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Plasticity of the neuromuscular system in response to changes in dactyly

Berki, Bianka Anna. Plasticity of the neuromuscular system in response to changes in dactyly. 2021, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

Changes in limb morphology enabled the terrestrial lifestyle of tetrapods, as they adapted to different types of locomotion. The distal part of the limb – called the autopod, and encompassing the carpals/tarsals, metacarpals/metatarsals, and digits (fingers and toes) – in particular, went through drastic morphological changes during tetrapod evolution. Both digit patterns, as well as digit numbers, were modified during this process. While fossil data of stem-group tetrapods show that some ancestors possessed up to eight digits on one extremity, this number was reduced to five at the basis of the crown tetrapod group. Today, there is no known natural tetrapod population that surpasses the so-called ‘pentadactyl state’, and having more than five fingers is considered a pathology referred to as ‘polydactyly’.
In the limb, not only the skeletal structure has to be modified during digit gains or losses, but also the soft tissues – like, e.g., the neuromuscular system - has to follow the skeletal changes, to form a fully functional unit. While polydactyly and the molecular alterations leading to it have been extensively studied at the skeletal level, little is known about the accompanying changes in the neuromuscular system.
The present work aims to bridge this gap, by studying the plasticity of the neuromuscular system in response to changes in digit numbers. First, we evaluated muscle and nerve patterns in the periphery of polydactyl limbs. Then, on a molecular and cellular level, we attempted to understand how muscle-specific motor neuron pools are modified in polydactyl individuals. We used chicken embryos, where in-ovo manipulation of the limb can result in mirror-image duplication of the digits, thus providing an ideal and well-established model for our study.
Using wholemount immunostaining of nerves and muscles followed by light sheet microscopy, we first reconstructed a 3D developmental time series of control wings and legs. There, our main finding uncovered a rotated pattern of the main nerve branches between wings and legs. Moreover, challenging the system with additional digits demonstrated a differential response of muscles and nerves in polydactyl limbs. Namely, while muscles seemed able to perfectly follow the pattern of skeletal mirror-image duplications, only two of the three main nerve branches responded and split to innervate the duplicated muscles.
These intriguing results motivated us to turn our attention toward the central nervous system. The cell bodies of limb-innervating motor neurons reside in the spinal cord and are organized into small subsets, so-called motor neuron pools. Each pool innervates one specific muscle, and the survival and maturation of these pools largely depend on target-muscle derived factors. The observed changes in innervation patterns pointed toward a potential modification of motor neuron survival and pool identity, a rationale we set out to explore in the second half of this thesis.
First, we showed that motor neuron numbers in the rostral LMC are decreased in the spinal cord of polydactyl individuals, due to increased cell death. This phenomenon was correlated to changes in the muscle patterns of the forearm, and the modification of fast versus slow muscle fibre composition in the limb, which might impair efficient innervation. In order to gain insights into the motor neuron pool compositions and transcriptomes, we performed, for the first time in chicken embryos, single-cell RNA-sequencing of spinal cord tissue. The resulting data identified putative digit-specific motoneuron pool markers but indicated only minor differences in cell-type distribution between polydactyl and control neural tubes. However, the traditional emulsion-based method might have failed to capture high enough numbers of motor neurons. Accordingly, we focused on a different, more direct approach, for an in-depth study of motor neuron pools innervating native and duplicated muscles in polydactyl embryos.
To this aim, we developed a method combining ex-ovo retrograde axonal labelling, manual purification of neurons, and the highly sensitive Smart-seq2 single-cell RNA-sequencing technique. Our results showed that our method is efficient in extracting embryonic motor neurons, that it captures a high number of genes per cellular transcriptome, and therefore represents a valuable tool for studying motor circuit formation. Preliminary comparative transcriptomic analysis of neurons coming from two motor neuron pools – innervating the EMR and FDQ wing muscles - revealed unique pool-specific signatures and further validated our technique.
Collectively, in the present thesis work, we describe the plasticity of the neuromuscular system in response to polydactyly, and how muscles and nerves integrate changes in the skeletal patterning. Furthermore, we show that changes in muscle identity and shifts in muscle fibre composition can affect motor neuron survival. Also, we developed a technique to study in great detail the transcriptomes of muscle-specific motoneurons, and we intend to use this methodology to better understand neuronal wiring and molecular muscle-neuron cross-talk during circuit formation. Our data provide the basis for further developmental studies and offer a framework for medical research, in order to better understand the etiologies of human polydactyl phenotypes.
Advisors:Tschopp, Patrick and Ebert, Dieter and Stoeckli, Esther
Faculties and Departments:05 Faculty of Science > Departement Umweltwissenschaften > Integrative Biologie > Evolutionary Biology (Ebert)
05 Faculty of Science > Departement Umweltwissenschaften > Integrative Biologie > Regulatory Evolution (Tschopp)
UniBasel Contributors:Tschopp, Patrick and Ebert, Dieter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14830
Thesis status:Complete
Number of Pages:xii, 118
Language:English
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss148308
edoc DOI:
Last Modified:29 Oct 2022 04:30
Deposited On:28 Oct 2022 08:49

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