BIG PICTURE: why the whole is bigger than the sum of the parts

In essence we study how cells integrate external and internal information in order to make decisions, and how the combination of individual decisions leads to higher-level outcomes, such as organogenesis.

The control of organ size during development and repair is one of the last frontiers of biology, as we lack a fundamental understanding of how organ size is encoded, sensed and regulated. 

 

We use limb development as a model to study this type of questions, with a focus on the long bones.

Growth of the long bones
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Bone elongation is driven by the growth plate (GP), a transient cartilage structure where chondrocytes transition from resting (quiescent, stem cell-like) to proliferative and then hypertrophic (panel A), until they die or transdifferentiate into osteoblasts (bone-forming cells), such that the cartilage template (laid down by chondrocytes) is replaced by bone. Bone growth is controlled by GP-intrinsic (panel A) and GP-extrinsic factors (panel B), but the extent to which these interact is quite unknown. Especially important for regenerative medicine are skeletal stem cells (defined by their ability to give rise to cartilage, bone and marrow). Their location, identity and fate are actively investigated, and recent studies showed that bone repair and development use different pools of stem cells that could be targeted by distinct therapies. The Figure has been adapted from our WIRES Dev. Biol. review.

Specific themes studied in the lab

Theme A. Catch-up growth (funded by NHMRC APP2002084 and HFSP CDA 00021/2019-C)

One powerful approach to study the regulation of organ size is analysing how organs recover a normal growth trajectory after a developmental insult, which is known as catch-up growth (see Figure on the left).

 

A related question is how body proportions are attained and maintained, especially after a local perturbation.

A new approach: unilateral perturbation of limb/bone growth
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Pitx2-Cre; R26R-LacZ

Right

Left

The asymmetric enhancer of Pitx2 drives Cre expression in the left lateral plate mesoderm, inducing recombination in the cells that will form the left-limb mesenchyme.

Courtesy of Dr Shiratori, from the Hamada lab (Shiratori et al. 2006, Development).

Model recovery_edited.png

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2

We use a unique genetic model (see below) to induce transient unilateral perturbation of long-bone growth. We then study the mechanisms that lead to recovery or maintenance of limb symmetry. In all cases, the main hypotheses (not necessarily exclusive) are:

1) There is a rejuvenation of the growth plate via increased proliferative potential of the remaining chondrocytes.

2) There is increased contribution of stem/ progenitor cells, possibly located outside the cartilage.

Our previous findings. Bidirectional bone-body crosstalk
Cell-nonautonomous local and systemic responses to cell-cycle arrest in the growth cartilage lead to adaptive growth in developing mice

A

B

For our PLoS Biol 2018 study, we developed a double-conditional approach that requires both Cre and (r)tTA activity to activate expression of the gene of interest (panel A). We called it Doxycycline-controlled and Recombinase Activated  Gene Overexpression (DRAGON). We combined a version of this allele that expresses the cell cycle suppressor p21 with the left-specific Pitx2-Cre described above, and a cartilage-specific Col2a1-rtTA (a kind gift of Karen Posey and Jacqueline Hecht) to achieve cell cycle arrest in the chondrocytes of the left limb at the desired time (panel B). Surprisingly, this proliferative blockade during the last gestational week did not have a major effect in bone length, due to local compensatory proliferation by the spared cells. To know more about our DRAGON strategy and related mouse lines, check our Development 2020 paper

Hypothesis: Three phases of catch-up growth

We hypothesise that catch-up growth is divided into three phases, which define our projects:

 

1) Initiation of the response. Either because the abnormal bone size is somehow detected, or because the injury releases an alarm signal.

 

2) Execution. Certain stem/progenitor cells alter their behaviour in response to the perturbation, and drive the catch-up growth response.

 

3) Termination. Once the normal growth trajectory has been resumed, the overgrowth needs to stop.

Theme A projects

1) Identify the alarm signals that initiate catch-up growth

This project will address the mechanisms that trigger the compensatory response upon insult. We hypothesise that this phenomenon involves feedforward control based on production of an alarm signal. We will determine the requirement of candidate alarm signals produced by insulted cells and the role of the subsequently activated signalling pathways.

For this project, we are using RNA-seq and spatial lipidomics via MALDI Imaging, which has revealed some interesting differences in Left vs Right limbs (see Figure below)

2) Characterise the cell populations and signalling pathways that underlie catch-up growth

By combining lineage tracing with our models of tissue specific injury, we have been able to identify at least one cell population that increases its contribution to the cartilage in response to cell cycle arrest in the cartilage. We are characterising this population at the single-cell level, and determining its role in the catch-up growth process.

A different injury model (mosaic cell death) is starting to reveal a different response, in which existing cells in the cartilage activate a stem cell program that is not normally activated until later in life. We are characterising the signalling events that lead to this 'in situ reprogramming'.

3) How do chondrocytes know when a certain size for age has been achieved?

When one of the two growth plates in a bone is damaged, the other one can partially compensate for its absence (Hall-Craags 1968), suggesting that there is a target size for age, and that a feedback mechanism informs cartilage cells of bone size, so that a response is generated in the spared growth plate.

To discover the identity of this feedback factor, we have generated a transgenic quail model in which one of the growth plates can be genetically ablated, so that the response of the other one can be studied. This will allow us to uncover and manipulate the mechanisms mediating this compensatory response.

Theme B. Genetic and epigenetic determinants of limb size

A classic approach to study limb size determination is to cross-graft limb primordia (a.k.a. buds) between close species that differ in size. Initial experiments suggested that the size of the donor limb was unaffected or only slightly modulated by the host. However, we and others showed that when the whole limb bud is taken, its own epithelium exerts a strong instructive influence, so that limb bud cells can only be repro­grammed by extrinsic host signals when they are taken from the undifferentiated limb region of young donors and isolated from the influence of the epithelium (Rosello-Diez et al. 2011, Science). Therefore, the question remains as to whether the outcome of the inter-species transplantation would be different if donor limb cells developed from the beginning within the environment of the host.

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In this project, we will generate for the first time chimeric animals in which stem cells of species that differ in size from the host are incorporated early on in the host embryo, giving rise to the limb connective tissues (bones, tendons, dermis, etc) in the context of the host’s signals (See Figure on the left). Detailed anatomical characterisation will assess whether mechanical loading, hormones and other extrinsic signals can modulate the genetically-encoded limb size and proportions. Gene expression and chromatin accessibility will be compared for the donor limb cells in their endogenous vs. the chimeric context, to uncover the key molecular events underlying this modulation. Another innovative aspect is that besides using mice as hosts, we will capitalise on the unique Australian capability of generating transgenic quails to use them as hosts. This is crucial to expand the range of species that can be used as donors to chicken and marmoset, as avian embryos are better hosts for avian and primate stem cells. In summary, we will address the following aims:

1. Determine systemic effects on mouse limb growth when all limbs derive from rat or marsupial stem cells.

2. Generate and characterise quail models in which one of the limbs is derived from chicken or primate stem cells.

One way to generate developmental chimeras is to inject or aggregate pluripotent stem cells (PSCs) into early embryos. If done into an embryo genetically incapable of forming an organ of interest, the injected cells will mostly contribute to form such organ. This process, when done in the rodent blastocyst, is called blastocyst complementation, and we will use it to generate mice in which the limb mesenchyme is derived from PSCs of other species (see left Figure). To that goal, we have generated a unique mouse model in which the limbs cannot form, and have established several international collaborations to complement this model with PSCs from multiple species.

 

In addition, we will also generate a transgenic quail model in which the limb field cannot form, in order to use it as host for the injection of primate stem cells.