December 1, 1998;
The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation.
) encodes a transmembrane protein expressed initially in Spemann''s organizer
and then in paraxial mesoderm
. Together with another member of the protocadherin family, Axial Protocadherin
), it subdivides gastrulating mesoderm
into paraxial and axial domains. PAPC
has potent homotypic cell adhesion activity in cell dissociation and reaggregation assays. Gain- and loss-of-function microinjection studies indicate that PAPC
plays an important role in the convergence and extension movements that drive Xenopus gastrulation. Thus, PAPC
is not only an adhesion molecule but also a component of the machinery that drives gastrulation movements in Xenopus. PAPC
may provide a link between regulatory genes in Spemann''s organizer
and the execution of cell behaviors during morphogenesis.
[+] show captions
Fig. 2. pcdh8 / protocadherin 8 / PAPC is expressed during mesodermal mantle
morphogenesis. (A) PAPC expression in the dorsal marginal zone at
late blastula (stage 9G). (B) Expression expands around the marginal
zone at early gastrula (stage 10G). (C) Repression of PAPC
expression in the axial mesoderm prefiguring the segregation
between axial and paraxial mesoderm (stage 11G). (D) A sharp
anterior border separates head and trunk mesoderm (stage 13). (E) At
early neurula (stage 14), stripes of PAPC expression prefigure the
future somites. (F) As the first mature somite is segmented (stage
17), the anterior stripe disappears and new posterior stripes appear.
PAPC expression is also found in lateral plate mesoderm and in the
forming otic vesicle (not shown). (G) Transverse section of stage 13
embryo stained for PAPC in blue and chordin in brown by double in
situ hybridization; note PAPC expression exclusively in paraxial
mesoderm. (H) PAPC stripes during somite segmentation; the most
anterior PAPC stripe is in the mature somite (s), the second stripe
marks the forming somite (fs) and the more intense two posterior
stripes are located in the segmental plate (sp). (I) Transverse section
of stage 18 embryo showing AXPC expression exclusively in the
notochord. (J) Dorsal view of AXPC expression in the notochord
(stage 14), anterior to the left. (K) Lateral view of AXPC expression
in the notochord (stage 22). At later tailbud stages AXPC was
transiently expressed in developing heart and pronephros (data not
shown). Image from Kim et al., 1998.
pcdh8 (protocadherin 8) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage13, dorsal view, anterior left.
Image from Kim et al., 1998.
pcdh1 (protocadherin 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 14, dorsal view, anterior left.
Image from Figure 2. Kim et al., 1998. XB-ART-14006.
Fig. 3. PAPC causes homotypic cell sorting in reaggregation experiments. (A) Experimental design of cell sorting assays, which are described in more detail in Materials and Methods. (B) Complete mixing between cells injected with prolactin control mRNA (800 pg per blastomere) and GFP mRNA (100 pg) and uninjected cells (12/12 aggregates). (C) FL-PAPC mRNA (800 pg)-injected cells (labeled with GFP) sort out from uninjected cells (20/21). (D) Bisection of the FL-PAPC aggregates showing that FL-PAPC- injected cells tend to migrate to the center of the aggregates (10/12). (E) Bisection of M-AXPC (100 pg) aggregates showing cohesive patches in the periphery of the aggregates (11/11). (F-K) Aggregates containing M-PAPC (100 pg, in red), M-AXPC (green) and uninjected cells demonstrating homotypic cell sorting (12/12). (F-H) External views; (I-K) bisected aggregates.
Fig. 4. Dominant-negative PAPC affects paraxial mesoderm morphogenesis. (A) Control prolactin mRNA (200 pg) injection into an animal blastomere at the 32-cell stage showing cell mingling at stage 12. (B) M-PAPC (100 pg) injection generated a cohesive patch with a sharp clonal boundary (54/54). (C) DN-PAPC (1.6 ng) interfered with the formation of the cohesive patch induced by 100 pg M-PAPC (38/43). (D) Additional FL-PAPC mRNA (400 pg) rescued formation of cohesive patches (48/51), indicating that the dominant-negative effects are reversible. (E) M-AXPC (200 pg) generated cohesive patches with sharp clonal boundaries (36/38). (F) Coinjection of DN-PAPC mRNA (1.6 ng) did not interfere with AXPC patches (3/32); coinjection of 1.6 ng of DN-AXPC dispersed M-AXPC patches (31/38, not shown). (G) Unilateral injection of control prolactin mRNA (1.6 ng) and lacZ tracer (100 pg) at the 2- cell stage does not affect paraxial mesoderm marked by MyoD (n=44). (H) DN-PAPC (1.6 ng) caused defects in anterior somites on the injected side at stage 20 (105/151). (I) Rescue of paraxial mesoderm morphology by co-injection of FL-PAPC (600 pg) together with DN-PAPC (42/47). (J) When injected posteriorly, DN- PAPC (1.6 ng) caused defects in midline convergence of paraxial mesoderm on the injected side. (K) Transverse section showing effect of DN-PAPC on MyoD convergence, but not on notochord tissue marked by chordin. In G-K, MyoD is marked in blue, lacZ in red and chordin in brown.
Fig. 5. DN-PAPC mRNA inhibits animal cap extension by activin. (A) Animal caps without any treatment (n=32, three independent experiments). (B) High Activin (10 ng/ml) induced elongation of the explants (33/35). (C) DN-PAPC mRNA (1.5 ng per blastomere at the 4-cell stage) injection interfered with explant elongation (31/35). (D) Coinjection of FL-PAPC mRNA (400 pg per blastomere) together with DN-PAPC mRNA (1.5 ng) rescued the elongation of animal caps (36/38). (E) RT-PCR of RNA extracted from these caps showed no significant effect of PAPC mRNA injection on the expression of mesodermal markers such as Xbra, α−actin, MyoD, chordin and collagen 2; EF1(serves as loading control.
Fig. 6. FL-PAPC can promote animal cap elongation and changes in cell morphology. Embryos were injected with or without PAPC in each animal blastomere at the 8-cell stage and animal cap explants were treated with low activin at stage 8 (1 ng/ml). (A) Control animal cap explants with no activin treatment (26/26). (B) Microinjection of FL- PAPC mRNA (400 pg) without activin treatment (18/18). (C) Low activin (1 ng/ml) treatment was not sufficient to induce explant elongation (21/24). (D) FL-PAPC mRNA (400 pg) promoted elongation of animal caps treated with low activin (22/27, three independent experiments). (E) Microinjection of M-PAPC (400 pg) did not promote explant elongation (18/18), suggesting a requirement for the intracellular domain. (F) RT-PCR showed no significant effect of FL- PAPC mRNA on the expression of the mesodermal markers, MyoD, α- actin, Xwnt8 and collagen 2. (G-J) Confocal microscopy of open-faced animal caps (see Materials and Methods). (G) Low activin treatment as in C did not induce changes in cell polarity (41/43). (H) FL-PAPC mRNA (400 pg) injection as in B had no effect on ectodermal cell morphology (10/10). (I) FL-PAPC mRNA (400 pg) with low activin treatment, as in D, caused cells to elongate into fusiform or (J) monopolar cells (34/51) at stage 12 (the inset shows a particularly asymmetric cell with membrane vesicles within the thin end).
Fig. 7. Multiple regulatory steps control PAPC expression. (A) Wild- type PAPC expression at stage 10.5 in dorsal mesoderm. (B) Ventralizing UV treatment eliminates early dorsal expression. (C) Dorsalizing LiCl treatment expands PAPC expression around the marginal zone. Thus, initial PAPC expression is activated by factors from Spemann organizer. (D) Wild-type PAPC expression during midgastrula (stage 11.5). (E) Dorsal injection of Xombi mRNA (50 pg) at 32-cell stage induces ectopic PAPC expression in the dorsal midline (arrowhead) at stage 11.5. (F) Lateral injection of Xnot-2 mRNA (100 pg) induces ectopic repression of PAPC (arrowhead) suggesting that Xnot-2 may be a upstream repressor of PAPC in the dorsal midline. (G) Wild-type PAPC expression (blue) and chordin (brown) at stage 13. (H) Microinjection of Xcer mRNA (50 pg) coinjected with lacZ as lineage tracer (arrowheads) into a vegetal site at the 4-cell stage displaces posteriorly the anterior border of PAPC expression. (I) Model summarizing spatial regulation patterns suggested by the mRNA injection studies.
pcdh1 (protocadherin 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 18, transverse section, dorsal up.
Image from Figure 2. Kim et al., 1998. XB-ART-14006.