New finding in ADAM 13 that plays a crucial role in CNC migration in Xenopus laevis embroys

Introduction

African clawed frog (Xenopus laevis) as a model organism

As a model organism, Xenopus laevis has been used in many studies and researches of cell biology, developmental biology, neurobiology, and et cetra. In particular, using Xenopus laevis embryos has a great advantage for its ease of manipulation, despite it has long generation time and genetic complexity.

Cranial neural crest(CNC) migration

  • Population of cells that arises from the lateral part of the developing brain
  • The most anterior neural crest
  • Migrates ventrally and coordinates the entire cranio facial development of vertebrates.
  • The work of the transmembrane, cell surface metalloproteases ADAM 13 and ADAM 19
  • Orginated from the midbrain and hindbraine
  • Has a function of coordinating craniofacial development

Figure 0. Neural crest formation

Source: Campbell biology, 9th edition

So…Why we consider CNC migration?

  • To discover new molecules that may involve in its process
  • To understand cell fates ultimately determined by corresponding molecules
  • For human sake, to understand what cause craniofacial abnormalities

Currently known molecules that participate in CNC migration

  • α5β1
  • Fibronectin
  • Syndecan4
  • The Planar Cell Polarity pathway
  • ADAMs family (transmembrane proteins) – New category

ADAM 13 and other ADAM proteins

  • Contain a disintegrin & cysteine rich domain
  • Half of known ADAMs (e.g. ADAM 9, 12, 13, 19 and 33) are predicted to have metalloprotease activity (Alfandari etal.,2009).
  • Cleave a number of extracellular proteins.
  • Required for CNC cell migration
  • Modify the transcription of a number of genes
  • Important for cell migration
  • They are cell surface metalloprotease with a domain rich of a disintegrin and cysteine
  • They are critical in various biological processes, such as cell signaling and cell adehesion

ADAM 13

  • Its metalloprotease domain is capable of cleaving many substrates, including fibronectin and Cadherin-11
  • In a related species, Xenopus tropicalis, ADAM13 cleaves ephrin B ligands and contributes to neural crest cell induction but this role does not appear to be conserved in Xenopus laevis (Cousin etal.,2011)

Methods & Results

The paper stated several hypotheses regarding the effect of ADAM 13 for its role in CNC migration as well as other proteins, such as Cadherin-11, that may involve. More specifically, interaction of ADAM 13 with its environment was the main focus in this paper. This made authors to come up with following experiments that might answer their questions.

For one of the experiment, the authors used two different techniques, including the graft assay and the targeted  injection assay, to test if each generates similar result for the embryos that were injected with mRNA encoding GTP alone (Ctl) or together with antisense morpholino for ADAM 13 alone (MO13) or ADAM 13 and 19 (2MO) (Fig 1A). The compounds were injected in both cell (graft and targeted injection)at the 8–16 cell stage(for targeted injection) and the procedure was carried out at the cell stage 15 and then the embryos were scored for their lack of CNC migration at tail bud stage (26–28) for both assays.

As a result, the 2MO inhibited the CNC migration similarly in both assays. However, MO13 inhibited CNC migration with 5 times more efficient in the targeted injection assay than in the graft assay (Fig 1B)

Separating CNC from the surrounding tissue somewhat rescued CNC migration that was induced by ADAM 13 knockdown. After injecting either GFP mRNA alone or a mixture of GFP mRNA and ADAM13 morpholino the dorso-animal cell at the 8-cell stage, embryos were sorted for their expression of GFP and underwent one of the following procedures at stage 15 (Fig 2A):

  1. The ectoderm peeled off
  1. The ectoderm peeled off and the crest lifted away from the underlying mesoderm
  1. The CNC excised from the embryo and placed back in
  1. The ectoderm pulled back on and left to heal.

These embryo were develop until tail-bud stage and scored for their lack of CNC migration.
The result showed that ADAM13 knockdown significantly inhibited CNC migration in the targeted injection assay. The ectoderm peel procedure did not affect this inhibition. But the step 2 (ectoderm/mesoderm peel) and the step 3 procedures rescued CNC migration to levels not significantly different from the GFP control (Fig 2B).

To test the ability of morphant crests to migrate in vitro on a two-dimensional fibronectin substrate, embryos were injected in one cell at the 2-cell stage with a mixture of GFP mRNA and either control morpholino (Ctl), ADAM13 morpholino (MO13) or ADAM13 and 19 morpholino(2MO), which then sorted at stage 15. The CNC explants were placed on the fibronetin-coated plates. Typical explants were shown before migration (t=0), at the end of the sheet migration phase (t=6 h) and at the end of the single cell migration phase (t=16 h). Morphant CNC cells were capable of migrating in vitro in a pattern indistinguishable from the control CNC and therefore indicating ADAM 13 and 19 knockdown has no effect on CNC migration in vitro (Fig 3.1). On the other hand, ADAM 13 knockdown in CNC was sufficient and prevented Cadherin-11 cleavage (Fig 3.2).

For this result, CNCs that were dissected, placed on fibronectin substrate, and biotinylated had their proteins extracted and immunoprecipitated sequentially using a goat anti-ADAM13 antibody, the mouse mAb to Cadherin-11 (1B4), and the mouse mAb to integrin β1. Both antibody and 1B4 recognized the cytodomains of ADAM 13 and Cadherin-11, respectively. ADAM13 knockdown completely abolished the expression mature ADAM13 at the cell surface. The full length Cadherin-11 protein is still strongly expressed but is no longer cleaved.

Another experiment showed that ADAM13 function is not cell autonomous. For this experiment, embryos were injected in a two-cell stage embryo with both GFP mRNA and morpholino (GFP+2MO) or with RFP mRNA. At stage 15, CNC were dissected and grafted into non-injected embryos; either the RFP expressing explant was grafted ventrally to the morphant crest (Wt leader and 2MO follower) or the RFP expressing crest was grafted dorsally to the morphant crest (Wt follower and 2MO leader).  The pictures of grafted embryo before (stage 15) and after migration (stage 26) and histograms (Fig. 4.) show the failed migration of each type of CNC (Wt or 2MO). This indicates that the co-grafting of wildtype CNC significantly rescues morphant CNC migration, the single configuration of the graft has no impact on the efficiency of the rescue, and the co-grafted morphant CNC had no effect on the ability of wildtype CNC to migrate.

The authors also questioned if the function of ADAM 13 metalloprotease is required crest. Donor embryos were injected in a cell with RFP and either the morpholinos (2MO) or the two morpholinos with the mRNA encoding the cytoplasmic domain of ADAM13 (2MO+C13). The recipient embryos were injected with a mixture of GFP and either a form of ADAM13 lacking the cytoplasmic domain (ΔCyto) or the cleaved form of Cadherin-11 (EC1-3). At stage 15, embryos were sorted for their expression, CNC explants taken out from donor embryos (RFP, 2MO or 2MO+C13) and grafted into various recipient embryos (NI, ΔCyto or EC1-3). The histogram (Fig. 5.) and the result indicate that morphant crest migration cannot be rescued if CNC doesn’t have the metalloprotease activity of ADAM13 (2MO->ΔCyto). The expression of the cyto domain of ADAM 13 in the morphant crest somewhat rescued CNC migration (2MO+C13->NI). By comparison with the prior one, the rescue was slightly lesser when the CNC were grafted in embryos with ΔCyto (2MO+C13->ΔCyto) showing that ADAM 13 can exert its metalloprotease function only when it is expressed by the CNC. The rescue was almost complete when the 2MO+C13 CNC are grafted in embryos with EC1-3 (2MO+C13->EC1-3).

The effect of Cadherin-11 knockdown in ADAM13 was also considered. Embryos were injected at the 1 cell stage with either ADAM13 morpholino (MO131ng) alone or together with various doses (5ng ~ 25ng) of morpholino against Cadherin-11 (MOC11). At stage 20,  the proteins were extracted from embryos,  immunoprecipitated, and blotted. The knockdown of ADAM13 alone led to an increase of the full-length form of Cadherin-11 that can be decreased by a partial cadherin-11 knockdown using 5 ng of MOC11.

At the 8-cell stage, embryos were injected with either MO131ng only or together with various doses (1ng~5ng) of MOC11 as well as the mRNA encoding GFP co-injected or singly injected, which then were raised until stage 28 and scored for the inhibition of CNC migration. The histogram and pictures (Fig. 6.) show that the partial knockdown of Cadherin-11 with MOC11 (1ng or 2.5ng) did not rescue CNC migration from inhibition induced by ADAM13 knockdown. Also, the co-injection of 5ng of MOC11 inhibited CNC migration more than the knockdown of ADAM13 alone.


Conclusions

To sum up, all conclusions were made as follows:

  1. ADAMS 13 is not required CNC migration in vitro, however it is essential in vivo.
  2. Manually separating the CNC from the underlying mesoderm in vivo can rescue migration of CNC lack of ADAM13.
  3. The expression of ADAM13 metalloprotease domain in the migrating pathways cannot rescue morphant crest migration
  4. Other than its role in digesting ECM, ADAM13 metalloprotease activity is also required specifically in the CNC itself.
  5. Reducing Cadherin-11 protein level does not rescue migration, and therefore Cadherin-11 does not have a main role of ADAM13

Strength & Weakness

  • Strength
    • Paper has several supporting ideas on the role of ADAM 13. Particularly, the authors themselves continued to support their studies by doing experiments on same subject matter, such as ADAM 13 in this paper. This make the paper more concrete and add more clarity to what they do.
  • Weakness
    • Paper is long and it is required for readers to know the knowledge of previous studies done by authors made or models that they suggested

References

  • Cousin H, Abbruzzese G, McCusker C, Alfandari D. ADAM13 function is required in the 3 dimensional context of the embryo during cranial neural crest cell migration in Xenopus laevis. Developmental Biology [serial online]. August 15, 2012;368(2):335-344. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed March 8, 2013.
  • Dominique Alfandari, et al. “Extracellular Cleavage Of Cadherin-11 By ADAM Metalloproteases Is Essential For Xenopus Cranial Neural Crest Cell Migration.” Molecular Biology Of The Cell 20.1 (2009): 78-89. MEDLINE. Web. 2 Apr. 2013.
  • http://nationalzoo.si.edu/Animals/ReptilesAmphibians/Facts/FactSheets/Africanclawedfrog
  • Cousin, .,Abbruzzese, G., Kerdavid, E.,Gaultier, A.,Alfandari, D.,2011. Translocation of the cytoplasmic domain of ADAM13 to the nucleus is essential for Calpain8-a expression and cranial neural crest cell migration. Dev. Cell20, 256–263.
  • McCusker, C., Cousin, H., Neuner, R., Alfandari, D., 2009. Extracellular cleavage of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest cell migration.Mol.Biol.Cell20,78–89.

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