Catch-up growth and the establishment of body proportions

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, The basic underlying question is common to most biological fields: how do cells process intrinsic and extrinsic cues such that their combined behaviours give rise to collective outcomes? We study this topic by analysing how organs recover a normal growth trajectory after a developmental insult, which is known as catch-up growth (see Figure on the left). We are also interested in how body proportions are attained and maintained, a process that is important for evolution and health, and that may involve inter-organ communication.

Growth defects are particularly taxing when they affect the limbs or the back, as they hamper locomotion and most daily activities. Musculoskeletal disorders are in fact the main cause of disability in modern societies and represent a major burden for healthcare systems. We use the limbs, with a focus on the long bones, to study the compensatory mechanisms that are triggered by different types of injuries.

Growth of the long bones

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 End Rev 2015 review.

A handy approach: unilateral perturbation of limb/bone growth

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 Hidetaka Shiratori, from Hiroshi Hamada's lab. See also Shiratori et al. 2006, Development.

Our previous findings. Bidirectional bone-body crosstalk
Tissues in the knee joint modulate postnatal bone growth in mice

The toxin does not reach the cartilage!

In our Elife 2017 study, we used Pitx2-Cre to activate expression of diphtheria toxin receptor (DTR) in the left-limb mesenchyme, so that we could trigger cell death by injecting DT at the desired time. Due to the size of the toxin and the fact that the cartilage is avascular, DT did not reach the growth cartilage when injected postnatally in mice. Bone growth was however affected, due to altered paracrine signalling from the injured knee, where a strong inflammatory response was observed. These results revealed that the tissues surrounding the bones can influence bone growth, and we

identified the infrapatellar region and the articular cartilage as key players in this influence. In future studies, we would like to explore whether this growth defect after injury is permanent, and whether mechanical factors such as shortening of the patellar tendon are also involved.

Cell-nonautonomous local and systemic responses to cell-cycle arrest in the growth cartilage lead to adaptive growth in developing mice

Pit-Col-p21

model

C

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 (see panel A above). 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 the activation of two adaptive growth mechanisms: local compensatory proliferation by the spared cells, and a systemic growth reduction due to inter-organ communication and impaired placental activity (panel C). We are currently exploring what kind of signals are involved in both effects, and whether stem cells external to the cartilage are recruited to help cope with the insult.

Towards a holistic understanding of organ growth

In conclusion, based on our results and the accumulated evidence, our vision is that there are three levels of growth regulation (see Figure):

 

1) At the cell level, external inputs are integrated and interact with the genetic growth program, such that a context-dependent response is generated upon physiological or pathological perturbation. Feed-forward and feed-back mechanisms likely modulate the response.

 

2) At the organ level, growth of tissues such as cartilage is controlled not only by intrinsic regulators, but also by extrinsic information released by adjacent tissues (e.g. the fat pad), such that growth of intimately linked organs remains coupled. This modulation is probably reciprocal.

 

3) Most or all organs produce signals that convey their growth status to other organs, presumably via a central relay. This central system integrates inputs from all the organs to generate a proportional hormonal response, its effect ranging from slowed-down growth to developmental arrest (e.g. delayed puberty, metamorphosis). As different organs may require differential regulation by systemic signals, one possible strategy is that the intrinsic properties of each organ (e.g. expression levels of the signal receptor) determine its specific response to a common systemic signal. A less likely alternative is that each organ produces a specific growth-related signal that triggers a response specific to that organ by the central integrator.

Current projects

1) Molecular, cellular and tissular mechanisms of catch-up growth

This project will address the mechanisms driving chondrocyte hyperproliferation upon insult. We hypothesize that this phenomenon involves feedforward control based on production of an alarm signal, whose effect depends on the type of insult (panel A). We will determine the cell-autonomous requirement of candidate alarm signals produced by insulted cells and the role of the density and distribution of the insult in the activation and propagation of the response (short-range vs self-propagated, panel B). Some of these experiments will be performed confronting populations of chondrocytes with different growth properties in vitro, varying their proportion and distribution (see video below)

Col2-CFP+ and WT micromass cultures merging together

Based on the fact that overall proliferation does not surpass normal levels, we also propose that an external tissue exerts a top-down control over the cartilage, in a negative feedback proportional to bone growth rate. We will thus explore the control of chondrocyte proliferation by negative feedback from the perichondrium wrapping the cartilage, testing the role of candidate molecules.

2) Inter-organ communication in the establishment of body proportions

This project will explore the role of inter-organ communication in the adjustment of body proportions in response to a local insult during development. We will use our Pit-Col-p21 model in which cell cycle arrest in the left-limb cartilage leads to two effects in distant organs (panel A): systemic growth reduction due to impaired placental activity, and direct left-right limb communication that leads to reduced right-limb growth (independently of placental activity). We hypothesize that certain insults can trigger release of a stress signal that travels via the vascular or nervous system to other organs, affecting their growth directly (case of the limbs) or their production of hormones/growth factors, such that the rest of the body is also affected. As in Project 1, we will use transcriptomics and proteomics to identify candidate stress signals and we will test their effect in other organs, and their role in the systemic effect (panel B). As potential conduits of the signal, we will explore the role of the nervous and the vascular system.

3) Tracing and probing the role of external progenitors in bone growth and repair

It is unclear whether cells external to the growth cartilage can participate in bone growth, and whether this contribution varies in response to injuries or growth defects. We have designed both targeted and unbiased (panel A) lineage tracing approaches to follow the contribution of external cell populations to the growth cartilage during bone elongation (normal and catch-up growth, see potential outcomes in panels B and C). Once we identify cell populations with a consistent contribution (as recently described by the Iwamoto lab), we will ablate those cells during normal and catch-up growth, to determine their requirement during growth compensation.

(Before 2019, the Elife, PLoS Biol and End Rev papers have Alberto as (co)corresponding author)