Expression of some notochord genes are unaffected in <i>Foxa1</i>;<i>Foxa2</i> double knockouts but the notochord sheath is abnormal.

<p>A–D: E9.5 and E–H: E10.5 whole-mount <i>in situ</i> hybridization for <i>Noto</i>. I–L: E10.5 whole-mount <i>in situ</i> hybridization for <i>T</i> (<i>Brachyury</i>). Both genes are expressed in the posterior notochord and expression extends through the tail of the embryo. <i>Noto</i> expression at E9.5 in single (B,C) and double mutants (D) was indistinguishable from control embryos (A). However, at E10.5, <i>Noto</i> was barely detectable in the tail of double mutants (H). I–L: <i>T (Brachyury)</i> expression at E10.5. There was no detectable differences in <i>T</i> expression in control (I), single mutant (J,K) and double mutant embryos (L). E11.5 alcian blue and nuclear fast red staining of the notochordal sheath at the forelimb level (M–P). Scale bars are 10 µm. Adjacent or near-adjacent sections of the notochord are pictured in the inset of M–P and show <i>T (Brachyury) in situ</i> hybridization of the notochord. At the forelimb level, the notochord sheath is clearly visible as a thin blue ring around the notochord in control (M) and single mutant (N,O) embryos (red arrows). In double mutants, the notochord is visible but the sheath is barely apparent (P, red arrow).</p

<em>Foxa1</em> and <em>Foxa2</em> Are Required for Formation of the Intervertebral Discs

<div><p>The intervertebral disc (IVD) is composed of 3 main structures, the collagenous annulus fibrosus (AF), which surrounds the gel-like nucleus pulposus (NP), and hyaline cartilage endplates, which are attached to the vertebral bodies. An IVD is located between each vertebral body. Degeneration of the IVD is thought to be a major cause of back pain, a potentially chronic condition for which there exist few effective treatments. The NP forms from the embryonic notochord. <em>Foxa1</em> and <em>Foxa2</em>, transcription factors in the forkhead box family, are expressed early during notochord development. However, embryonic lethality and the absence of the notochord in <em>Foxa2</em> null mice have precluded the study of potential roles these genes may play during IVD formation. Using a conditional <em>Foxa2</em> allele in conjunction with a tamoxifen-inducible <em>Cre</em> allele (<em>ShhcreER^T2</em>), we removed <em>Foxa2</em> from the notochord of E7.5 mice null for <em>Foxa1</em>. <em>Foxa1^−/−</em>;<em>Foxa2^c/c</em>;<em>ShhcreER^T2</em> double mutant animals had a severely deformed nucleus pulposus, an increase in cell death in the tail, decreased hedgehog signaling, defects in the notochord sheath, and aberrant dorsal-ventral patterning of the neural tube. Embryos lacking only <em>Foxa1</em> or <em>Foxa2</em> from the notochord were indistinguishable from control animals, demonstrating a functional redundancy for these genes in IVD formation. In addition, we provide <em>in vivo</em> genetic evidence that <em>Foxa</em> genes are required for activation of <em>Shh</em> in the notochord.</p> </div

<i>Foxa1</i>;<i>Foxa2</i> double mutants have severe defects in intervertebral disc formation.

<p>A–H: Alcian blue and picrosirius red staining of disc sections. Scale bar is 200 µm. Thoracic sections at E19.5 show a normal sized nucleus pulposus (NP) surrounded by a red-stained annulus fibrosus (AF) in control (A) <i>Foxa1</i> null (B) and <i>Foxa2</i> notochord knockout (C) embryos. Asterisk in <i>Foxa1^−/−</i>;<i>Foxa2^c/c</i> column denotes that embryo was E17.5. Disc morphology was also normal at the lumbar region in these mice (E, F, G). In <i>Foxa1</i>;<i>Foxa2</i> double mutants (D and H) nuclei pulposi were abnormal. Nuclei pulposi were observed to be much smaller than nuclei pulposi present in control or individual single mutants. I and J: A fatemap of notochord cells using the <i>R26R</i> reporter allele demonstrated proper nuclei pulposi formation in control (I) and <i>Foxa2</i> notochord knockouts (J). In contrast, <i>Foxa1</i>;<i>Foxa2</i> double mutants (K) had severely deformed nuclei pulposi and blue cells were dispersed. Defects were more severe in the posterior. (I) is an embryo of the genotype <i>Shhgfpcre</i>;<i>R26R</i>. White asterisk marks dorsal root ganglia. The <i>Shhgfpcre</i> allele is constitutively active, therefore all <i>Shh</i>-expressing cells, including dorsal root ganglia, and their descendants are <i>LacZ</i>-positive. All LacZ positive cells in J and K are derived exclusively from the notochord <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055528#pone.0055528-Choi1" target="_blank">[3]</a>. In our mating it was not possible to obtain a <i>Foxa1^−/−</i> notochord fatemap without also removing the floxed <i>Foxa2</i> allele, therefore the <i>Foxa1^−/−</i> fatemap is not shown (N/A = not applicable). L, M and N: Sections of LacZ-stained tissue at the lumbar level. Inset in N shows an additional section outside of the plane of the NP; numerous cells are scattered throughout the disc region and vertebral bodies. L–M: Scale bar is 100 µM. Arrow in L denotes a notochord remnant, which is a notochord cells that does not reside in the NP.</p

Cell Death is increased in the tail of E11.5 but not E10.5 <i>Foxa1</i>;<i>Foxa2</i> double mutant embryos.

<p>Cell death was assayed using LysoTracker on live embryos at E10.5 and E11.5. A–D. Control (A), <i>Foxa1</i> null (B), <i>Foxa2</i> notochord knockouts (C), and <i>Foxa1</i>;<i>Foxa2</i> double knockout embryos (D) had similar amounts of cell death at E10.5. Inset is a brightfield image of an E10.5 embryo shown for reference. Scale bar for the LysoTracker is 1 mm. E–H: At E11.5, control (E and I), <i>Foxa1</i> null (F, J) and <i>Foxa2</i> notochord knockout (G and K) embryos had LysoTracker positive cells in the somites, dorsal tail, and end of the tail but were indistinguishable from one another. In contrast, double mutant embryos (H and L) had numerous LysoTracker positive cells in the somites and midline of the tail (white arrows). Images I–L are zoomed in areas denoted by the boxes in E–H. The insets in E–H are brightfield images of E11.5 embryos. The scale bar for the LysoTracker images in E–H is 1 mm.</p

Hedgehog signaling is decreased in <i>Foxa1</i>;<i>Foxa2</i> double knockouts.

<p>A–H: <i>Shh</i> whole-mount RNA <i>in situ</i> hybridization. At E9.5, <i>Shh</i> mRNA is found in the brain, midline, and hindgut of control (A) <i>Foxa1</i> null (B) and <i>Foxa2</i> knockout embryos (C). In <i>Foxa1</i>;<i>Foxa2</i> double mutants, midline expression terminates at the posterior end of the embryo (D, arrow) while it remains in control and single knockout embryos (A–C, arrows). At E10.5, <i>Shh</i> mRNA was robustly expressed in the posterior limbs and midline of control and single knockout embryos (E, F, and G). In <i>Foxa1</i>;<i>Foxa2</i> double mutants, <i>Shh</i> was expressed in the posterior limbs but absent from the midline of the tail (H). I–L: Scale bars are 50 µm. At the hindlimb level, <i>Shh</i> was expressed in the notochord (arrow) and floorplate (arrowhead) in control and single mutant embryos (I, J, and K). No <i>Shh</i> expression was observed in the floor plate of E10.5 double mutants (L). Expression was downregulated in the notochord (arrow). I–L are transverse sections at the hindlimb level of E10.5 embryos. M–P: <i>Ptch1</i> whole-mount <i>in situ</i> hybridization. <i>Ptch1</i> expression was decreased in the tail of <i>Foxa1</i>;<i>Foxa2</i> double mutants (P) compared to control (M), <i>Foxa1</i> null (N) and <i>Foxa2</i> mutants (O). Insets in A–D, E–H and M–P are close-up views of the boxed region shown in the respective figures.</p

Strategy to remove FOXA2 from the mouse notochord.

<p>A: Mice heterozygous for the <i>Foxa1</i> null allele (<i>Foxa1^+/−</i>), homozygous for the <i>Foxa2</i> floxed conditional allele (noted as <i>Foxa2^c/c</i> in this report) and that contained the <i>ShhcreER^T2</i> allele were crossed to <i>Foxa1^+/−</i>;<i>Foxa2^c/c</i> mice to create double knockouts. The pregnant dam was given tamoxifen by oral gavage (4 mg) in corn oil when embryos were E7.5. * indicates that the <i>R26R</i> allele was crossed into some animals for fate-mapping. B: Exposure of E7.5 embryos to tamoxifen that contained the <i>ShhcreER^T2</i> and CRE-inducible <i>R26R LacZ</i> reporter resulted in reporter expression in the notochord (NC) but not the floorplate. The <i>ShhcreER^T2</i> allele is not active in the floor plate at E7.5. Scale bar is 100 µm. The ventral neural tube is outlined in black for clarity. C–F: Confirmation of removal of <i>Foxa2^c/c</i> from the notochord using a FOXA2-specific antibody. C and D: FOXA2 protein was present in the notochord and floorplate of control embryos (C) but absent from the notochord of <i>Foxa2^c/c</i>;<i>ShhcreER^T2</i> embryos (D; the notochord is outlined in white). E and F: The presence of a notochord in <i>Foxa2^c/c</i>;<i>ShhcreER^T2</i> embryos was confirmed by staining for Laminin protein. Dissociation of the notochord from the neural tube in D and F is likely a result of tissue processing. G and H: DAPI staining of a similar E9.5 section to show nuclear staining. White arrows in C–H denote the notochord. C–H: Scale bar is 50 µm.</p

The sclerotome is initially unaffected in <i>Foxa1</i>;<i>Foxa2</i> double knockouts.

<p>A–D: <i>Pax1</i> expression at the hindlimb level (transverse sections are shown). <i>Pax1</i> is expressed in the sclerotome at E10.5. In controls (A), <i>Foxa1</i> nulls (B), <i>Foxa2</i> notochord knockouts (C), and double mutants (D) there was no difference in <i>Pax1</i> expression. E–H: E10.5 sagittal sections showing <i>Tbx18</i> expression at mid-trunk level. <i>Tbx18</i> is confined to the anterior sclerotome. No differences in expression of <i>Tbx18</i> were observed in single (F and G) and double (H) mutants compared to control embryos (E). Scale bars are 200 µm.</p

Proposed role for Bmp signaling in the AER.

<p>(A) In wild type limb buds, <i>Bmps</i> are initially expressed in the AER and the anterior and posterior mesenchyme. At later stages of development, <i>Bmp</i> expression is maintained in the AER and in the underlying mesenchyme. (B) Removal of <i>Bmp</i> ligands in the AER resulted in an abnormal expansion (early) and then loss (late) of the central AER. This resulted in defects in autopod patterning but not in limb outgrowth. During early limb bud development, BMP proteins produced in the anterior and posterior limb bud mesenchyme may partially rescue BMP signaling in these regions of the AER (arrows). The central AER appears to require early Bmp expression within this structure. During later development BMP proteins are expressed in the mesenchyme underneath the AER but expression at this time point in not sufficient to maintain a functional AER. Red  =  <i>Bmp</i> expression, green  =  limb bud mesenchyme, white  =  mutant AER.</p

Removal of <i>Bmp2</i>, <i>Bmp4</i> and <i>Bmp7</i> from the same limb bud using the <i>Msx2-cre</i> allele.

<p>RNA <i>in situ</i> hybridization of <i>Bmp2, Bmp4</i> and <i>Bmp7</i> in wild type (A, C, E) and a triple mutant (B, D, F) hindlimb. <i>Bmp2, Bmp4</i> and <i>Bmp7</i> expression was removed in the triple mutants. Triple mutants (B, D, F) contained a thinner AER than wild type littermates. Whole mount and section RNA <i>in situ</i> hybridizations for <i>Msx1</i> and <i>Msx2</i> in E11.5 hindlimb buds (G-K). Anterior (section a), central (section b) and posterior (section c) sections of wild type (G and J) and the triple mutant (H and K) hindlimb buds are shown. In the anterior (G’-K’) and central (G”-K”) AER <i>Msx1</i> expression was downregulated and <i>Msx2</i> expression was absent. <i>Msx1</i> and <i>Msx2</i> expression persisted in the posterior AER (G”’-K”’). 20 µm transverse sections of E10.5 and E11.5 hindlimb buds are shown. Insets in A-F are close-up views of the respective AER.</p

Removal of <i>Bmp2, Bmp4,</i> and <i>Bmp7</i> from the AER resulted in polydactyly, interdigital webbing, and split hand foot malformations.

<p>Skeleton preparation (A-H) of control and mutant fore- and hindlimbs of newborn mice (P0). Removal of either five of the six <i>Bmp</i> alleles from the forelimb or all known <i>Bmp</i> alleles from the forelimb AER (triple mutants) produced autopod patterning defects (A-C). Removal of five of the six <i>Bmp</i> alleles expressed in the AER (<i>Bmp7^f/+</i>; <i>Bmp2^f/f, Bmp4^f/f, Msx2-cre</i>) resulted in the truncation of medial digits in hindlimbs (E and F). Removal of all six <i>Bmp</i> alleles from the AER (<i>Bmp7^f/f</i>; <i>Bmp2^f/f, Bmp4^f/f, Msx2-cre</i>) produced severe truncation of medial and/or anterior digits (G and H). In the hindlimbs two examples are shown for each genotype. Truncation of the tibia was observed in 3/10 (30%) of triple mutant hindlimbs (“*” in H’). No other defects in proximal/distal patterning were observed in either the fore- or hindlimbs. A’-H’ are images of the entire limbs.</p