Figure 1.
Conditional expression of RockDN in NCC.
A) Strategy for expressing RockDN construct in NCC. B) Quantitative real time PCR for the CAT gene cassette, using RNA extracted from pharyngeal arches 1 and 2, from E11.5 mutant RockDN+;Wnt1-cre+ and control embryos. There is a statistically significant (P<0.05 *), 13 fold decrease, in the expression of the CAT box in the mutant sample, as calculated using the one-way Anova test.
Figure 2.
Craniofacial malformations in RockDN;Wnt1-cre embryos.
A,B) Severe frontonasal (arrow), maxilla and mandible hypoplasia (arrowhead) and exencephaly (red arrow) in E17.5 RockDN;Wnt1-cre embryo (B), compared with control littermate (A). Note also the abnormally shaped eye and absence of eyelids in the mutant embryo (magnified inserts in A and B). C,F) Lateral views show the abnormal development of the snout in mildly affected RockDN;Wnt1-cre embryos at E18.5. Mild truncations of the frontonasal processes (arrow) and mandible (arrowhead) are apparent. D,G) Clefting of the upper lip and nose are apparent in a frontal view of an E18.5 RockDN;Wnt1-cre embryo (G; same fetus as in F). Double arrows are the same length in D and G highlighting the increased intra-ocular distance in RockDN;Wnt1-cre embryos. E,H,I,J) Measurements were taken from E15.5 heads (11 control embryos, 6 severely affected RockDN;Wnt1-cre mutants and 3 mildly affected RockDN;Wnt1-cre mutants). The distance between the eyes and the size of the angle between the nose and the forehead was measured (double arrow in E,H) and the results are shown in graphs I and J, respectively. In both cases, the measurements in the severely affected mutant embryos were significantly different from the controls (P<0.0001 *). There were no significant differences between controls and the mildly affected mutants for either measurement. Scale bar in A–D,F,G = 500 µm; E,H = 375 µm.
Figure 3.
Abnormalities in formation of the craniofacial bones in RockDN;Wnt1-cre embryos.
A,B) In severely affected RockDN;Wnt1-cre embryos at E14.5, the frontonasal bones (stained with alcian blue) are absent (arrow in B, compare to A). Meckel's cartilage is also reduced in size (arrowhead in B, compare with A). C,D) Bone (red) and cartilage (blue) staining of a mildly affected RockDN;Wnt1-cre embryo at E18.5 (D), shows that the maxilla (arrowhead) and mandibular (arrow) bones are well formed, although the hyoid bone (red arrow) is reduced in size in mutant embryos. E,F) Inferior views of the base of the skull in mildly affected embryos shows that the basisphenoid and the presphenoid bones are hypoplastic in RockDN;Wnt1-cre embryos, whereas the nasal septum is completely missing. Moreover, the maxillary bones are widely separated in mutant embryos (double arrow in F), compared to control littermates (E). bs = basisphenoid; n = nasal septum; ps = presphenoid. Scale bar = 500 µm.
Figure 4.
Expression of Rock1 in the developing head.
A–F) D–F are magnified regions from A–C, respectively. At E9.5, Rock1 protein (red) is expressed in the neural tube and the dorsal root ganglia, where it colocalises with Wnt1-cre+ve NCC (green, detected by GFP antibody, arrows in A,D), and in the pharyngeal tissue (B,E). In the frontonasal processes (C,F) Rock1 is expressed at the boarders of NCC rich ectomesenchyme and the surface ectoderm (arrows) and neural ectoderm (arrowheads), which are devoid of NCC. G) The position of the transverse sections shown in A–C are illustrated on a cartoon of an E9.5 embryo. Line i represents the position through the neural tube (A) and pharyngeal arch 1 (B) and line ii is the level of the fronotnasal processes (C). drg = dorsal root ganglia; fnp = frontonasal processes; mes = mesenchyme; ne = neural ectoderm; nt = neural tube; pa1 = pharyngeal arch 1; se = surface ectoderm. Scale bar in A–C = 45 µm.
Figure 5.
β-gal staining of NCC (blue) in RockDN;Wnt1-cre embryos.
A–D) At E8.5, the numbers of NCCs migrating towards pharyngeal arches 1 and 2 (arrows in A,C) and within pharyngeal arch 1 (B and D) are similar. There is a reduction in NCCs populating the region anterior to the developing eye (asterisks in A,C). E–H) At E9.5, there is increased distance between the frontonasal regions and the first pharyngeal arch (double arrows in E,G). There is a reduction in the numbers of NCC in the head (asterisks in G, compare to E) and the first pharyngeal arch (F,H). The surface ectoderm (arrowheads in F,H) is thickened and uneven in the mutant (compare H with F). I,J) At E10.5, the hypoplasia of the frontal region is more obvious in severely affected RockDN;Wnt1-cre embryos and the pharyngeal arches are frequently hypoplastic (numbered 1, 2 and * (posterior pharyngeal arches 3–6)). The cranial ganglia are misshapen and smaller in size in RockDN;Wnt1-cre embryos (white arrowheads in E,G,I,J). K,L) By E13.5, abnormalities in the distribution of NCC are seen even in the more mildly affected embryos, with reduced β-gal staining observed in the midline of the forming calvarias bones (arrows) and below the eye (white arrowheads). The frontonasal region is mildly truncated. pa1 = pharyngeal arch 1; 1 = pharyngeal arch 1; 2 = pharyngeal arch 2. Scale bar in A,C = 200 µm; B,D = 2 µm; E,G = 160 µm; F,H = 40 µm; I–L = 600 µm.
Figure 6.
Ectopic and excessive cell death in E9.5 RockDN;Wnt1-cre embryos.
A–D are sections through the neural tube (line i in M), E–H through the pharyngeal arch 1 (line i in M) and I–L through the frontonasal processes (line ii in M). A,C,E,G,I,K show caspase 3-expressing cells (red) and Wnt1-cre+ve NCCs (green). B,D,F,H,J,L are the same sections but only showing the caspase 3-expressing cells. A–D) Whereas only very occasional activated caspase 3-expressing, dying, cells (red) are seen in the neural epithelium in control embryos, there are many dying cells observed in the dorsal part of the neural tube, from which the NCC emerge, in RockDN;Wnt1-cre mutant embryos (arrows in C and D). E–L) Very few activated caspase3-expressing cells are observed in the NCC-derived ectomesenchyme of pharyngeal arch 1 (E,F) and the frontonasal region (I,J) in control embryos. In contrast, many dying cells are seen in corresponding regions from RockDN;Wnt1-cre mutants (G,H,K,L). The surface ectoderm in the mutant is more irregular, compared to controls (arrowheads in E,G,I,K) and the inner NCC-derived ectomesenchyme is loosely arranged with gaps between the cells (arrows in G,K). N,O) The mean apoptotic and mitotic indexes were calculated for NCC within E9.5 pharyngeal arches. There is a significant increase in cell death in the mutant samples compared to controls (P = 0.019; * in N). There is no significant difference in cell proliferation between the two samples (P = 0.433; O). Scale bar = 50 µm.
Figure 7.
Disruption of the actin cytoskeleton and vinculin-containing focal contacts in E9.5 RockDN;Wnt1-cre embryos.
A,B,E,F,I,J,M,N (line i in Q) are sections from first pharyngeal arch and C,D,G,H,K,L,O,P (line ii in Q) are from the frontonasal processes. A–H show phalloidin (red) and caspase 3 (green) immunofluorescence, with E–H being magnified regions as shown by the boxes in A–D, respectively. I–L show vinculin (red) and caspase 3 (green) dual immunofluorescence, with M–P being magnified regions as shown by boxes in I–L. The dotted lines in C,D,G,H,K,L,O,P indicate the boundary between the inner ectomesenchyme and the neural ectoderm and the surface ectoderm. A–H) Filamentous actin, labelled with phalloidin (red) outlines the cells in NCC-derived ectomesenchyme and neural ectoderm in the first pharyngeal arch (A,E) and frontonasal processes (C,G) of control embryos. Cortical phalloidin staining is lost in the ectomesenchyme from RockDN;Wnt1-cre mutants (F,H) but is maintained in the neural ectoderm (compare G with H). In addition, intense phalloidin-labelled foci are observed throughout the ectomesenchyme of the RockDN;Wnt1-cre mutants (dense red foci, blue arrow in F,H). Green caspase 3-positive cells are interspersed (white arrow) and overlapping with the phalloidin-intense cells (arrowheads in F,H). I–L) Vinculin and caspase 3 staining. In the pharyngeal arch the vinculin staining is not restricted to the centre of the arch in the mutant (compare J with I). In the frontonasal processes vinculin outlines the boundary between the surface ectoderm and the neural ectoderm with the inner NCC-derived ectomesenchyme (dotted lines in K,L). This discrete vinculin staining is lost in the RockDN;Wnt1-cre mutants (compare P with O). cas3 = activated caspase-3; mes = mesenchyme; ne = neural ectoderm; phall = phalloidin; se = surface ectoderm; vin = vinculin. Scale bar = 50 µm.
Figure 8.
Disruption of focal adhesions and extracellular matrix in E9.5 RockDN;Wnt1-cre embryos.
A,B,E,F are sections of the first pharyngeal arch (line i in O) and C,D,G,H,I–N are from the frontonasal processes (line ii in O). A–H) E–H are magnified regions shown in the boxes on A–D, respectively. Paxillin has a cortical distribution in the first pharyngeal arch in the control embryos (A,E) and also marks the boundary between the neural ectoderm and NCC-derived ectomesenchyme (arrow in C,G) in the frontonasal processes. This boundary staining is lost in the mutant (arrow in D,H) and there are intense paxillin positive foci in the pharyngeal arch and frontonasal processes (arrowheads in B,F,D,H), confirming loss of cell-substrate adhesion. I–L) Laminin was lost from the ectomesenchyme-neural ectoderm boundary (compare arrowhead in J with L) and was abnormally distributed in the surface ectoderm in the frontonasal processes in the RockDN;Wnt1-cre embryos (compare arrow in I with K). The H&E staining of the same sections are shown in M and N, allowing visualisation of the different cellular layers. fnp = frontonasal process; pa1 = first pharyngeal arch; ne = neural ectoderm; se = surface ectoderm; mes = mesenchyme. Scale bar in A–D,I,K = 50 µm; M,N = 40 µm.