Final Report Summary - PATTERN AND GROWTH (Coordination of patterning and growth in the developing neural tube)
In the developing spinal cord, several types of neuronal progenitors are specified and spatially arranged along the dorso-ventral axis in response to the morphogen gradient of Sonic hedgehog (Shh). This process of establishment of the neural tube pattern involves the transcriptional specification of progenitor identity, as well as proliferation and terminal differentiation. How these processes are coordinated is poorly understood.
The main goal of this project is to describe the mechanisms that coordinate growth and patterning of the vertebrate neural tube. As outlined in section B1 of this proposal, the project is separated in three main steps:
1) construct a model of spatiotemporal dynamics of pattern and growth in the neural tube;
2) determine the relationship between cell fate and proliferation;
3) determine how Shh signaling relates to cell fate specification and growth at the single cell level.
Summary of progress towards each objective:
1. The work performed during this period pertaining to objective 1 includes data acquisition and mathematical analysis. In addition to the time-series data from fixed transverse section acquired in chick (as outline in the midterm report), we have now collected data from mouse neural tubes. The type of data includes measurements of the proliferation rate, differentiation rate, apoptosis, the rate of cell fate change and the increase in size of different progenitor domains. This time-series data from fixed sections was complemented by lineage tracing experiments where single cells were marked by inducing ubiquitous YFP expression using the Tamoxifen-inducible Sox1Cre line. All data was analysed according to the formulated mathematical model for spatio-temporal growth of the neural tube.
2. To complement previous experiments on the second objective, we have performed chick electroporation experiments where cell proliferation or differentiation in the chick neural tube were blocked using p21[CIP] or Notch Intracellular Domain. These experiments have confirmed that the rate of differentiation is key for determining the patterning proportions at later stages of development. Consistent with this, we have also confirmed that the rate of cell fate change decreases over time by using conditional tracing of the Olig2 lineage using Tamoxifen-inducible Olig2Cre to drive GFP reporter expression.
3. Towards objective 3, we analysed Shh null mice in a fashion as outlined in objective one. We found that in this mutant cell fate specification occurs early and later the formed domains are expanded by proliferation, differentiation and apoptosis. To extend this to a single cell level, we are currently performing experiments aimed at lineage tracing analysis in Shh null mutants. In addition, we have developed conditions for whole embryo culture and 2-photon in-vivo imaging of electroporated chick embryos, which allows us to track the growth in distinct dorsoventral domains of the neural tube with single cell resolution. We are currently working on automating the analysis of this data.
Summary of main results achieved so far:
We measured the spatio-temporal dynamics of gene expression, proliferation and differentiation rates during three days of development. We found that all domain sizes change over time at different rates. In addition, the data shows that the proliferation rate is spatially uniform, whereas the differentiation rate progressively increases in time and differs between progenitors. Before the onset of differentiation, the changes in the sizes of progenitor domains correlate with the increasing amplitude of the Shh gradient and occur faster than the tissue grows, implying that progenitor identity is actively specified in response to signaling. At later times, the change in domain sizes can be explained by the proliferation and differentiation of different progenitor types alone.
These findings suggest that scaling of pattern with tissue size does not occur throughout development and the reproducibility of pattern between embryos cannot be explained with a simple gradient scaling model. It suggests a role for the temporal regulation of the differentiation rate in regulating the formation of patterns in developing organs.
Expected final results and potential impact and use:
Our data suggests a 2-phase model of patterning: early domain size depends on switches of cell identity, which are instructed by Shh signaling, whereas the late elaboration of pattern depends on proliferation and differentiation. Ongoing experiments are designed to test and validate this hypothesis. Potentially, this finding would have impact for understanding the mechanisms of establishment of spatial patterns of cell differentiation during development. Our data suggests that for the emerging morphology of developing organs, the initial establishment of a pre-pattern and the subsequent temporal control of the differentiation rate are key. This could be applicable to medically relevant tissue-engineering approaches and the field of regenerative medicine, as well as to stem-cell research.
The main goal of this project is to describe the mechanisms that coordinate growth and patterning of the vertebrate neural tube. As outlined in section B1 of this proposal, the project is separated in three main steps:
1) construct a model of spatiotemporal dynamics of pattern and growth in the neural tube;
2) determine the relationship between cell fate and proliferation;
3) determine how Shh signaling relates to cell fate specification and growth at the single cell level.
Summary of progress towards each objective:
1. The work performed during this period pertaining to objective 1 includes data acquisition and mathematical analysis. In addition to the time-series data from fixed transverse section acquired in chick (as outline in the midterm report), we have now collected data from mouse neural tubes. The type of data includes measurements of the proliferation rate, differentiation rate, apoptosis, the rate of cell fate change and the increase in size of different progenitor domains. This time-series data from fixed sections was complemented by lineage tracing experiments where single cells were marked by inducing ubiquitous YFP expression using the Tamoxifen-inducible Sox1Cre line. All data was analysed according to the formulated mathematical model for spatio-temporal growth of the neural tube.
2. To complement previous experiments on the second objective, we have performed chick electroporation experiments where cell proliferation or differentiation in the chick neural tube were blocked using p21[CIP] or Notch Intracellular Domain. These experiments have confirmed that the rate of differentiation is key for determining the patterning proportions at later stages of development. Consistent with this, we have also confirmed that the rate of cell fate change decreases over time by using conditional tracing of the Olig2 lineage using Tamoxifen-inducible Olig2Cre to drive GFP reporter expression.
3. Towards objective 3, we analysed Shh null mice in a fashion as outlined in objective one. We found that in this mutant cell fate specification occurs early and later the formed domains are expanded by proliferation, differentiation and apoptosis. To extend this to a single cell level, we are currently performing experiments aimed at lineage tracing analysis in Shh null mutants. In addition, we have developed conditions for whole embryo culture and 2-photon in-vivo imaging of electroporated chick embryos, which allows us to track the growth in distinct dorsoventral domains of the neural tube with single cell resolution. We are currently working on automating the analysis of this data.
Summary of main results achieved so far:
We measured the spatio-temporal dynamics of gene expression, proliferation and differentiation rates during three days of development. We found that all domain sizes change over time at different rates. In addition, the data shows that the proliferation rate is spatially uniform, whereas the differentiation rate progressively increases in time and differs between progenitors. Before the onset of differentiation, the changes in the sizes of progenitor domains correlate with the increasing amplitude of the Shh gradient and occur faster than the tissue grows, implying that progenitor identity is actively specified in response to signaling. At later times, the change in domain sizes can be explained by the proliferation and differentiation of different progenitor types alone.
These findings suggest that scaling of pattern with tissue size does not occur throughout development and the reproducibility of pattern between embryos cannot be explained with a simple gradient scaling model. It suggests a role for the temporal regulation of the differentiation rate in regulating the formation of patterns in developing organs.
Expected final results and potential impact and use:
Our data suggests a 2-phase model of patterning: early domain size depends on switches of cell identity, which are instructed by Shh signaling, whereas the late elaboration of pattern depends on proliferation and differentiation. Ongoing experiments are designed to test and validate this hypothesis. Potentially, this finding would have impact for understanding the mechanisms of establishment of spatial patterns of cell differentiation during development. Our data suggests that for the emerging morphology of developing organs, the initial establishment of a pre-pattern and the subsequent temporal control of the differentiation rate are key. This could be applicable to medically relevant tissue-engineering approaches and the field of regenerative medicine, as well as to stem-cell research.