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Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo

Adam H. Hart¹, Lynne Hartley¹, Koula Sourris^1,, Elizabeth S. Stadler^1,, Ruili Li¹, Edouard G. Stanley^1,, Patrick P. L. Tam², Andrew G. Elefanty^1,*, and Lorraine Robb^1,*,

¹ The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Vic 3050, Australia
² Embryology Unit, Childrens’ Medical Research Institute, Wentworthville, NSW 2145, Australia
^* These authors contributed equally to this work
Present address: Centre for Early Human Development, Monash Institute of Reproduction and Development, 27-31 Wright St, Clayton, Vic 3168, Australia

View larger version (61K): [in a new window]   Fig. 1. Targeted mutation of Mixl. (A) Genomic organization of the Mixl1 locus and structure of the targeting vector. Boxes indicate exons, coding sequence is filled, with the homeodomain indicated by lighter shading. A mutant allele (Mixl1–) is produced by replacing exon 1 downstream of the ATG with an EGFP loxP-MC1-neo-loxP cassette. Excision of the floxed MC1neo sequence by Cre-recombinase generates the allele Mixl1neo. Positions of 5' and 3' genomic DNA probes and PCR primers are indicated. The positions of restriction enzymes are shown, together with the sizes of diagnostic restriction fragments (HIII, HindIII; RI, EcoRI). (B) Southern blot analysis of HindIII-digested DNA purified from wild-type, Mixl1+/– and Mixl1+/neo mice, probed with the 3' probe. The 11 kb band corresponds to the wild-type Mixl1 allele. The 4.7 kb band identifies the Mixl1 allele after homologous recombination with the targeting construct. The targeted allele, after CRE-mediated excision of the neomycin resistance cassette, gives rise to a 3.7 kb band. WT, wild type. (C-F) GFP expression in Mixl1+/– (+/–) (C,E) and Mixl1–/– (–/–) (D,F) 7.5 dpc embryos visualized by confocal microscopy. High levels of GFP expression are found in the primitive streak and lateral mesoderm of the Mixl1–/– embryo. By contrast, a lower and more uniform expression of GFP in the mesoderm is observed in the Mixl1+/– 7.5 dpc embryo. (E) Transverse section of a Mixl1+/– 7.5 dpc embryo through the middle level of the primitive streak, showing GFP expression in the mesoderm. (F) Transverse section of a Mixl1–/– embryo reveals GFP expression in the ingressing cells (arrowhead) in the thickened primitive streak (ps) and the nascent mesoderm (lm) located lateral to the primitive streak.

View larger version (44K): [in a new window]   Fig. 2. Mixl1–/– embryos show an accumulation of mesenchymal cells in the thickened primitive streak and adjacent mesoderm. Lateral views of an early-bud stage wild-type (+/+) embryo (A) and a mid-streak stage Mixl1–/– (–/–) (F) embryo. The Mixl1–/– embryo has a thickened primitive streak, lacks a node (n) and shows delay in closure of the amnion (paf, posterior amniotic fold). Transverse sections through wild-type (B-E) and Mixl1–/– (G-J) embryos at the levels indicated in A,F, showing the congregation of cells in the anterior segment of the primitive streak (ps) and adjacent mesoderm (lm) in the mutants. The allantois (al) and chorion (ch) are morphologically similar in the wild type and mutant. Scale bar: 25 µm.

View larger version (61K): [in a new window]   Fig. 3. Early somite stage Mixl1–/– embryos exhibit multiple developmental abnormalities. (A-C) Lateral view of a five-somite wild-type (+/+) embryo (A), and lateral (B) and dorsal (C) views of a three-somite Mixl1–/– (–/–) littermate. The Mixl1–/– embryos do not adopt the lordotic morphology of the wild-type embryo, develop no heart and form a large allantois. The asterisk indicates the midline tissue mass separating the somites (s). (D) A 9.0 dpc Mixl1–/– embryo showing disorganization of the neural folds and the formation of a bulbous caudal protrusion (double asterisks). (E,F) Anterior (E) and lateral (F) views of 8.25 dpc wild-type and Mixl1–/– conceptuses. Note the absence of clearly defined midline structures (ml) in the Mixl1–/– embryo. The silhouette of collapsed head folds of the mutant is outlined by a broken line. (G-M) Histological sections of 8.25 dpc wild-type and Mixl1–/– conceptuses, at planes indicated in F. Note the poorly developed neural folds and foregut portal in the mutant. A compact core of cells (ax, J) is found in the midline in place of the notochord. Abbreviations: allantois (al), blood islands (bi), foregut portal (fg), heart (h), notochord (nc), neural folds (nf) and paraxial mesoderm (pm). Scale bar for histological sections: 50 µm.

View larger version (42K): [in a new window]   Fig. 4. Excessive growth of axial mesoderm and bifurcation of the embryonic axis in 9-9.5 dpc Mixl1–/– embryos. (A) Dorsolateral view of a mutant embryo showing Brachyury (T)-expressing tissues in the bulge (arrow) formed at the caudal end of the embryonic axis and (B) ventral view of the same embryo showing the protrusion derived from the axial tissue mass (arrow). (C-E) Histological sections at the planes indicated in B show (C) T expression in the flat notochord-like structure, (D) the protruding tissues on the ventral side of the embryo (arrow indicates the same structure shown in B) and (E) the bulge that extends dorsally through a split in the neural plate. hf, head fold; al, allantois; nc, notochord-like structure; ne, neuroectoderm; nf, neural fold.

View larger version (93K): [in a new window]   Fig. 5. Brachyury (T) and Nodal expression domains are expanded in Mixl1–/– embryos. (A-D) T is expressed in a wider domain in 7.5 dpc late-bud stage Mixl1–/– (–/–) mutant embryos (B) than in the wild-type (+/+) embryo (A). Transverse sections (C,D), at the levels shown in A,B reveal that T expression in the mutant embryo is extended more laterally from the primitive streak (ps) to encompass the adjacent lateral mesodermal (lm) cells. (E) Lateral view of an early somite stage wild-type embryo, and dorsal (F) and lateral (G) views of the mutant embryo showing the expanded T expression domain in the mutant embryo, which may be accentuated by the foreshortening of the anterior part of the embryo. (H,I) Histological sections of the mutant embryo at planes shown in G, showing the T-expressing cells in the notochord-like structure (nc) under the neural folds (nf), and in the dorsal superficial cells in the primitive streak (ps) and the ventral mesendodermal tissue sheet that resembles the notochordal plate (ncp). (J) Nodal lacZ expression is expanded in the posterior tissues of the late-streak stage Mixl1–/– embryos. In the wild-type 8.5 dpc embryo (K), Nodal lacZ is expressed in the perinodal tissues of the primitive streak (ps) and the left lateral plate mesoderm (lpm). In the mutant embryo (L), which displays small head folds (hf) and an enlarged allantois (al), NodallacZ is expressed strongly in an expanded domain in the anterior segment of the primitive streak (ps) and in the axial tissues immediately anterior to the primitive streak.

View larger version (80K): [in a new window]   Fig. 6. Expression of markers of axial mesendoderm, paraxial mesoderm and cardiac mesoderm is maintained in Mixl1–/– embryos. (A,B) Lateral views of wild-type (+/+) (A) and Mixl1–/– (–/–) (B) 7.5 dpc embryos showing the expanded Foxa2 expression domain in the tissues anterior to the primitive streak. (C-F) Frontal views of early somite stage wild-type (+/+) and Mixl1–/– (D) embryos, and histological sections (E,F) taken at the planes indicated in D. Foxa2 is expressed in the floor plate (fp) and notochord of the wild-type embryo and in the compact tissue mass in the midline of the mutant embryo (arrows in D,F). (G,H) Lateral views of wild-type (G) and Mixl1–/– (H) 7.5 dpc embryos showing that Shh is expressed in a cell cluster near the anterior end of the streak in the mutant embryo, but expression is not extended to the more anterior axial tissues as in the wild-type embryo. At 8.5 dpc (I-L), Shh is expressed only in the ventral part (arrow in L) of the midline axial mass of the mutant embryo. (I,J) Lateral views, (K) anteroventral view, (L) histological section (plane indicated in J). (M,N) Twist expression is maintained in Mixl1–/– embryos (N) in the mesoderm at the base of the allantoic bud and the presumptive lateral plate mesoderm, similar to that in the early somite stage wild-type embryos (M). However, unlike wild-type embryos, Twist expression is absent from the mesoderm underneath the rudimentary cranial neural plate. (N) Dorsal view showing Twist expression in the lateral mesoderm (arrows). (O,P) Expression of the Nkx2.5 in the heart tube of wild-type embryos (O, lateral view) and in a poorly defined crescent-shaped patch of cells in Mixl1–/– embryos (P, anterior view), but no heart is formed. (Q,R) Ventral views of early-somite stage wild-type and Mixl1–/– embryos showing Six3 expression in the prospective ventral forebrain.

View larger version (81K): [in a new window]   Fig. 7. Endoderm is specified in Mixl1–/– embryos. (A-D) Expression of Afp in 7.5 dpc wild-type (+/+) (A,B) and Mixl1–/– (–/–) embryos (C,D). Lateral (A,C) and anterior views (B,D) showing Afp expression in the visceral endoderm of the vitelline yolk sac, but not in the definitive endoderm (de). The Afp expression domain seems to extend more distally in the mutant embryo. (E,F) Cer1 is expressed in anterior definitive endoderm of the late bud/early head fold stage Mixl1–/– embryo (F) and of the early bud stage wild-type embryo (E). (G,H) Sox17 is expressed in the anterior definitive endoderm of the early-bud wild type and Mixl1–/– embryo (arrows).

View larger version (84K): [in a new window]   Fig. 8. Expression of Cer1 and Sox17 in the presumptive foregut of Mixl1–/– embryos. (A-C) Lateral (A,B) and dorsal (C) views of Cer1 expression in wild-type (+/+) (A) and Mixl1–/– (–/–) embryos (B,C, same specimen). Expression is seen in the two newly formed somites and anterior presomitic mesoderm in both wild-type and mutant embryos, but ectopic expression (arrows) is found in the endoderm of the presumptive foregut of the mutant embryo. (D) Histological section at the plane shown in C shows Cer1 expression in the rudimentary foregut pocket. Lateral (E), anterior (F) and dorsal (G) views of early-somite stage wild type (E) and Mixl1–/– embryos (F,G) showing Sox17 expression in the endoderm of the presumptive foregut (arrows in G,H) but not in the hindgut of the mutant embryo. (H) Transverse section at the plane shown in G.

View larger version (73K): [in a new window]   Fig. 9. Mixl1–/– ES cells (identified by the lack of lacZ activity) display reduced potency to colonize the embryonic endoderm. (A) A chimera with more than 90% contribution of Mixl1–/– ES cells recapitulates the phenotype of Mixl1–/– embryo. (B) A chimera with low Mixl1–/– ES cell contribution shows widespread distribution of the mutant cells in the embryonic tissue (C, plane of sectioning indicated in B), but poor contribution to the endoderm of the foregut (fg) and hindgut (hg). (D) A chimera with high mutant ES cell contribution shows extensive colonization of most embryonic tissues by the mutant cells, but sparse presence in the foregut endoderm (E) and complete absence from the hindgut endoderm (F). Planes of sectioning for E,F are indicated in D.