Axial protocadherin (AXPC) regulates cell fate during notochordal morphogenesis

Abstract

The separation and specification of mesoderm into the notochord and somites involves members of the non-clustered δ-protocadherins. Axial (AXPC) and paraxial (PAPC) protocadherins are expressed in the early dorsal mesoderm and later become refined to the developing notochordal and somitic mesoderm, respectively. The role of PAPC in this process has been studied extensively, but the role of AXPC is poorly understood. Partial knockdown of AXPC causes a specific bent-axis phenotype, while more severe knockdown results in the loss of notochord formation. The inability of these embryos to develop a notochord is not due to a cell-sorting event via changes in cell adhesion during gastrulation, but rather this defect is manifested through the loss of axial mesoderm specification, but not general mesoderm induction. The results presented here show that AXPC functions in notochord morphogenesis by directing cell-fate decisions rather than cell-cell adhesion.

Figure 1

A newly identified allele of AXPC is predominantly expressed during early development. A. Comparison of the sequence of the two alleles of AXPC surrounding the ATG start codon. MO-1 refers to a previously published construct (orange). MO-2 is a newly designed morpholino (green). Red box indicates the ATG start codon. Differences between the sequences denoted by an *. B. Western blot detection of both exogenous and endogenous expression of AXPC in lysates from embryos collected at the indicated stage. Embryos over-expressing AXPC (lane: AXPC) were collected at stage 12.5. C. Western blot of endogenous AXPC in embryo lysates at stage 12.5 injected with the indicated amount of control morpholino or AXPC morpholino. For co-injections, morpholino-1 and morpholino-2 were mixed in equal parts to give the indicated final amount. In B and C, AXPC was detected by western blotting with anti-AXPC antibody. Each lane represents the equivalent of 1 embryo.

Figure 2

Morpholino to the second AXPC allele causes developmental axial defects. A–D. Embryos were injected on one side of 2-cell embryo with 10, 20, or 40ng (shown) of morpholino along with a Dextran-Ruby tracer. Embryos were cultured and observed at stages 22 (C,D) and 30 (A,B). Fluorescent Dextran-Ruby (red) images were superimposed onto brightfield images in C and D. E. Embryos were scored at stage 30 as a strong (>45°), mid/weak (0°<X<45°), or no (0°) phenotype. Anterior is up. F,G. Whole mount immunofluorescence on embryos bisected sagitally at stage 30, stained with anti-AXPC (green) and anti-fibronectin (red). Asterisks denote side of morpholino injection. Confocal images obtained from ventral aspect at 600X magnification. Scale bar = 50μm.

Figure 3

Loss of AXPC expression perturbs notochordal morphogenesis A. 2-cell stage embryos were injected bilaterally in the dorsal marginal zone with 40ng control morpholino (a), 20 or 40ng AXPC morpholino (c,d) or 1ng AXPC mRNA (d) and cultured until uninjected controls reached stage 12.5. Stage was determined by blastopore size and percentage was calculated by: # in given stage/total # embryos (A′, n>50). B. Whole mount immunofluorescence of bisected embryos injected with either control morpholino (CtlMO, a–c) or AXPC morpholino-2 (AXPC MO2, d–i). Sections were stained to detect C-cadherin to outline cell borders (a,d,g) and fibronectin to delineate tissue boundaries (b,e,h). Dextran tracer can be observed as red in c,f, and i. Arrowhead in h marks a localized loss of FN staining. Confocal images acquired at 200X magnification. Scale bar = 150μm.

Figure 4

AXPC does not mediate cell sorting. A–C. In cell dispersal assays, embryos were injected into one cell at 32-cell stage with nuclear-GFP alone (A), GFP+PAPC (B) or GFP+AXPC (C) and cultured to stage 9. Scale bar = 0.5mm. D–F. Reaggregation assay performed using blastomeres isolated from excised animal caps expressing RFP, GFP, AXPC-mCherry, and PAPC+GFP. Cells were mixed as indicated and images were acquired once aggregates formed (5–6 hours). Scale Bar = 1mm. G,H. AXPC constructs have functional activity in perturbing notochordal morphogenesis. Sagittal cryosections of stage 12.5 embryos from injected dorsally at 2-cell stage, with 1ng RFP (I) or 1ng AXPC (J). C-cadherin, fibronectin (FN), dextran-TRITC (red). Confocal images taken at 200X magnification. Scale bar = 150μm.

Figure 5

AXPC is specifically required for specification of axial mesoderm but not general mesoderm induction. A. Embryos were injected bilaterally in the dorsal marginal zone at the 2-cell stage with 40ng of either control (CtlMO) or AXPC morpholino (AXPCMO), then cultured until stage 12.5 and prepared for in situ hybridization with either chordin or MyoD. A′. Embryos were scored by using the categories of ‘normal’, ‘reduced’, and ‘absent’ to describe the staining intensity. A. Examples of ‘normal’ and ‘reduced’ are depicted (a,b or c,d respectively) (n > 30). Anterior is up. B. Embryos were prepared as described above for the detection of chordin at stage 10.5. B′. The categories ‘strong’, ‘mid’, and ‘weak’ represent the degree of chordin staining and used to score the control and AXPC MO injected embryos. Lower image is a higher magnification of the dorsal lip (boxed region) (n>30). C. RT-PCR analysis of animal caps from embryos injected with either control or AXPC MO and treated with or without 100ng activin to induce mesodermal gene expression. Data was normalized to histone 4 (XH4). D. Mesoderm is not respecified to nueroectoderm or endoderm due to loss of AXPC. Embryos were injected as described in A and probed for Xbra, Sox17 (endoderm), or Sox2 (neuroectoderm). Asterisks denote the blastopore. Arrowhead indicates dorsal lip. Scale Bar in A and B = 250μm.