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The Foxh1-dependent autoregulatory enhancer controls the level of Nodal signals in the mouse embryo

Dominic P. Norris^*, Jane Brennan, Elizabeth K. Bikoff and Elizabeth J. Robertson

Department of Molecular and Cellular Biology, Harvard University, The Biological Laboratories, Cambridge, MA 02138, USA
^* Present address: Medical Research Council Mammalian Genetics Unit, Harwell OX11 0RD, UK

View larger version (60K): [in a new window]   Fig. 1. Foxh1 is essential for ASE activity in vivo. (A) The 600 ASE deletion was engineered in the context of both the wild-type and NodalLacZ loci, resulting in the Nodal600 and Nodal600.LacZ alleles, as indicated. The PEE, Node and ASE enhancers are indicated by colored circles, and the exons by black boxes. (B-I) ß-Galactosidase staining patterns in 6.5 dpc embryos with anterior towards the left. (B) Wild-type (WT) embryos express the ASE lacZ transgene in the epiblast (more strongly in the posterior than the anterior) and overlying VE. (C) By contrast, the ASE lacZ transgene is not expressed in Foxh1-deficient embryos. (D) NodalLacZ is expressed in the epiblast and overlying VE of WT embryos. By contrast, in Foxh1-deficient embryos (E), expression is proximally restricted within the epiblast and lost from the overlying VE. (F) A proportion (25%) of Foxh1–/–, NodalLacZ/+ embryos show a more severe phenotype, are rounded in appearance with a uniformly thickened VE, and display ß-galactosidase staining throughout the epiblast but not in the VE. (G) Nodal600.LacZ/+ embryo showing expression confined to the proximal posterior epiblast, and undetectable in the VE. (H) In Foxh1–/– embryos, Nodal600.LacZ expression is similarly restricted to the proximal epiblast but lacks AP asymmetry. (I) A rare Foxh1–/–, Nodal600.LacZ/+ embryo, with a more severe phenotype showing ß-galactosidase staining throughout the epiblast. PEE, posterior epiblast element; ASE, asymmetric element; Node, node-specific enhancer.

View larger version (55K): [in a new window]   Fig. 2. Defective PD to AP rotation in Nodal600/– embryos. Primitive streak (B-D,F-I), AVE (E-I) and extra-embryonic ectoderm (K,J) marker analysis in 6.5 dpc embryos. Wild-type controls are shown above Nodal600/–mutants. Cartoons indicate primitive streak/mesoderm markers in blue and AVE markers in pink. Anterior is towards the left. (A) Nodal600/– embryo. Mesoderm (m) is visible proximal to the epiblast (epi), while the chorion (ch) is proximally displaced. The VE shows a distinct thickening at the distal tip (indicated by *). The embryonic/extra-embryonic boundary is indicated by the arrowhead. (B) Cripto, which is normally localized to the posterior epiblast, is expressed throughout the proximal epiblast. (C) T (brachyury) marks the nascent mesoderm and primitive streak. In Nodal600/– mutant embryos, T is expressed symmetrically in the proximal epiblast and in the extra-embryonic mesoderm. (D) Wnt3, which is normally confined to the posterior proximal epiblast and overlying VE just prior to and following the initiation of gastrulation, is expressed symmetrically throughout the proximal epiblast in Nodal600/– mutant embryos, but is absent from the VE (data not shown). (E) Foxa2 is expressed in the anterior primitive streak (AS) and the AVE. In Nodal600/– mutant embryos, Foxa2 is expressed in distal VE and epiblast. (F) Hex is expressed in the AVE both prior to and following the initiation of gastrulation in wild-type embryos. Hex is also expressed in newly formed definitive endoderm (DE) by mid streak stages. Hex expression in Nodal600/– mutant embryos is restricted to the distal most VE. (G) Lhx1 is expressed in mesoderm emerging from the anterior primitive streak (AS) and in the AVE at gastrulation, but in Nodal600/– mutant embryos expression is present in the proximal epiblast and the distal VE. (H) Goosecoid (Gsc), a putative target of Nodal signaling, is expressed in AVE and anterior primitive streak. In Nodal600/– mutant embryos, Gsc expression is only weakly detected in the proximal epiblast and the distal VE in a proportion of embryos and is undetectable in others. (I) Lefty1 and Lefty2 expression, examined using a common in situ hybridization probe that detects both transcripts, shows that Lefty1 (L1) is expressed in the AVE, and Lefty2 (L2) in nascent mesoderm. In Nodal600/– mutant embryos, Lefty1 expression in the AVE is not detected. Lefty2 expression is detected in the proximal epiblast of mutant embryos. (J) The T box gene eomesodermin (Eomes) is expressed in the chorion (ch) and the posterior epiblast. In Nodal600/– mutant embryos, expression is maintained in the chorion, while epiblast expression is proximal. (K) Expression of Bmp4, which marks extra-embryonic ectoderm, is unaffected in Nodal600/– embryos.

View larger version (62K): [in a new window]   Fig. 3. Disturbed Nodal expression patterns in Nodal600/– embryos. Genotypes are indicated towards the left of each panel. Black boxes indicate functional Nodal alleles and null alleles are shown as white boxes. The blue box represents the lacZ insertion into exon 2 of Nodal. Enhancers are indicated as circles, the PEE in green and the ASE in red. The 600 deletion is indicated by the cross. In all panels, anterior is towards the left. (A) As assessed by ß-gal staining, punctate Nodal600.LacZ expression observed at 5.5 dpc throughout the epiblast becomes restricted to the proximal epiblast by 6.5 dpc and gradually resolves to the posterior. Expression continues in the primitive streak after the onset of gastrulation, but is undetectable in the VE. (B) Nodal expression directly assessed by whole-mount in situ hybridization. In contrast to wild type (WT), expression in Nodal600/– embryos is noticeably reduced, restricted to the proximal epiblast, and fails to show obvious AP asymmetry. Section analysis confirms the loss of expression within the VE (data not shown). (C) Nodal expression in NodalLacZ/600 embryos is proximally restricted. The embryo shows a characteristic constriction at the embryonic/extra-embryonic boundary (arrowhead). The position of the section shown is indicated by the box. ß-Gal staining is clearly detectable in the VE of these embryos (arrows). (D) ß-Gal staining and tissue morphology in Nodal600.LacZ/600 embryos resemble that in C. Arrowhead indicates embryonic/extra-embryonic boundary. However no lacZ expression is detected in the VE. VE, visceral endoderm; m, mesoderm; epi, epiblast.

View larger version (90K): [in a new window]   Fig. 4. Nodal signals are required for definitive endoderm specification. (A-E) Nodal600/– mutant embryos at 7.5 dpc (A,B) and 8.5 dpc (C-E). Hematoxylin and Eosin stained sections of the embryos are shown in the bottom of each panel. Two classes of embryo are apparent by 7.5 dpc (Table 1); those that gastrulate inside the visceral yolk sac (VYS) (A), and those that gastrulate externally (B). Twenty-four hours later, embryos remaining inside the VYS form an AP axis, but show defects in heart looping and anterior truncations (C). Embryos that grow external to the VYS develop an AP axis with somites along the trunk; however, fused somites indicate the midline may be defective in many embryos. The posterior is closely associated with the VYS in approximately half of these embryos (D), and in the others both the anterior and posterior are fully external to the VYS (E). end, endoderm; neur, neurectoderm; PS, primitive streak; som, somite. (F-M) Whole-mount in situ hybridization of 7.5 dpc (F,J) and 8.5 dpc (G-I,K-M) embryos. Anterior views (F,H) and lateral views with anterior towards the left (G,I-M). (F) Sonic hedgehog (Shh) is expressed in the midline anterior definitive endoderm (ADE) of WT embryos. Nodal600/– mutant embryos show highly reduced Shh expression. (G) One day later, Shh expression in the midline extends into the ventral forebrain in the WT embryo (arrow); however, this expression domain is absent in Nodal600/– mutant embryos. The anterior extent of the expression is indicated by the arrowhead. (H) Anterior view of the embryos shown in G, underscoring the absence of ventral forebrain expression and greatly reduced anterior foregut (fg) expression domain in the Nodal600/– mutant embryo. (I) In severely affected Nodal600/– mutant embryos, Shh expression is confined to the posterior midline. The embryo on the right expresses Shh along the length of the midline and in hindgut endoderm (hg). There is no obvious anterior gut endoderm population in either embryo. (J) Foxa2 is expressed in the node, midline and ADE in the WT embryo, but is highly downregulated in Nodal600/– mutant embryos at a similar stage. (K) Later in development, Foxa2 expression in the CNS and ADE extends into the ventral forebrain of the WT embryo. By contrast, Foxa2 expression is restricted to the level of heart (indicated by arrowhead) in Nodal600/– mutants. (L,M) Nodal600/– mutant embryos stained with Otx2 to assess the presence of forebrain/midbrain tissue. A severely affected embryo (L) and an embryo with the less severe phenotype (M) both express Otx2 in a distinct, but highly reduced, anterior domain.

View larger version (62K): [in a new window]   Fig. 5. Normal AP patterning and endoderm specification in Nodal600/600 embryos. (A) Whole-mount analysis of endogenous Nodal mRNA expression in WT embryos at 6.5 dpc. The box indicates the position of the sagittal section shown in B. (B) Nodal expression in the epiblast (epi) and visceral endoderm (VE) is indicated by black arrowheads. (C) Nodal 600/600 mutants show markedly reduced expression within the epiblast. (D) Sagittal section shows loss of expression within the VE (white arrowheads). (E) In WT embryos at 7.5 dpc, Nodal mRNA is confined to the node (n). (F) Nodal 600/600 mutants are morphologically indistinguishable from WT and display robust Nodal expression in the node. (G) Asymmetric Nodal expression in the node and left lateral plate mesoderm (llpm) of WT embryos at 8.5 dpc. (H) Posterior views of embryos shown in G. (I) Transverse section shows asymmetric Nodal expression. (J) Nodal 600/600 mutant embryos strongly express Nodal in the node. A proportion of embryos exhibit a very low level of expression in the left LPM. Posterior view (K) and section (L) of the Nodal 600/600 mutant embryo shown on the right in J, confirming node expression is equivalent on both sides. s, somite. All panels are lateral views with anterior towards left, except H and K, which show posterior views.

View larger version (82K): [in a new window]   Fig. 6. Loss of Lefty gene expression and delayed activation of Pitx2 activation in Nodal600/600 embryos. Whole-mount in situ analysis of Lefty1 (L1) and Lefty2 (L2) (using a common lefty probe; A,B) and Pitx2 (C-H) in Nodal600/600 embryos (B,D,F,H) and wild-type controls (A,C,E,G). Lateral views with anterior towards left, except for A (posterior view). (A) Lefty1 expression on the left side of the prospective floorplate of the neural tube and Lefty2 in the left LPM in wild-type embryos. (B) Lefty1 is not expressed in Nodal600/600 mutants, except in a few midline cells close to the node. Lefty2 is expressed in 30% of mutant embryos but at significantly lower levels (left-hand embryo in B). (C) Pitx2 expression in the head and left lateral plate mesoderm (llpm) from the three-somite stage in WT embryos. (D) Nodal600/600 mutant embryos maintain the anterior expression domain seen in WT embryos, but have a reduced level of expression in the llpm. Note the llpm expression is more posteriorly restricted compared with that in WT. (E) At 8.75 dpc, asymmetric Pitx2 expression in the llpm extends behind the sinoatrial region of the heart (anterior extent of expression indicated by arrowheads). Remnants of the visceral yolk sac (VYS) expressing Pitx2 are seen lateral to the llpm. (F) The anterior extent of Pitx2 expression in the llpm is posteriorly restricted in Nodal600/600 mutant embryos and is absent from tissue lying behind the sinoatrial region of the heart (arrowheads). (G) After embryonic turning, the difference in the anterior extent of llpm expression of Pitx2 is particularly obvious (compare positions of arrowheads in G and H). The expression in the Nodal600/600 mutant embryos (H) is anteriorly restricted by approx. two somite widths compared with the wild type (G). This results in Pitx2 not being expressed behind the sinoatrial region of the heart in the mutants. Aberrant heart looping is also obvious within this mutant embryo, although the direction of embryonic turning is normal. Ant, anterior.

View larger version (82K): [in a new window]   Fig. 7. Nodal600/600 embryos exhibit heart and lung situs defects. In wild-type (WT) embryos at 9.5 dpc (A), the heart loops laterally across the body of the embryo, while in Nodal600/600 mutant embryos (B), the heart loops ventrally. Hearts are visualized by expression of the myocardial marker -cardiac actin. Looping differences are summarized in A’ and B’. (C) In WT 12.5 dpc embryos (left), the lungs branch to give four lobes on the right and one lobe on the left. By contrast, Nodal600/600 mutant embryos (right) have four right lobes and two or three left lobes, indicating partial right isomerism. In WT embryos, the apex of the heart (H) points to the left, while in mutant embryos it is more medially positioned. These differences are summarized in the cartoon in C’. Lung lobes are labeled R1, 2, 3 and 4 to indicate the cranial, middle, caudal and accessory lobes, respectively, and L1, 2 and 3 to indicate the normal left lobe or duplicated cranial lobe (L1), the duplicated middle lobe (L2) and the duplicated caudal lobe (L3). Hearts from WT (D,E) and Nodal600/600 mutant (F,G) embryos collected 12 hours before birth. The thoracic cavity has been dissected away to reveal the heart and the major blood vessels leading from it. The view is from the ventral side of the embryo in D and F, and of the same embryos rotated onto their right sides in E and G. In all panels, rostral is upwards. (D) WT heart. The aorta (ao) emerges from behind the pulmonary artery (pa) and arches to the left. The subclavian and common carotid arteries ascend from the aortic arch. The pulmonary artery emerges form the heart and at this stage of development empties through the ductus arteriosus (indicated by *) into the aorta. The left and right pulmonary arteries that deliver blood to the lungs carry very little blood prior to birth and are not visible in these embryos. (E) View of embryo in D from the left hand side. The pulmonary artery (pa) can be clearly seen to emerge from in front of the aorta (ao). (F) Nodal600/600 mutant embryo. A large aorta (ao) is present in this embryo that divides into four ascending vessels. The pulmonary artery is not visible from this angle lying directly behind aorta. (G) View of embryo in F from the left-hand side. In addition to the aorta, from this angle it is possible to see the pulmonary artery (pa) that empties into the aorta through the ductus arteriosus (indicated by *). (D’-G’) Schematics of D-G, indicting the position of the aortic arch and ascending arteries in red and the pulmonary artery in blue.

View larger version (30K): [in a new window]   Fig. 8. An autoregulatory feedback loop controls dosage dependent Nodal signals responsible for embryonic patterning. Nodal null embryos fail to induce an AVE at 5.5dpc. Cripto-null, and some Nodal600/– and Foxh1–/– embryos induce but fail to rotate the AVE at 6.0 dpc, owing to lowered Nodal signaling. Other Nodal600/–, and Foxh1–/– embryos rotate the AVE but fail to induce the ADE at 6.5 dpc, resulting in reduction of the forebrain at 8.5 dpc. Nodal600/600 embryos have yet higher level of Nodal signaling and correctly induce and rotate the AVE and induce the ADE at 6.5 dpc, yet they fail to establish the LR axis correctly at 8.5 dpc, resulting in defects in heart looping visible at 9.5 dpc.