primary embryonic MEDICAL SCIENCES cloned into the BamHI and XhoI sites of p galpAloxneo (12). Ϫ/Ϫ ϩ/Ϫ ͞ fibroblasts from each of SOCS3 , SOCS3 , and wild-type This targeting vector was electroporated into C57BL 6 embry- embryos were stimulated for1hwith100ng͞ml IFN-␥. Cells onic stem cells (19). Transfected cells were selected in G418, and were harvested for extraction of total cellular RNA and assess- resistant clones were picked and expanded. Hybridization of ment of SOCS3 expression by Northern blotting as described HindIII-digested genomic DNA with a 1.5-kb EcoRV-HindIII Ј (20) using a full-length SOCS3 coding region fragment as fragment situated 6-kb 3 of the SOCS3 gene (Fig. 1) was used hybridization probe. to differentiate between the endogenous (20-kb) and targeted (9-kb) SOCS3 alleles. Two independent targeted embryonic Results ͞ stem cell clones were injected into BALB c blastocysts to Embryonic Lethality in Mice Lacking SOCS3. A targeting vector for generate chimeric mice. Male chimeras were mated with the deletion of the SOCS3 coding sequence was constructed C57BL͞6 females to yield heterozygotes for the targeted SOCS3 ϩ ϩ (Fig. 1) and transfected into embryonic stem cells. The vector allele, which were interbred to produce wild-type (SOCS3 / ), was designed so that homologous recombination with an endog- ϩ Ϫ Ϫ Ϫ heterozygous (SOCS3 / ), and mutant (SOCS3 / ) mice on a enous SOCS3 locus would delete the entire SOCS3 coding C57BL͞6 background. The deletion of SOCS3 coding sequence sequence and place the lacZ gene under the transcriptional and subsequent inability to produce SOCS3 mRNA in cells from control of the SOCS3 locus. When offspring of mice heterozy- mutant mice was confirmed in nucleic acid blots, which were gous for the disrupted SOCS3 allele were genotyped at weaning, performed as described (20). Northern blots were probed with no SOCS3Ϫ/Ϫ mice were present consistent with embryonic or a full-length SOCS3 coding region probe and then with a 1.2-kb perinatal lethality. Over 1,000 weanlings from these crosses have

Table 1. Embryonic lethality in SOCS3؊/؊ mice No. of embryos* Proportion of SOCS3Ϫ/Ϫ Viability of SOCS3Ϫ/Ϫ Age of embryos SOCS3ϩ/ϩ:SOCS3ϩ/Ϫ:SOCS3Ϫ/Ϫ embryos, % embryos, %

E9.5 20:38:19 25 100 E10.5 5:10:8 35 100 E11.5 52:69:26 18 33 E12.5 41:72:25 18 50 E13.5 12:19:2 6 0 E14.5 and 15.5 6:10:0 0 0

*Number of embryos of each genotype from matings of SOCS3ϩ/Ϫ parents are shown.

Roberts et al. PNAS ͉ July 31, 2001 ͉ vol. 98 ͉ no. 16 ͉ 9325 Downloaded by guest on October 1, 2021 Fig. 2. Placental abnormalities in SOCS3Ϫ/Ϫ embryos. (a) Viable E9.5, E10.5, and E12.5 SOCS3 null embryos and their wild-type littermates. Embryos were stained for lacZ expression. The SOCS3 null embryos are to the right of their wild-type littermates. (b–m) Histological sections of the placentas from these embryos. Placentas were stained for lacZ and then sectioned and further stained with hematoxylin and eosin (H&E). The lacZ-staining pattern was detectable in the H&E-stained sections and was conﬁrmed by staining near-adjacent sections with nuclear fast red (not shown). Temporal proﬁle of placental development in wild-type (b, d, f, h, j, and l) and SOCS3 null (c, e, g, k, i, and m) placentas at E9.5 (b–e) and E10.5 (f, g, j, and k) and E12.5 (h, i, l, and m) is shown. There is a paucity of spongiotrophoblast in the SOCS3 null placentas. Higher power (d, e, and j–m) reveals lacZ staining of the labyrinthine trophoblast and the allantois in the SOCS3 null placentas. Some lacZ staining is also detected in the walls of embryonic vessels penetrating the labyrinth. LacZ staining is not seen in wild-type embryos. At E12.5, embryonic (arrowheads) and maternal (arrows) vessels within the labyrinth are dilated. Al, allantois; gi, trophoblast giant cells; la, labyrinth; lt, labyrinthine trophoblasts; ma, maternal decidua basalis; sp, spongiotrophoblast. Scale bars for b, c, and f—I ϭ 100 ␮m; for d, e, and j—m ϭ 10 ␮m.

now been genotyped, all of which were SOCS3ϩ/Ϫ or wild type. 1). Reverse transcription–PCR analysis of RNA purified from Examination of SOCS3ϩ/Ϫ mice revealed no apparent abnor- E11.5 embryos confirmed the absence of SOCS3 expression in malities. When embryos from SOCS3ϩ/Ϫ crosses were genotyped SOCS3Ϫ/Ϫ mice (data not shown). at E10.5, viable mice of each genotype were present in numbers consistent with normal Mendelian inheritance of the disrupted Placental Abnormalities in SOCS3؊/؊ Embryos. Initial microscopic SOCS3 allele (Fig. 1 and Table 1). Similar analysis at E11.5 to inspection and histological analysis of E9.5–E13.5 SOCS3Ϫ/Ϫ E15.5 revealed selective death of SOCS3Ϫ/Ϫ embryos between embryos indicated that, from E10.5, many SOCS3Ϫ/Ϫ embryos E11 and E13 (Table 1). To confirm that gene targeting had were slightly smaller than their wild-type or SOCS3ϩ/Ϫ litter- generated a null allele, primary embryonic fibroblasts were mates (Fig. 2). However, no gross defects were seen, and derived from SOCS3Ϫ/Ϫ and control embryos and then stimu- extensive histological analysis failed to reveal any anatomical lated with IFN-␥. Although SOCS3 RNA was induced in wild- abnormalities that might have been responsible for the embry- type embryos, consistent with previous data (23), no SOCS3 onic lethality. This result prompted us to examine placentas from RNA was present in IFN-␥-stimulated SOCS3Ϫ/Ϫ fibroblasts, SOCS3Ϫ/Ϫ embryos and their littermates. Embryos and placen- and an intermediate level was induced in SOCS3ϩ/Ϫ cells (Fig. tas from timed matings were stained for lacZ activity as a

9326 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.161271798 Roberts et al. Downloaded by guest on October 1, 2021 Table 2. Yolk sac hematopoiesis in SOCS3؊/؊ mice E10.5 E11.5

SOCS3ϩ/ϩ SOCS3ϩ/Ϫ SOCS3Ϫ/Ϫ SOCS3ϩ/ϩ SOCS3ϩ/Ϫ SOCS3Ϫ/Ϫ

Number analyzed 3 2 2 10 19 3 Cellularity, ϫ10Ϫ6 0.24 Ϯ 0.15 0.14 Ϯ 0.01 0.18 Ϯ 0.15 0.35 Ϯ 0.15 0.33 Ϯ 0.16 0.20 Ϯ 0.22 Myeloid-CFC, per 1 ϫ 104 7 Ϯ 3 9.5 Ϯ 5 10.5 Ϯ 16Ϯ 24Ϯ 25Ϯ 1 BFU-E, per 1 ϫ 104 12 Ϯ 523Ϯ 24 29 Ϯ 57Ϯ 35Ϯ 318Ϯ 15 Mix-CFC, per 1 ϫ 104 4 Ϯ 14Ϯ 54Ϯ 32Ϯ 1.5 1 Ϯ 11Ϯ 1

Results represent the mean Ϯ SD of data from multiple embryos. Triplicate cultures were stimulated with SCF, IL-3, and EPO and incubated for 7 days in a humidiﬁed atmosphere of 5% CO2/air at 37°C.

surrogate marker of SOCS3 transcription and then serially SOCS3Ϫ/Ϫ tissues compared with those in SOCS3ϩ/Ϫ mice, sectioned for morphological analysis. Only placentas obtained although patterns of expression were the same. from viable embryos, as judged by the presence of a beating heart and absence of signs of necrosis, were studied. Embryonic Hematopoiesis in SOCS3؊/؊ Mice. To determine whether At E9.5, there was a marked decrease in the spongiotropho- SOCS3 plays an essential role in hematopoiesis, embryos were Ϫ Ϫ blast layer in SOCS3 / placentas and an accompanying increase analyzed at E10.5, E11.5, and E12.5. Blood islands developed in the number of giant trophoblast cells. The allantois was normally in the yolk sacs of SOCS3Ϫ/Ϫ embryos. Yolk sac attached to the chorion normally, but there was a delay in cellularity and erythroblast morphology were similar in mice of chorioallantoic branching and a decrease in vascularity of the all genotypes, and the hematopoietic progenitor content of yolk labyrinth (Fig. 2 b–e). The defects in trophoblast giant cell, sacs at both E10.5 and E11.5 was also normal in SOCS3Ϫ/Ϫ mice spongiotrophoblast and labyrinthine layers persisted in E10.5– (Table 2). Ϫ/Ϫ 12.5 SOCS3 placentas (Fig. 2 f–m). Instead of the sparse, The cellularity of fetal livers from viable SOCS3Ϫ/Ϫ embryos discontinuous trophoblast giant cell layer present in control was similar to that of wild-type and SOCS3ϩ/Ϫ embryos at E11.5 placentas, giant trophoblast cells were increased in number. (Table 3). As judged by cytology, erythroblast content was Spongiotrophoblast cells were markedly reduced. The labyrin- similar between genotypes at this time point. Semisolid culture thine trophoblast layer was thickened, but the labyrinth was assays revealed that the content of hematopoietic progenitors thinner. Embryonic vessels were present in the labyrinthine layer Ϫ Ϫ (CFU-E, BFU-E, myeloid-CFC, and mixed lineage-CFC) was of SOCS3 / mice. However, unlike placentas from wild-type Ϫ Ϫ also unaltered in SOCS3 / fetal livers (Table 3). At E12.5, livers littermates, in which the embryonic blood vessels were elongated Ϫ Ϫ in viable SOCS3 / embryos appeared small, and the total and radiated uniformly from the chorionic plate into the laby- cellularity of the organ was reduced 10-fold (Table 3). However, rinthine layer, the network of embryonic vessels and maternal the frequency of hematopoietic progenitors among fetal liver sinuses was poorly developed in mutant placentas. By E12.5, ϩ/Ϫ there was obvious dilation of maternal and embryonic vessels cells was similar to that observed for wild-type and SOCS3 MEDICAL SCIENCES and pooling of maternal blood in the labyrinthine layer of the embryos (Table 3). In addition, the size of the colonies formed mutant placentas (Fig. 2 h, i, k, l, and m). This phenotype was from individual progenitor cells was indistinguishable between similar on inbred C57BL͞6 or hybrid C57BL͞6 ϫ 129Sv (B6129) genotypes. To exclude the possibility of a strain effect interfering strain backgrounds. with the interpretation of these results, embryos on a mixed Whole mount lacZ staining of SOCS3Ϫ/Ϫ placentas indicated B6129 genetic background were also examined, and similar SOCS3 transcription in the allantoic mesenchyme and the results were observed. In particular, the frequency of hemato- endothelium of embryonic vessels penetrating the chorionic poietic progenitor cells was similar between genotypes (Table 3). plate. Expression was also seen in the chorionic plate tropho- To further assess whether the absence of SOCS3 had conse- blast. No lacZ staining was seen in wild-type placental sections quences for hematopoietic progenitor cell growth in vitro, cul- (Fig. 2). Extensive staining was also evident in the embryo (Fig. tures of fetal liver cells were maintained for 3 or 4 weeks. Again, 2a), including the yolk sac and embryonic vasculature, neuro- no differences were observed between genotypes with respect to epithelium, nerve ganglia, precartilagenous condensations, and either the number of colonies present, or the sizes of individual limb buds (L.R., unpublished results). Staining was stronger in colonies in the cultures (data not shown).

Table 3. Fetal liver hematopoiesis in SOCS3؊/؊ mice C57BL/6 C57BL/6 ϫ 129/Sv

E11.5 E12.5 E11.5

SOCS3ϩ/ϩ SOCS3ϩ/Ϫ SOCS3Ϫ/Ϫ SOCS3ϩ/ϩ SOCS3ϩ/Ϫ SOCS3Ϫ/Ϫ SOCS3ϩ/ϩ SOCS3ϩ/Ϫ SOCS3Ϫ/Ϫ

Number analyzed 18 33 7 15 23 3245 Cellularity, ϫ10Ϫ6 0.41 Ϯ 0.24 0.47 Ϯ 0.23 0.32 Ϯ 0.28 3.27 Ϯ 1.52 2.87 Ϯ 1.79 0.27 Ϯ 0.10 0.46 Ϯ 0.11 0.6 Ϯ 0.19 0.40 Ϯ 0.3 CFU-E, per 5 ϫ 103 294 Ϯ 143 285 Ϯ 172 278 Ϯ 4 110 Ϯ 44 117 Ϯ 38 254 (1) 356 Ϯ 112 464 Ϯ 31 278 Ϯ 4 Myeloid-CFC, per 16 Ϯ 614Ϯ 612Ϯ 614Ϯ 515Ϯ 49Ϯ 718Ϯ 218Ϯ 216Ϯ 4 1 ϫ 104 BFU-E, per 1 ϫ 104 36 Ϯ 14 34 Ϯ 16 32 Ϯ 12 15 Ϯ 819Ϯ 822Ϯ 26 28 Ϯ 446Ϯ 20 40 Ϯ 4 Mix-CFC, per 1 ϫ 8 Ϯ 26Ϯ 42Ϯ 24Ϯ 47Ϯ 43Ϯ 26Ϯ 06Ϯ 44Ϯ 0 104

Results represent the mean Ϯ SD of data from multiple embryos as indicated. Replicate cultures were stimulated with EPO alone, or SCF, IL-3, and EPO, and incubated for 2–3 days for CFU-E and 7 days for CFC in a humidiﬁed atmosphere of 5% CO2/air at 37°C.

Roberts et al. PNAS ͉ July 31, 2001 ͉ vol. 98 ͉ no. 16 ͉ 9327 Downloaded by guest on October 1, 2021 until E10, but died between E11 and E13 (Table 1). No reproducible embryonic abnormalities were observed in SOCS3Ϫ/Ϫ mice. Rather, in all SOCS3Ϫ/Ϫ embryos examined, defects were evident in the developing placenta that would account for their developmental arrest and death. The chorioallantoic placenta links the embryo with the maternal circulation and is required for embryonic growth and development from E9.5. In mice, there are three distinctive trophoblastic cell structures in the mature placenta that are formed by E10 and persist throughout gestation: an inner labyrinthine layer, an intermediate spongiotrophoblast layer, and an outermost layer of trophoblast giant cells (24). The histology of SOCS3Ϫ/Ϫ placentas demonstrates that absence of SOCS3 affects both the formation of the spongiotrophoblast and the morphogenesis of the placental labyrinth (Fig. 2). The reduction in spongiotrophoblast suggests an early misallo- cation or differential proliferation of diploid trophoblast stem cells. In the labyrinthine layer of SOCS3 placentas, vessels enter the labyrinth, but do not sustain further branching. The labyrinth trophoblast remains thickened and poorly differentiated (Fig. 2). Given evidence of SOCS3 expression in labyrinthine trophoblasts and in the allantoic mesenchyme (Fig. 2), defects in either, or both, may affect the normal branching and͞or survival of the embryonic labyrinthine vascular system. Abnormalities of the labyrinthine layer can arise as a consequence of abnormalities of the allantoic mesenchyme or of the trophoblast (reviewed in ref. 25). Studies of Mash2 null mutant placentas also showed a reduced spongiotrophoblast layer and increased numbers of giant cells, together with a reduction in the labyrinthine layer (26). Interestingly, chimera studies revealed the absence of spongiotrophoblast to be a primary, intrinsic cellular defect, implying that an intact spongiotrophoblast layer is required for normal development of the labyrinthine trophoblast layer (27). A similar analysis, combining SOCS3Ϫ/Ϫ with diploid or tetraploid wild-type embryos, should help better define the key roles of SOCS3 in the different placental components affected in SOCS3Ϫ/Ϫ mice. Biochemical analyses and studies of SOCS3 action in overex- pression and cell line models provide compelling evidence that Fig. 3. Erythropoietin responsiveness of SOCS3Ϫ/Ϫ erythroid progenitor cells. SOCS3, like other SOCS family members, can act as a negative (a and b) Data from two independent experiments are shown. E11.5 fetal liver feedback regulator of cytokine signaling (2). Knockout studies of cells (5 ϫ 103) were cultured for 3 days in various concentrations of erythro- SOCS1 and SOCS2 have demonstrated key physiological roles poietin. Individual results represent means of duplicate cultures, normalized for these family members in modulation of IFN-␥ and GH͞ for CFU-E number in supramaximal concentrations of erythropoietin (2 units͞ insulin-like growth factor-I signaling, respectively (14, 15). These ml). The numbers of CFU-E in cultures containing maximal concentrations of data imply that the defects in SOCS3Ϫ/Ϫ mice may arise from 4 erythropoietin ranged between 264 and 684. (c) E11.5 fetal liver cells (10 ) excess signaling from one or more cytokines acting in placental were cultured for 7 days in triplicate in stem cell factor, IL-3, and various ﬁnal development. Although few studies have examined the effects of concentrations of erythropoietin, and the number of BFU-E (including mix- CFC) enumerated as above. The numbers of BFU-E in cultures containing cytokine overproduction on the development of the placenta, maximal concentrations of erythropoietin ranged between 26 and 50. gene targeting studies have implicated several cytokines in this process. Mice lacking hepatocyte growth factor (HGF) or its receptor c-Met die at E11.5 with defects in the labyrinthine layer, Normal EPO Responsiveness in SOCS3؊/؊ Erythroid Progenitor Cells. and fibroblast growth factor receptor 2 (FGFR)-deficient mice Because SOCS3 has been implicated as a negative regulator of exhibit lethality at E10.5, with defective chorioallantoic fusion or erythropoietin signaling (6, 18), experiments were performed to absence of the labyrinthine layer (reviewed in ref. 25). Leukemia determine whether this role for SOCS3 was of physiological rele- inhibitory factor receptor-null embryos exhibit abnormal, dis- vance in primary cells. The growth of CFU-E from cultures of E11.5 organized placentas (28), and mice lacking the epidermal growth fetal liver cells stimulated by titrated concentrations of erythropoi- factor receptor also display genetic background-dependent defects in the placental labyrinth (29). Future studies to precisely etin was monitored in parallel for each genotype. In two indepen- Ϫ Ϫ define the physiological role of SOCS3 will require analysis of dent experiments, the responsiveness of SOCS3 / CFU-E to Ϫ Ϫ cytokine signaling in placentas of SOCS3 / mice. erythropoietin was identical to that observed for wild-type and Ϫ/Ϫ ϩ/Ϫ Previous studies of SOCS3 mice reported a marked eryth- SOCS3 cells (Fig. 3). Similar results were obtained when erythrocytosis associated with the embryonic lethality (18). From our ropoietin responsiveness of BFU-E was assayed in the presence of own analyses, we were unable to find evidence for excess the synergizing growth factors, stem cell factor and IL-3 (Fig. 3). erythroid cell production. Microscopic examination revealed no erythrocytosis in SOCS3Ϫ/Ϫ embryos examined from E9.5 to Discussion E13.5. Yolk sac erythropoiesis was normal, and normal numbers We have used gene targeting to generate mice unable to produce of erythroblasts, BFU-E, and CFU-E were evident in the fetal SOCS3. Consistent with a previous report (18), we find that livers of SOCS3Ϫ/Ϫ mice at E11.5 and E12.5 (Tables 2 and 3). SOCS3 is essential for survival during embryogenesis. In the Although the SOCS3Ϫ/Ϫ fetal liver was abnormally small at absence of SOCS3, embryos developed apparently normally E12.5, the frequencies of hematopoietic progenitors were nor-

9328 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.161271798 Roberts et al. Downloaded by guest on October 1, 2021 mal, suggesting that the hypocellularity of the liver reflected the those reported previously is unclear. We conclude from our developmental arrest of the embryos rather than a specific analysis that SOCS3 is essential for placental development but hematopoietic defect. The absence of an intrinsic defect in dispensable for normal hematopoiesis in the mouse embryo. SOCS3Ϫ/Ϫ hematopoietic progenitors is consistent with the report of normal hematopoiesis in irradiated adult recipients of We thank Janelle Mighall, Sally Cane, Sandra Mifsud, and Ladina SOCS3Ϫ/Ϫ fetal liver cell transplants (18). Moreover, we could DiRago for technical assistance, Steven Mihajlovic for histology, and find no evidence that erythroid progenitor cells have any greater Kathy Hanzinikolas for animal husbandry. This work was supported by responsiveness to EPO in the absence of SOCS3: both BFU-E the National Health and Medical Research Council, Canberra, the Ϫ/Ϫ Anti-Cancer Council of Victoria, an Australian Government Coopera- and CFU-E from SOCS3 mice exhibit identical EPO quan- tive Research Centres Program Grant, the National Institutes of Health titative responsiveness for colony formation in vitro,inthe (Grant CA22556), the J. D. and L. Harris Trust, the Sylvia and Charles presence or absence of IL-3 or stem cell factor (Fig. 3). The basis Viertal Charitable Foundation, and AMRAD Operations Pty. Ltd., for the discrepancies in erythropoiesis between our mice and Melbourne. A.R. holds a Neil Hamilton Fairley Fellowship (977211).

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