Projects

CONTENTS

Introduction

Research Opportunities

Additional project information


Introduction

The Zebrafish model
The fundamental question of how different populations form within an embryo has until now, been extremely difficult to address in conventional systems purely due to logistical constraints; mammalian embryos develop in- utero, and direct visual observation of living muscle is all but impossible. In contrast, the zebrafish develops ex-utero and is optically clear during the embryonic and juvenile stages- yielding a unique possibility to examine development in vivo.

Techniques
Projects will involve developmental and molecular biology, incorporating modern research techniques (in-situ hybridisation, confocal and electron microscopy, PCR, bioinformatics, fish husbandry, transgenic fish technology, immuno-histochemistry, histology, in-vivo cell lineage tracking) and utilising the zebrafish model system.

Observable development
I focus upon the precursor specification, migration and proliferation that is deployed to generate vertebrate limbs and muscle. The fundamental question of how different populations form within an embryo has until now, been extremely difficult to address in conventional systems purely due to logistical constraints; mammalian embryos develop in- utero, and direct visual observation of living muscle is all but impossible. In contrast, the zebrafish develops ex-utero and is optically clear during the embryonic and juvenile stages- yielding a unique possibility to examine development in vivo.The muscle structure of zebrafish represents a relatively simple paradigm where muscle precursors specification and subsequent myoblast elongation, fusion and attachment can be followed in real time using time-lapse photo microscopy. Just as in human embryos, the appendicular muscles of zebrafish are formed from populations of long-range migrating precursors that originate in the somites and express the gene lbx1. In addition, our limbs evolved from the paired fins of ancestral fish, such that initiation and outgrowth of fins is genetically similar to early limb formation. These characteristics make zebrafish a powerful and genetically tractable model system for the analysis of vertebrate limb initiation and muscle development.

Outcomes
The long-term outcome of this work will enhance our understanding of limb formation and how stem cell-driven muscle formation and repair occurs in vertebrate embryos. This knowledge will have profound implications for our understanding of the pathology and treatment of limb developmental defects and degenerative muscle disease.

Research Opportunities

Current projects include but are not limited to the research listed below.

MOTORNEURON DISEASE
Creating and curing fish models of human motorneuron disorders (in particular ALS)

In collaboration with Professor Garth Nicholson, ANZAC Research Institute
Inherited neuropathies are one of the most common human genetic disorders and produce life long disability. There are more than 50 different genes known to produce inherited neuropathies. This program uses powerful molecular and cell biology techniques for the first time to create fish models of inherited neural disorders. The effects of mutations in peripheral nerve genes will be examined to determine the their effect on mobility and development of pathology using fluorescent labeled nerves in living Zebra fish. Nerve degeneration can be seen in real time. The new fish models developed will later be used in high throughput drug screens to develop effective treatments for these previously untreatable diseases. Professor Nicholson's laboratory and clinic has discovered a number of new genes and mutations causing neural disorders. Discovery of the gene mutations causing these disorders continues to proceed rapidly with about 50% of neuropathy genes so far discovered. Each new discovery uncovers new areas of cell biology, leading to publications in leading international journals. This program will allow the effect of the neural gene mutations to be seen for the first time in living organisms and will allow development of high throughput drug screens aiming for future cures.

fish mated with transgenic RFP (KillerRed) neuron

Transgenic FUS 521C:GFP fish mated with transgenic RFP (KillerRed) neuron fish enable us to examine spinal neuron phenotype in vivo as the fish develop and grow in real time (above).

MUSCLE AND LIMB/FIN DEVELOPMENT AND EVOLUTION
Initiation, specification and control of vertebrate limb and muscle development

The general aim of my research is to generate a detailed understanding of the morphological and genetic control of precursor specification, migration and proliferation that is deployed to generate vertebrate limbs and muscle. Our limbs evolved from the paired fins of ancestral fish, such that initiation and outgrowth of fins is genetically similar to early limb formation. These characteristics make zebrafish a powerful and genetically tractable model system for the analysis of vertebrate limb initiation and muscle development.
The muscle structure of zebrafish represents a relatively simple paradigm where muscle precursors specification and subsequent myoblast elongation, fusion and attachment can be followed in real time using time-lapse photo microscopy. Just as in human embryos, the appendicular muscles of zebrafish are formed from populations of long-range migrating precursors that originate in the somites. The long-term outcome of this work will enhance our understanding of limb formation and how stem cell-driven muscle formation and repair occurs in vertebrate embryos. This knowledge will have profound implications for our understanding of the pathology and treatment of limb developmental defects and degenerative muscle disease.

pelvic finless zebrafish

Light microscopy image showing the loss of pelvic fins and no other abnormalities in pelvic finless zebrafish A. Wild-type and B. pelvic finless zebrafish. pec: pectoral fin; pel: pelvic fin; d: dorsal fin; a: anal fin; c: caudal fin

Additional project information

Embryonic origins of vertebrate muscle
Limb muscles are formed by the long-range migration of precursor cells from the developing embryonic somites. Zebrafish fin muscle precursors possess molecular and morphogenetic identity with these limb muscle precursors. The mechanisms controlling precursor specification, initiation, migration and differentiation are yet to be determined. In addition the embryonic origin of many other muscle groups is still unknown. We now have a unique opportunity to utilise the resolving power of novel transgenic tools to permanently in vivo track the derivatives of muscle precursors in real time and therefore determine the spatial and temporal origins of migratory muscles. A deeper understanding of muscle lineage specification will provide insights into the normal, as well as pathological, aspects of skeletal muscle, heart and craniofacial development.

Determining the position and timing of limb initiation
The developmental origins and molecular processes that generate our legs and associated musculature have not been fully defined. To date, only two hind-limb specific genes have been discovered (Pitx1 & Tbx4). Tetrapod hind-limbs evolved from the pelvic fins of ancestoral fish and the signaling centres involved in limb formation are similarly involved in fin formation, for example, Pitx1 and Tbx5 are required for pelvic fin development. Therefore, examining the genetic control of pelvic fin development will shed light upon the developmental mechanisms of correct hind limb formation. We will utilize the power of the zebrafish vertebrate model to investigate the genes responsible for pelvic fin specification, initiation and outgrowth. In addition, we have pelvic fin and pectoral fin (evolutionary forerunner to tetrapod hind and fore-limb respectively) deficient zebrafish. Elucidating the gene or signaling centre responsible for these morphologies will highlight genes involved in limb development and disease.

Motor neuron disease
Amyotrophic Lateral Sclerosis (ALS), also called motor neuron disease (MND) is a rapidly progressive neurodegenerative disorder that leads to loss of motor neurons and death within 3-5 years of first symptoms. Fused in Sarcoma (FUS) and TDP-43 proteins are highly related, predominantly nuclear proteins that share similar structure and common role of RNA-binding. Mutations in genes encoding these proteins have been identified in ALS patients, providing compelling evidence of a putative role for these aberrant proteins in the pathogenesis of ALS. We are investigating known ALS genes and the functional consequences of previously unknown disease causing mutations. Molecular and cell biology approaches will be used to create fish models of inherited neural disorders. This will allow us to investigate the effect of neural gene mutations in living organisms and gain insight into the mechanisms and pathways that lead to motor neuron death.

Evolutionary origins of vertebrate limb musculature and the tetrapod transition
Locomotor strategies in terrestrial tetrapod species have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapod species, a hind limb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition on to land. A number of fossil forms have provided information on the evolution of the appendicular skeleton of the hind limbs within early tetrapods. Although the fossil record has, in part, charted the evolution of the skeletal framework of the load bearing limbs of tetrapods, it can shed little light on how the accompanying dramatic alterations of the limb musculature required to drive locomotion in terrestrial tetrapods have arisen, as soft tissues are rarely preserved within the fossil record. In order to examine this question it is necessary to uncover the mechanisms that generate limb and fin muscles within extant species strategically positioned within the vertebrate phylogeny. We are examining this question by describing the mechanisms utilised to generate fin muscles within extant fish species positioned at critical points within the vertebrate phylogeny (sharks, paddlefish and lungfish).