Seetharaman, A., Selman, G., Puckrin, R., Barbier, L., Wong, E., D’Souza, S., & Roy, P. (2011). MADD-4 Is a Secreted Cue Required for Midline-Oriented Guidance in Caenorhabditis elegans. Developmental Cell, 21 (4), 669-680 DOI: 10.1016/j.devcel.2011.07.020
A newly characterised protein, MADD-4, involved in attracting muscle membrane extension and axons in nematodes is the founding member of a new family of secreted axon guidance proteins.
Bilaterally symmetrical animals use the left-right midline to organise development along the dorsal-ventral (DV) axis. Secreted molecules, such as Slits and Netrins, create gradients that migrating cells or cell extensions (such as axons) use as either attractive or repulsive guidance cues. In the nematode worm Caenorhabditis elegans, a ventral nerve cord is the site of highest Netrin (UNC-6) concentration, whilst Slit (SLT-1) is most concentrated in the dorsal midline. These molecules then act to guide cell migration and axon guidance on the DV axis. Commissural motor neurons originate on the ventral midline where their cell bodies are located. Their axons travel down the UNC-6 gradient to the dorsal midline where they extend longitudinally (see image). The locomotory body muscles of C. elegans are arranged in longitudinal rows, flanking the dorsal and ventral nerve cords. The muscles extend actin based membrane projections called muscle arms to the motor axons in the nearest nerve cord where they synapse producing neuromuscular junctions.
Seetharaman et al have used muscle arm extension to investigate midline oriented guidance. The group’s previous work had shown that the Netrin receptor, UNC-40 was required in the body muscles to mediate muscle arm extension. Interestingly however, UNC-6 (Netrin, it’s canonical ligand) was not required. To dissect muscle arm extension further the researchers carried out a genetic screen to identify mutants with muscle arm defective (madd) phenotypes. They found that animals with loss of function alleles of madd-4 have extensive dorsal muscle arm extension defects and weaker ventral extension defects. The alleles are semidominant suggesting dose dependency (consistent with secreted signalling molecules). The worms do not show serious disturbances to commissural axon guidance or dorsal cord formation.
Using transgenic reporter constructs the group showed that the two isoforms of MADD-4 were only expressed in the commissural motor neurons, hence showing localisation in the ventral and dorsal cords (a similar pattern to that in the image). Misexpressing MADD-4 in neurons that extend along the lateral line of the worm caused redirection of muscle arm extension (towards this lateral expression). This phenotype could be suppressed by loss of unc-40 function, suggesting that MADD-4 interacts with the netrin receptor (either directly, or via another receptor whose function is dependent on UNC-40. Using transgenic worms in which MADD-4 cannot be secreted, the researchers showed that it’s function is dependent on extracellular secretion and diffusion. The paper goes on to show that MADD-4 is not just involved in muscle arm extension but also has roles in axon guidance.
MADD-4 is the founding member of a new family of guidance proteins. Orthologues of madd-4 are present in the human and Drosophila genomes. In the case of Netrins and Slits, after their identification in invertebrate model organisms it has been shown that they are widely used in neural development for axon guidance and cell migration in vertebrates. It is likely by analogy that MADD-4 orthologues are playing important roles in vertebrate development. Interestingly a human orthologue ADAMTSL3 has been linked to colorectal cancer, as has the Netrin receptor DCC.