Françoise Helmbacher explores mechanisms regulating the establishment of neuromuscular circuits during embryogenesis, and how molecular players exert distinct complementary activities in motor neurons, muscles and connective tissues.

During vertebrate embryonic development, neuromuscular morphogenesis involves a large array of tissue interactions driving the generation of skeletal muscles and the production of the motor neurons that innervate them. Both processes are based on the execution of an intrinsic regulatory program governing how cells acquire their fate and how they differentiate. Meanwhile, both cells types dynamically explore the surrounding tissues in order to define muscle shapes and adequately wire the connections between the nervous system and muscles. Whereas muscle progenitors migrate from their initial location to their final destination, motor neurons emit axons that navigate across tissues, to establish synaptic connections with their partner muscles in their final position. Although numerous signals acting on either cell type have been identified, the mechanisms that allow the two processes to be accurately coupled have not been studied. Françoise Helmbacher’s team has a longstanding interest for the mechanisms of neuromuscular development. Previous work by the team has consistently outlined the tight coupling between muscle and motor neuron development. The team recently identified the Fat1 Cadherin, an adhesion molecule, as a novel regulator of muscle morphogenesis, and showed that disrupting the Fat1 gene in mice resulted in morphological alterations of subsets of muscles in the face and shoulder region, partly as a consequence of defective migration polarity (Caruso N. et al., PLoS Genetics, 2013). In a new study published recently in PLoS Biology (Helmbacher F., 2018), she has applied a combinatorial genetic approach to explore how Fat1 operates in distinct tissue types to couple development of motor neurons and their target muscles.

In this study, F. Helmbacher has used the cutaneous maximus (CM) muscle, emblematic of Fat1-driven morphogenetic activities, as a powerful developmental model to explore the tissue cross-talks regulating neuromuscular morphogenesis. The CM is a flat subcutaneous muscle, in which myogenic progenitors migrate collectively under the skin in a planar manner, spreading radially from their point of origin, accompanied by elongating axons of their partner motor neurons. In this muscle, myogenic differentiation occurs with a delay with respect to progenitor migration and axon elongation, such that the front of progression of myogenic progenitors and axons is physically separated from the front of elongation of muscle fibers. The three processes can be followed quantitatively to assess how they are affected by genetic modifications. The study uncovers that constitutive Fat1 disruption interferes with both expansion and differentiation of the CM muscle, as well as with its motor elongation and with specification of its partner MN pool. Remarkably, Fat1 is expressed in all the cell types outlining this developing neuromuscular system, including the subset of motor neurons innervating the CM, the myogenic cells that constitute it, and the surrounding connective tissues across which the muscle migrates. This study establishes that Fat1 disruption in connective tissue robustly alters CM muscle morphogenesis, affecting not only progenitor migration and myofiber expansion, but also subsequently impairing axon growth and specification of cognate motor neurons. This identifies connective tissue as a cell type in which Fat1 activity is required for the non–cell-autonomous control of CM circuit morphogenesis. In parallel, results of this study show that Fat1 is also required in motor neurons to promote their axonal growth and specification, modestly influencing muscle progenitor progression in a non-cell autonomous manner. Together, these results show that Fat1 coordinates the coupling between muscle and neuronal development by playing complementary functions in mesenchyme, muscles and MNs. These findings could inspire research on muscle pathologies associated with FAT1 alterations in humans.

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Top image : microscope image featuring a section across a mouse embryo, showing the CM muscle progenitors appearing in red (anti-β-galactosidase, GdnfLacZ), and the connective tissue in which Fat1 activity is necessary for muscle morphogenesis appearing in green (expresses YFP, driven by Prx1-Cre-mediated activation of the conditional reporter R26Lox-Stop-Lox-YFP. anti-GFP antibody). The mirror-duplicated image is fused on its ventral side for esthetical reasons, not reflecting the biological situation.
Lower image: schematic summary of the activities played by Fat1 in connective tissues, muscle progenitors and motor neurons, during morphogenesis of the CM muscle and circuit.