QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fujiwara, T. Articles by Hogan, B. L. M. Search for Related Content PubMed PubMed Citation Articles by Fujiwara, T. Articles by Hogan, B. L. M.

Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

Takeshi Fujiwara^1,*, Deborah B. Dehart^2,3, Kathleen K. Sulik^2,3 and Brigid L. M. Hogan^1,

¹ Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical Center, 1161 21st Ave. South, Nashville, TN 37232-2175
² Department of Cell and Developmental Biology, The University of North Carolina, Chapel Hill, NC 27599-7178, USA
³ Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, NC 27599-7178, USA
^* Present address: Dana-Farber Cancer Institute, Department of Pediatric Oncology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

View larger version (102K): [in a new window]   Fig. 1. Gene expression and node morphology in Bmp4tm1 mutants. (A) Posteroventral views of five-somite stage wild-type (parts a,d,g,j) and four- to five-somite stage Bmp4tm1 mutants (all other panels). (a) Nodal expression in the node periphery and left LPM. (b,c) Nodal expression is either patchy or greatly reduced in the node (arrowheads) and absent in the LPM. (d-f) Nodal (purple) and Foxf1 (blue) expression by double whole-mount in situ hybridization. (d) Nodal expressed in the left LPM (arrow), and Foxf1 bilaterally in the LPM (arrowheads) (n=5/5). (e,f) These show the same five-somite stage embryo. There is no Nodal expression in either the node or the LPM (e), but, after second hybridization, Foxf1 transcripts are seen bilaterally in the LPM (f, arrowheads; n=3/3). (g-l) Scanning electron micrograph (SEM) of the embryos in a-c after in situ hybridization. (j-l) Higher magnification of the node (yellow brackets). (g,j) Wild-type embryo with well-formed primitive streak and a concave node. (h,i,k,l) Mutant embryos show abnormal bulge (h,i; arrows) in the posterior region and flat node morphology with irregular periphery. (B) Node morphology. SEM of embryos at the six somite (a-h) and presomite stage (i). (a-d,j) Wild-type embryos; all other panels, Bmp4tm1 mutants. (b,f,d,h) Higher magnification of the node (b,f), indicated by yellow brackets in a,e, and the amniotic cavity (d,h), indicated by white brackets in c,g. (a,b,e,f) Ventral views of wild-type embryo with well-formed primitive streak (a) and a concave node (b), and a mutant embryo with abnormal posterior bulge (e, arrowhead) and flat node morphology with irregular periphery (f). (c,d,g,h) Embryos were cut through the plane (black line) indicated in a,e and viewed posteriorly (c) or dorsoposteriorly (g). (c,d) Amnion extends over the amniotic cavity in wild-type embryo; (g,h) mesodermal cells (arrows) abnormally fill up the amniotic cavity and extend to the surface of the ectoderm in mutant embryo; (i) mutant embryo with abnormal large endoderm-like cells inside the node (arrows). (j,k) Sagittal sections of three- to four-somite stage embryos showing Evx1 expression (blue). Anterior is towards the left. Note abnormal accumulation of Evx1-positive mesodermal cells in the posterior of mutant embryo (k, arrow) compared with wild-type (j, arrow). (C) Gene expression. (a-h) Lefty2 (a-c) and cryptic (d-h) expression by whole-mount in situ hybridization. (a,c) Lefty2 in the left LPM (arrowheads) of the 3- to 4-somite stage wild type (a, n=2/2) and some mutants (c, n=3/9). (b) Other mutants show no Lefty2 expression at the three- to four-somite-stage (n=6/9). (d-f) Cryptic is bilaterally expressed in the LPM and node (arrowheads) of wild-type (d, n=2/2), but not of mutants at the two- to four- (e; n=3/3) and five- to six- (f; n=4/4) somite stage. (g,h) Cryptic is expressed in the LPM and node (arrowheads) of wild type (g, n=3/3), whereas expression in the node (arrowhead) is reduced in mutants (h, n=4/4) at the headfold to one-somite stage. (i,j,l,m) ß-galactosidase staining to detect Bmp4lacZ reporter expression in six-somite stage Bmp4lacZ/+ heterozygous (i,j) and seven-somite stage Bmp4lacZ/lacZ homozygous-null (l,m) embryos. (j,m) Transverse sections in the plane indicated (yellow bar) in i and l. Arrows indicate the Bmp4lacZ-positive LPM. Note that no Bmp4 expression is detected in either the primitive streak (bracket), or the node (arrowhead) in i. (k,n) Foxf1 expression by whole-mount in situ hybridization. Foxf1 is expressed bilaterally in the LPM (arrowheads) in both two- and six-somite stage wild type (k, n=4/4) and mutants at the same stage (n, n=8/8). a, allantois; ac, amniotic cavity; am, amnion; e, ectoderm; nc, notochord; nf, neural fold; s, somite stage; ys, yolk sac. Scale bars: in A, 10 µm for parts g-i; in B, 100 µm for parts a,c,e,g,j,k and 10 µm for parts b,d,f,h,i; in C, 100 µm for parts i,j,l,m.

View larger version (108K): [in a new window]   Fig. 2. Reduced expression of Fgfs and Dte and maintenance of midline tissues in Bmp4tm1 mutants. Posteroventral views of wild-type embryos (A,D,G,I,K), and mutants (all other panels). (A-C) Fgf8 is expressed in the primitive streak of wild-type (A), but strongly reduced (n=5/6) in the posterior (arrowheads) of mutant embryos (B,C), at the five- to seven-somite stage. (D-F) Fgf4 is expressed in the primitive streak of wild-type (D) and one mutant (arrowhead; n=1/4) (F), but strongly reduced (arrowhead; n=3/4) in posterior of mutant embryos (E), at the one- to two-somite stage. (G-J) Shh is expressed in the node and midline of wild type at the headfold (G) to four-somite (I) stage, and mutant embryos at the headfold (H) to six-somite (J) stage (n=10/11). (K-M) Asymmetric Dte expression, enriched on right side of the node (arrowheads), in the wild-type (K) and one mutant (M; n=1/5), but low and patchy in the node (arrowheads) of other mutant embryos (L; n=4/5), at the four- to six-somite stage.

View larger version (93K): [in a new window]   Fig. 3. Abnormal heart development in Bmp4tm1 mutants. (A,D) Ventral views of nine-somite-stage wild-type (upper panels) and Bmp4tm1 mutant (bottom panels) stained with anti-VCAM-1 antibody. (B,C,E,F) Transverse section of embryos counterstained with Eosin in planes indicated by white (B,E) and yellow (C,F) bars in A,D. VCAM1 is expressed in the myocardium mostly in the primitive ventricle (purple) of both the wild-type and Bmp4tm1 mutant, although levels are reduced in the latter. (A) Rightward looping of the heart (arrow). (D) Failure of looping, with midline heart tube (arrow). (B) Anterior heart showing clear boundary of the primitive ventricle to the left of the embryo and the bulbus cordis to the right. (C) Clear distinction between primitive ventricle and atrial chamber in the posterior heart. (E,F) Abnormal positioning of the primitive ventricle and bulbus cordis caused by looping failure. ac, atrial chamber; bc, bulbus cordis; pv, primitive ventricle. Scale bars: 100 µm for B,C,E,F.

View larger version (94K): [in a new window]   Fig. 4. Development of Bmp4lacZ/tm1 ES cellxwild-type tetraploid chimeras. (A-F) SEM (A-D) and Shh expression (E,F) by whole-mount in situ hybridization. Posteroventral views of wild-type embryos (A,E) and tetraploid chimeras (B,F), and higher magnification of the node (C,D) indicated by brackets in A and B. (A-D) Concave node morphology (C,D), well-formed primitive streak (B, arrow), and well-defined notochord in the presomite-stage wild-type embryo (A,C) and tetraploid chimera (B,D). (E,F) Shh expressed in the node (arrowheads) and midline of the five- to six-somite-stage wild-type embryo (E) and tetraploid chimera (F). (G,J) Ventral view of the heart after staining with anti-VCAM1 antibody. Nine-somite stage wild-type embryo shows normal rightward looping (G) and ten-somite stage tetraploid chimera showing abnormal midline heart tube (J, n=4/4). (H,I,K,L) Transverse sections stained with Hematoxylin and Eosin taken at levels indicated in white for H,K, and yellow for I,L. ac, atrial chamber; bc, bulbus cordis; nc, notochord; pv, primitive ventricle. Scale bars: 100 µm in A,B,H,I,K,L; 10 µm in C,D.

View larger version (109K): [in a new window]   Fig. 5. Gene expression in tetraploid chimeras. (A-C) In wild-type embryos, Nodal is expressed in the node at the late-streak stage (A, bracket; lateral view), symmetrically in the node at presomite stage (B, bracket; posteroventral view), and asymmetrically, higher on the left, in the node and left LPM at the five-somite stage (C, bracket and arrow). (D-F) Tetraploid chimeras show no Nodal expression in the node (brackets) at all three stages (D-F; n=2/2 each) or in the left LPM (n=2/2) at the five-somite stage (F). (G,H) Fgf8 is expressed in the primitive streak (arrow) of the seven-somite stage wild-type embryos (G), and at a moderately lower level (arrow) in the 6-somite stage tetraploid chimeras (H; n=2/2). LS, late streak.

View larger version (87K): [in a new window]   Fig. 6. Effect of exogenous noggin on LR patterning. Whole-mount in situ hybridization of wild-type embryos, except for B (which detects NoglacZ reporter). (A-F) Posteroventral views. (A) Bmp2 is expressed bilaterally in the LPM (arrowheads) from the headfold to four-somite stage. (B) Noggin is expressed in the node (bracket) and the notochord (arrow). (C,D) Nodal expression in three- to five-somite stage embryos cultured with (D) or without (C) recombinant noggin protein. (C) In control embryos, Nodal is expressed in both the left LPM (arrowhead) and node (n=7/9). (D) No Nodal expression was observed in the LPM when embryos were cultured with noggin (n=17/17). However, Nodal expression (*) was detected in the node (n=17/17). (E,F) Cryptic and Shh expression in noggin-treated embryos at the three- to five-somite stage. Cryptic is expressed bilaterally in the LPM and midline (E, arrowheads; n=16/16), and Shh is expressed in the node and midline (F, arrowhead; n=7/7).

View larger version (36K): [in a new window]   Fig. 7. Model for independent functions of Bmp4 in node and primitive streak morphogenesis, and LR asymmetry. (A) Pre- to early-streak stage; lateral view. Bmp4 produced in the ExE patterns the proximodistal axis of the epiblast and thereby plays a role in specifying cell fates along the anteroposterior axis of the future primitive streak. The most proximal cells (black circle) will give rise (yellow arrows) to the precursors of allantois, PGCs and ExM, which will translocate posteriorly (blue/green striped arrow), while the more distal precursors of embryonic mesoderm move anteriorly and laterally (red arrows). Fgf8 is expressed in the posteroproximal epiblast. (B) The presomite stage; ventral view. Progenitors of the node are thought to lie in the mid-anterior primitive streak and move anteriorly to be incorporated into the node (red arrows). This morphogenetic process is dependent on Bmp4 expression in the ExE (see A) and Fgf8 in the primitive streak (Sun et al., 1999). We speculate that Bmp4 produced in the ExM and/or posterior mesoderm (PM) positively regulates an unknown gene X that initiates the expression of Nodal (broken black arrows) in the periphery of the node (purple). Nodal flow (left-pointing black arrows) generates enrichment of left-side Nodal expression in the periphery of the node. The future Nodal expression in the left LPM is possibly initiated by Nodal protein diffusing from the node (broken blue arrow). (C) The two- to eight-somite stage. Bmp4 in the LPM is required for Nodal expression in the LPM (broken blue arrow). Nodal produced in the node may maintain the expression of cryptic that permits the expression of left-side determinant genes such as Nodal in the left LPM (broken black arrows).