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First published online March 4, 2005
doi: 10.1242/10.1242/dev.01711

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The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse

Stéphane D. Vincent^1,*, N. Ray Dunn^1,, Roger Sciammas^3,, Miriam Shapiro-Shalef⁴, Mark M. Davis³, Kathryn Calame⁴, Elizabeth K. Bikoff^1,2,,¶ and Elizabeth J. Robertson^1,2,,¶

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Wellcome Trust Center for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
³ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
⁴ Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (17K): [in a new window]   Fig. 2. Generation of loss-of-function Blimp1 mutants by gene targeting. (A) Strategy used to engineer the prdm1BEH allele. Correctly targeted clones were initially identified with the 5' external probe (red) and subsequently confirmed with the 3' external probe (blue). A, ApaI; E, EcoRI; M, MscI; N, NcoI; P, PstI; X, XbaI. (B) Southern blot analysis of EcoRI-digested genomic DNA from individual drug-resistant ES cell clones. The 5' external probe detects 8.2-kb wild-type (WT) and 7.9-kb targeted prdm1BEH alleles. (C) A similar targeting strategy was used to generate the prdm1BAH allele, containing an additional 1-kb deletion immediately 5' to exon 1 (see Materials and methods). The selection cassette is not shown.

View larger version (141K): [in a new window]   Fig. 1. Blimp1 expression in the early mouse embryo. Whole-mount in situ hybridization identifies Blimp1 expression domains at (A) early streak, (B) mid-late streak, (C) late streak, (D) no allantoic bud, (E) early allantoic bud and (F) early headfold stages. (A) In the pre-gastrulation embryo, Blimp1 is expressed in a sub-population of visceral endoderm overlying the proximal epiblast. (B,C) At mid- to late streak stage, Blimp1 is detected in the anterior visceral endoderm (AVE, asterisk), and in the nascent primordial germ cells (PGCs) present in the incipient allantois (arrow). (D,E) At later stages, Blimp1 transcripts mark the prechordal plate and anterior definitive endoderm [labeled collectively as anterior axial mesendoderm (ame)], and PGCs present in the growing proximal posterior extraembryonic mesoderm (arrow). (F) At the early headfold stage (EHF), expression is lost from the midline but persists in the anterior definitive endoderm of the foregut pocket, and within the cluster of individual PGCs at the base of the allantois (arrow). ES, early streak; MS, mid streak; LS, late streak; OB, no allantoic bud; EB, early allantoic bud stages. Embryos are shown anterior to the left.

View larger version (107K): [in a new window]   Fig. 3. Loss of Blimp1 expression causes branchial arch patterning defects. Whole-mount in situ hybridization analysis of Blimp1 expression in wild-type embryos at (A) 7.75, (B) 8.5, (C) 9.5, (F) 10.5 and (G) 11.5 dpc. (A) Blimp1 transcripts are expressed in the anterolateral definitive endoderm, most abundantly in the tissue from which the first branchial arch (ba) will emerge (arrowhead). (B) This expression domain is maintained one day later (arrowhead), when Blimp1 transcripts also appear in the ventral anterior neurectoderm. (C) At 9.5 dpc, Blimp1 is expressed in the ventral forebrain, and in the regions of the first, second and third branchial arches. (D,E) Transverse sections of embryo shown in C showing expression confined to the pharyngeal endoderm of the first branchial arch (arrow in D), while Blimp1 is broadly expressed in the ectoderm, mesoderm and endoderm, more caudally in the region from which the second and third arches arise. From this stage onwards, Blimp1 transcripts are also detected in the foregut (fg) and otic vesicles (ot). (F) At 10.5 dpc, expression is downregulated within the branchial arches, and by 11.5 dpc (G), resolves to a subdomain within the first branchial arch. (H,K) Histological analysis of 9.5 dpc wild-type (+/+; H) and mutant (-/-; K) embryos. (H) Whole-mount view of a 9.5 dpc wild-type embryo and corresponding Hematoxylin-Eosin-stained transverse sections 1 and 2 (levels indicated by dashed lines). Three branchial arches are visible (outlined). (K) In a similarly staged mutant embryo, only the first branchial arch is present (outlined). Defects are restricted to the levels of the second branchial arch (K part 2, compare with H part 2). At this site (see high magnification image in K2'), apoptotic cells with pyknotic nuclei (arrow) accumulate within the mesenchyme (compare with H2'). (I,J,L,M) Fgf8 expression domains at 9.5 and 10.5 dpc. In 9.5-10.5 dpc embryos, Fgf8 is expressed at multiple tissue sites, including the ectoderm and endoderm of the branchial arches, the midbrain-hindbrain junction, the somites, the developing limb buds (arrows) and the tail bud. The branchial arch domain in the region of the second and third arches is largely missing in mutant embryos (compare brackets in I and L). (J,M) At 10.5 dpc, Fgf8 expression is more broadly expressed in the mutant than in the wild type, and extends into the epaxial lip of the rostral dermamyotome (short arrow). Fgf8 expression at all other sites, including the AER of the limb buds (arrow), is unperturbed. ba, branchial arch; ot, otic vesicle; pc, pharyngeal cleft; fg, foregut; tgg, trigeminal ganglia; op, optic vesicle.

View larger version (65K): [in a new window]   Fig. 4. Morphogenesis and tissue patterning are largely unaffected in Blimp1-deficient embryos. Whole-mount in situ hybridization analysis of Blimp1 expression at (A) 9.5, (B,C) 10.5 and (D,E) 11.5 dpc. (A-D) Blimp1 mRNA is strongly expressed throughout the mesenchyme of the emerging forelimb bud at 9.5 dpc (A), but becomes progressively restricted to the apical ectodermal ridge (indicated by the white arrowhead in D) and underlying mesenchyme by 10.5 dpc (B). One day later (D), at 11.5 dpc, expression is confined to the posterior side of the limb bud in a region encompassing the zone of polarizing activiy (ZPA, black arrowhead). At 10.5 dpc (B,C), Blimp1 transcripts are also expressed in the myotomal compartment of the somites, and in the intersegmentary arteries (arrow in C) forming between the somites. At 11.5 dpc (D,E), expression is detected in the endothelial cells of the capillary plexus (arrow in E). Blimp1 is not expressed in the developing heart at any stage examined. (F,G) Histological analysis of wild-type (+/+) and mutant (-/-) embryos fails to reveal differences in overall morphogenesis and patterning of the heart at 10.5 dpc. (F parts 1 and 2, G parts 1 and 2) Hematoxylin-Eosin stained transverse sections of embryos shown in F and G; planes of section indicated by the red dashed lines. ba, branchial arch; he, heart; st, septum transversum; da, dorsal aorta; cv, anterior cardinal vein; uv, umbilical vein; 2baa, second branchial arch artery.

View larger version (75K): [in a new window]   Fig. 5. Widespread hemorrhaging and defective placental development in Blimp1 mutant embryos. (A,C) The heart is still beating in 9.5 dpc mutant embryos, and injection of India ink into the outflow tract outlines the major arterial system. The dorsal aorta is visible in both wild-type (A) and mutant (C) embryos. Compared with wild type, only the first branchial arch artery (1) is visible in the mutant. (G) By 10.5 dpc, Blimp1-deficient embryos show widespread hemorrhaging. Numerous small blood pools form beneath the surface ectoderm and blood accumulates in the caudal dorsal aorta. (B,D,F,H) Hematoxylin-Eosin-stained mid-sagittal sections of wild-type (B,F) and mutant (D,H) placentas at 9.5 and 10.5 dpc. At 9.5 dpc (B,D), all the major tissue components of the wild-type placenta (B) have differentiated correctly. However, at this stage, the mutant placenta (D) displays defects in elaboration of the labyrinthine region; the luminal spaces of the labyrinth are much reduced when compared with the wild type. This defect is further exacerbated 24 hours later (H), when very little villous branching of the fetal blood vessels is apparent. Both maternal and embryonic erythrocytes are present. ebc, embryonic blood cells; mbc, maternal blood cells; stc, spongiotrophoblast; la, labyrinthine; tgc, trophoblast giant cells; ys, yolk sac; al, allantois; cp, chorionic plate; am, amnion; da, dorsal aorta; 1, first branchial arch; 2, second branchial arch; 3, third branchial arch.

View larger version (73K): [in a new window]   Fig. 6. Defective specification of primordial germ cells (PGCs) in Blimp1 mutant embryos. Endogenous alkaline phosphatase (AP) activity and SSEA1 antibody reactivity identifies PGCs. (A) Number of embryos in which AP-positive PGCs could be identified at the early headfold stage (EHF). Genotypes are indicated. Cell counts show that Blimp1 heterozygotes display less than 50% of the wild-type number of PGCs, while less than four AP-positive PGCs were observed in three out of six Blimp1 homozygous mutants. (B) AP staining in wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mutants at the indicated somite (s) stage. Reduced PGC numbers were found in the heterozygotes (arrow) compared with the wild-type siblings, and no migrating PGCs were detected in homozygous mutants embryos. (C) Evaluation of individual SSEA1 cell-surface-antigen-positive cells in the hindgut of wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mutants confirms the absence of migrating PGCs in Blimp1 null embryos, and reveals that significantly fewer SSEA1-positive cells are present in heterozygotes than in controls. Midline staining corresponds to SSEA1 reactivity with the hindgut lumen.

View larger version (11K): [in a new window]   Fig. 7. Linear regression analysis of PGC numbers versus somite numbers in embryos from Blimp1 heterozygous intercrosses. Combined PGC counts from prdm1BEH/+ and prdm1BAH/+ (C57BL/6x129/Sv//Ev) intercrosses. The values in the regression equation y=a+bx, for log PGC number (y) and somite number (x) were a=1.603 and b=0.0587, and a=0.9324 and b=0.0842, for wild type and Blimp1 heterozygotes, respectively. The variances between PGC number and somite number are not significantly different according to the F test (F=2.2x10-22 and F=5.4x10-23 for wild type and heterozygotes, respectively).