February 1, 2006;
A dominant-negative form of the E3 ubiquitin ligase Cullin-1 disrupts the correct allocation of cell fate in the neural crest lineage.
Selective protein degradation is an efficient and rapid way of terminating protein activity. Defects in protein degradation are associated with a number of human diseases, including potentially DiGeorge syndrome, which is characterised by abnormal development of the neural crest
lineage during embryogenesis. We describe the identification of Xenopus Cullin-1
, an E3 ubiquitin ligase, and show that blocking the function of endogenous Cullin-1
leads to pleiotropic defects in development. Notably, there is an increased allocation of cells to a neural crest
fate and within this lineage, an increase in melanocytes at the expense of cranial ganglia
neurons. Most of the observed effects can be attributed to stabilisation of beta-catenin, a known target of Cullin-1
-mediated degradation from other systems. Indeed, we show that blocking the function of Cullin-1
leads to a decrease in ubiquitinated beta-catenin and an increase in total beta-catenin. Our results show that Cullin-1
-mediated protein degradation plays an essential role in the correct allocation of neural crest
fates during embryogenesis.
neural crest cell development
Disease Ontology terms:
DIGEORGE SYNDROME; DGS
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Fig. 2. Expression analysis of Cullin-1. (A) RNA expression pattern of Cullin-1 during early X. tropicalis development. Cullin-1 is ubiquitous at blastula (1, lateral view) and gastrula (2, vegetal, 3, animal view) stages. At neurula stages (4, anterior view) it becomes enriched in neural tissue. At tadpole stages, it becomes enriched in the neural tube, eye and branchial arches (5+6, anterior towards the left, dorsal upwards). (B) Cullin-1 protein presence during development. Westerns with an anti-Cullin-1 antibody were carried out on protein extracts of whole X. laevis embryos at different developmental stages. Cullin-1 protein is found throughout development. Cullin-1 protein levels are slightly increased during late neurula/early tadpole stages.
Fig. 6. Phenotypes observed in Cul1-C75 and Cullin-1 injected embryos. (A) Wild-type embryo for comparison. The most striking phenotype observed was an increase in melanocytes (B), but eye defects (C, star represents where the eye should be) and secondary axes (D) were also commonly observed (see Fig. 5 for quantification). Embryos were hybridised in situ with a probe for N-tubulin, a marker for differentiated neurons (purple). Ectopic N-tubulin can be observed in the secondary axis (D). The lineage tracer lacZ (light-blue staining) indicates the area of injection. Lateral view with anterior towards the left and dorsal towards the top. The injected side is shown. Transverse sections of the embryos show clearly the large upregulation of melanocytes (E-G) and the ectopic tissue (white arrowhead) in the injected side. Neural differentiation in the neural tube is not affected (as marked by N-tubulin); however, cranial ganglia neurons (N-tubulin positive, arrows) are absent (compare B,C with A and E,F with control side). Transverse view with dorsal towards the top and the injected side on the right.
Fig. 8. Cul1-C75 RNA injections increase early neural crest markers. (A) Embryos were injected with 500 pg of Cul1-C75 or Cullin-1 (same phenotype, but not shown) plus the lineage tracer lacZ (light blue) into one blastomere at the two-cell stage. As a control, 500 pg lacZ RNA was injected alone. Embryos were grown to neurula stage and analysed for the early neural crest markers Sox9, Sox10, Slug and Zic3 (purple). Expression of all markers was expanded, predominantly into the anterior neural plate. In some cases, expression of Sox9 and Sox10 were reduced (not shown). Dorsal view with anterior towards the top. (B) When injections were targeted to the anterior neural plate, expression of Sox3 (a marker for proliferating neural tissue) was reduced. This was in the same region where the expansion of neural crest markers (as shown for Sox9) was seen. Dorsal view with anterior towards the top.