Angiomotin Regulates Visceral Endoderm Movements During Mouse Embryogenesis

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provided by Elsevier - Publisher Connector Current Biology, Vol. 13, 613–617, April 1, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00204-5 Angiomotin Regulates Visceral Endoderm Movements during Mouse Embryogenesis

Akihiko Shimono1,2 and Richard R. Behringer1,* frame of 1123 amino acids (Figure 1A). A human ortholog 1Department of Molecular Genetics that shares 84% amino acid identity was also identified University of Texas in the database. Both mouse and human SII6 protein M. D. Anderson Cancer Center have centrally located coiled-coil domains but no other Houston, Texas 77030 known protein motifs. A human cDNA encoding a shorter 2 Department of Developmental Biology isoform of SII6 was recently reported as angiomotin [8], National Institute for Basic Biology an Angiostatin binding protein (Figure 1A). Examination Myodaiji-cho of the Celera database mapped SII6 to a region of the Okazaki 444-8585 mouse X chromosome that is orthologous to human Japan Xq21.33-Xp11. We have renamed mouse SII6 as angiomotin. amot expression was initially detected in the AVE of early streak stage embryos at E6.5 (Figure 1B). Anterior Summary expression is maintained throughout gastrulation from early streak to late bud stages (Figures 1B–1D; [6]). At the In pregastrula stage mouse embryos, visceral endo- late streak to no-allantoic bud stage, amot expression is derm (VE) migrates from a distal to anterior position detected in the anterior and posterior embryonic and to initiate anterior identity in the adjacent epiblast [1, extra-embryonic visceral endoderm, anterior definitive 2]. This anterior visceral endoderm (AVE) is then dis- endoderm, and allantois (Figures 1C–1F). placed away from the epiblast by the definitive endo- To determine the role of amot during early mouse derm to become associated with the extra-embryonic embryonic development, amot mutant mice were gener- ectoderm and subsequently contributes to the yolk ated by gene targeting in mouse embryonic stem (ES) sac. Little is known about the molecules that regulate cells (see Figure S1 in the Supplemental Data available this proximal displacement. Here we describe a role with this article online). A gene-targeting vector was Ј for mouse angiomotin (amot) in VE movements. amot designed to delete the 5 two-thirds of the second cod- expression is initially detected in the AVE and subse- ing exon, including the splice acceptor site, whose dele- quently in the VE associated with the extra-embryonic tion should result in a frameshift and premature transla- ectoderm. Most amot mutant mice die soon after gas- tion termination. Potential transcripts generated from trulation with distinct furrows of VE located at the the mutant allele were analyzed by RT-PCR and DNA junction of the embryonic and extra-embryonic re- sequencing. Sequence analysis of the RT-PCR products gions. Mutant analysis suggests that VE accumulation verified the predicted frameshift and premature stop codon. Thus, the mutant allele cannot generate wild- in these furrows is caused by defects in cell migration type Amot protein. into proximal extra-embryonic regions, although dis- The genotypes of progeny weaned from crosses of tal-to-anterior movements associated with the epi- F1 heterozygous mutant females (Xamot X) with wild-type blast, definitive endoderm formation, and anterior males on a B6129 mixed genetic background demon- specification of the epiblast appear to be normal. strated that hemizygous mutant males (Xamot Y) were These results suggest that amot acts within subre- obtained at lower-than-predicted Mendelian frequen- gions of the VE to regulate morphogenetic movements cies, suggesting that half of the Xamot Y mutants probably that are required for embryo viability. died before birth (Table S1A). In addition, the genotypes of progeny weaned from crosses of Xamot X females with the surviving Xamot Y males confirmed that the embryonic Results and Discussion lethality was linked to the transmission of the mutant allele, with approximately 70% of the mutant females A differential screen between the anterior mesendoderm (Xamot Xamot) and (Xamot Y) males dying before birth. These of single wild-type and lhx1 (also called lim1) mutant results indicate that the penetrance of the amot mutant gastrula stage embryos yielded a cDNA clone (initially phenotype is sensitive to genetic background. designated SII6) that was expressed in the AVE of gas- Timed matings were established to investigate the em- trula stage wild-type embryos in a pattern that was simi- bryonic lethality. In crosses between surviving Xamot Xamot lar to lhx1 but absent or significantly reduced in lhx1 females and Xamot Y males examined at E7.5, only 38% of null embryos [3–7]. Screens of embryonic day 7.5 (E7.5) the decidua contained mutant embryos; the remainder cDNA libraries and 5’ RACE produced a cDNA of approx- contained hemorrhagic embryos (Table S1B). Thus, a imately 7.7 kb. No cDNA isoforms generated by alterna- significant number of mutant embryos fail to develop tive splicing or differential promoter usage were identi- around this stage. Morphological analysis revealed that fied in the screens. Sequence analysis of the SII6 cDNA nearly all (approximately 90%) of the mutant embryos identified a 3.4 kb region encoding an open reading recovered at E7.5 from crosses between Xamot Xamot females and Xamot Y males had excessive folds at the cranial limiting furrow (CLF) region that is located at the *Correspondence: [email protected] junction of the anterior epiblast and extra-embryonic Current Biology 614

Figure 1. Amino Acid Sequence of Amot Protein and Expression during Gastrulation (A) Predicted Amot amino acid sequence and comparison between mouse and human. Human AMOT was predicted by database analysis. A red star indicates methionine of human AMOT; orange lines indicate predicted coiled-coil domains. (B–F) amot expression during gastrulation. (B) AVE expression in pre- and early streak embryos. The scale bar represents 250 ␮m for (B) and (C). (C) Expression in the posterior VE and VE associated with the extra-embryonic ectoderm at the no-allantoic bud or early bud stage. (D) At the allantoic bud stage, expression is detected in the VE associated with the extra-embryonic ectoderm and also in a subregion of the anterior definitive endoderm. The scale bar represents 100 ␮m. (E and F) In situ hybridization of transverse sections of a no-allantoic bud stage embryo reveals amot expression in the anterior mesendoderm (E) and also in VE (between arrows in [F]) and allantoic mesoderm. The scale bar represents 100 ␮m in (E). Abbreviations are as follows: a, anterior; p, posterior; clf, cranial limiting furrow; and al, allantois. ectoderm (Table S1 and Figure 2B). The severity of this elevated in the CLF of amot mutant embryos relative to defect was variable (Figures 2 and S2; Table S1). How- controls (Figures 2K–2N). ever, all of the mutant embryos examined by histology Marker gene expression suggests that VE accumu- at E7.5 displayed an abnormal accumulation of cells in lates at the CLF of amot mutant embryos. In wild-type extra-embryonic tissues at the CLF (n ϭ 14) (Figure 2F late-streak to no-allantoic bud stage embryos, gata4 and Table S1B). In contrast, essentially all (98%) E7.5 and ␣-fetoprotein (afp) are expressed in the proximal wild-type embryos on a B6129 genetic background were VE associated with the epiblast and the VE associated morphologically normal (Table S1B). with the extra-embryonic ectoderm [9, 10]. afp expres- The embryos that displayed the mutant phenotype in sion is relatively restricted around the junction of the the CLF were always Xamot Xamot or Xamot Y mutants (re- embryonic and extra-embryonic regions (Figure S2). In ferred to hereafter as amot mutants). To gain insights amot mutant late-streak to no-allantoic bud stage em- into this defect, we examined tissue morphology in wild- bryos, the endoderm layer in the excessive folds ex- type, Xamot X, and amot mutant embryos at primitive presses gata4 and afp. Moreover, afp expression is re- streak stages. In wild-type embryos, the CLF consists stricted in the anterior region with the excessive folds of two cell layers at the anterior junction of the epiblast and in the posterior region, suggesting that a subpopula- and extra-embryonic ectoderm. The outer cell layer dis- tion of VE accumulated in the anterior region and also plays the typical morphology of visceral endoderm. The occasionally in the posterior region (Figures S2D and inner cell layer is likely to be definitive endoderm. In S2DЈ). These observations suggest an abnormality in amot mutant embryos, the CLF region consists of more the migration of amot mutant VE. To directly address than two cell layers. Moreover, the structure of the CLF this, we labeled the VE at the embryonic/extra-embry- is disorganized, with an accumulation of cells (Figures onic junction of wild-type and amot mutant late-streak 2C–2F). Histological analysis of mutant embryos indi- stage embryos with the lipophilic dye DiI and cultured cates that the accumulated CLF-region cells associated it in vitro for 10 hr (Figure 3). In wild-type embryos the with the unusual folding are visceral endoderm (Figures DiI-labeled VE cells migrated into proximal and lateral 2G–2J). This cell accumulation is apparently not due to regions (Figures 3A and 3C). Also, although distal-to- increased cell proliferation because BrdU incorporation proximal migration of DiI-labeled VE cells was observed assays indicated that cell proliferation ratios were not in the amot mutants, the lateral migration of DiI-labeled Brief Communication 615

Figure 2. Morphological Abnormalities in amot Mutant Embryos (A and B) Gross morphology. Arrows indicate CLF in a wild-type embryo (A) and “excess furrows” in an amot mutant (B). (C–F) Midsagittal sections of late streak stage wild-type (C) and mutant (E) embryos stained with haematoxylin and eosin (H&E). Regions of higher magnification of the wild-type (D) and amot mutant (F) are indicated in (C) and (E). The structure in the CLF is disorganized in the mutant embryo (asterisk). Arrows indicate “excess furrows.” The scale bar represents 100 ␮m in (C) and (E) and 25 ␮m in (D) and (F). (G–J) Cross-sections in the extra-embryonic region of late streak stage wild-type (G and I) and amot mutant (H and J) embryos stained with H&E. Regions of higher magnification of the wild type (I) and amot mutant (J) are indicated in (G) and (H). The scale bar represents 100 ␮m in (G) and (H) and 50 ␮m in (I) and (J). (K–N) Sagittal sections of late streak stage wild-type (G) and amot mutant (I) embryos visualized for the incorporation of BrdU into nuclei (brown). Regions of higher magnification of the wild type (H) and amot mutant (J) are indicated in (G) and (I). Unlabeled nuclei appear blue after counterstaining with haematoxylin. White arrows indicate “excess furrows.” Abbreviations are as follows: a, anterior; p, posterior; clf, cranial limiting furrow; ee, embryonic ectoderm; ve, visceral endoderm; and me, mesoderm. The scale bar represents 100 ␮m in (G) and (I) and 25 ␮m in (H) and (J).

VE cells was greatly reduced (Figures 3B and 3D). Thus, of most of the amot mutants may be caused by the VE cell migration is abnormal in amot mutant embryos. morphological abnormalities in the VE within the embry- In addition to showing morphological defects, the onic/extra-embryonic region and/or a VE-mediated nu- amot mutant embryos have a developmental delay. The tritional insufficiency and subsequent block in yolk sac gross morphology of embryos within the same litters formation. Interestingly, in humans a shorter isoform of from crosses between Xamot X females and Xamot Y males angiomotin that can bind Angiostatin has been identi- was compared. At E7.5, amot mutant embryos are usu- fied. Angiostatin is a circulating proteolytic cleavage ally delayed by approximately 6 hr and sometimes up product of plasminogen that acts as an inhibitor of angi- to 12 hr or more at E8.0 (Table S1B and Figures 4A–4F). ogenesis [8]. Angiostatin has been shown to inhibit en- Interestingly, the distal VE in amot mutant embryos ap- dothelial cell migration, and angiomotin appears to me- pears to migrate anteriorly during the early streak stage diate the cell migration-regulating activity of Angiostatin. because the expression of cer1 and lhx1, both markers In addition, we have isolated a plasminogen cDNA frag- of the AVE, are detected in the anterior region of E7.5 ment from E7.5 mouse embryos. The abnormalities in and 8.0 amot mutant embryos (Figures 4B, 4C, 4E, and the proximal VE of amot mutants suggest that Angio- 4F) [5, 11]. AVE specification of anterior fates in the statin-mediated regulation of cell migration may not be embryonic ectoderm of amot mutant embryos also oc- limited to endothelial cells. Finally, our findings suggest curs as judged by otx2 and six3 expression in delayed that there are independent mechanisms that operate amot mutant embryos recovered at E8.5 (Figures 4G–4J; within subregions of the VE to regulate morphogenetic [12–14]). movements essential for early mouse development. In the mouse, amot is expressed in a dynamic, region- specific pattern in the VE just before and during gastrula- Supplemental Data tion. Our findings suggest that amot is required in the Supplemental Data including the Experimental Proce- AVE for movement away from the epiblast and associa- dures, one Supplemental Table, and two Supplemental tion with the proximally located extra-embryonic ecto- Figures are available at http://images.cellpress.com/ derm. The developmental delay and embryonic lethality supmat/supmatin.htm. Table S1 documents the embry- Current Biology 616

References

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Acknowledgments

We thank N. Jenkins for initial gene mapping information, K. Mahon for the mouse embryo cDNA library, A. Bradley for AB1 ES and SNL 76/7 STO cell lines, T. Yagi for the pMCDTpA plasmid, H. Mizusaki, E. Jones, S.-L. Ang, and P. Gruss for the gata4, ␣-fetoprotein, otx2, and six3 probes, respectively. We also thank K. Morohashi (National Institute for Basic Biology, Okazaki, Japan) for sharing laboratory space with A.S, and we thank P. Tam for helpful comments on the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (No. 12680725) from the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research on Priority Areas (Nos. 13045053 and 13138203) from the Ministry of Education, Science, Sports, and Culture of Japan to A.S. and by grants from the National Institutes of Health and the Barnts Family to R.R.B.

Received: May 13, 2002 Revised: December 19, 2002 Accepted: February 14, 2003 Published: April 1, 2003 Brief Communication 617

Figure 4. Normal Distal-to-Anterior Migra- tion of VE in amot Mutant Embryos (A–C) cer1 expression in wild-type (A and C) and amot mutant (B) embryos. Gross morphology is compared in the same littermates (A and B). The embryo indicated in (C) is a wild-type embryo at a similar stage (early/ mid-streak) to the amot mutant embryo in (B). White arrows indicate “excess furrows”; a black arrow indicates migrating definitive endoderm. The scale bar represents 100 ␮m. (D–F) lhx1 expression in wild-type (D and F) and amot mutant (E) embryos. Gross morphology is compared in the same littermates (D and E). The embryo indicated in (F) is a wild-type embryo at a stage (mid-streak) similar to that of the amot mutant embryo in (E). White arrows indicate “excess furrow”. The scale bar represents 100 ␮m. (G–J) Anterior fate specification of the epiblast of amot mutant embryos. otx2 expression in wild-type (G) and arrested amot mutant embryo (H) recovered as E8.5. Abbreviations are as follows: a, anterior; and p, posterior. six3 expression in a wild-type (I) and arrested amot mutant embryo (J) recovered at E8.5. The scale bar represents 200 ␮m.