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NATIONAL INSTITUTE OF GENETICS
Mammalian Development Laboratory

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RESEARCH

Reserch

  1. Genetic technologies in the mouse
  2. Molecular mechanisms of somite segmentation
  3. Germ cell development
  4. The roles of Notch signaling in cardiovascular development

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2. Molecular mechanisms of somite segmentation

Purpose of study:

Animal bodies including the human body are composed of metameric structures, such as vertebra, ribs, spinal nerve system and blood vessels. How are these structures established? The question can be addressed by asking how somites are generated during embryogenesis, because somites provide the basic structural information to build body architectures.


Background:

  1. Importance of somites
    Somites are generated by a dynamic morphogenetic event called somitogenesis that occurs as a temporal event during embryogenesis (from embryonic day 8 to 13).

    Fig.1.Mouse somitogenesis
    Our body is primarily generated as a structure composed of only head and tailbud. The intermediate body region is filled in subsequently. The tailbud is the source that provide new cells (known as presomitic mesoderm: PSM). These cells are precursors of somites that are sequentially subdivided as somites from anterior end of the PSM to the posterior direction every 2 hours (Fig.1). The segmental structure acts as a reference for segmental patterns of blood vessels, neural networks and vertebra. This is the reason why mutations involved in somitogenesis are known as causable genes of genetic diseases. Especially, defects in vertebral column may induce life-threatening severe symptom. Thus somitogenesis has to be under a strict and robust genetic control.
  2. Segmentation clock
    What is measuring the timing of the clock and how is it being kept precisely? There are many mathematical models to explain this interesting clock mechanism. The most famous and convincing model is the so-called “Clock and wavefront” model. This model predicts two components; one is the clock that measures the times, which determines the period for somite formation. The other is spatial information (wavefront) which determines the place of segmentation and is defined as a constant distance from the posterior end of the tailbud. The model is proposed in 1977, well before any molecular studies were feasible, but has now been validated as the molecular mechanism. Notch signal works as a switch to turn on the timer and wavefront is defined by FGF gradient. Next important question is what happens once the switch is turned on? We know the answer that a transcription factor called Mesp2 is turned on. Actually Mesp2 is the most important and critical gene to trigger somite formation (boundary formation).
    Fig.2.Clock and wavefront model
    Without Mesp2 (Mesp2-KO mouse), no segmental border is generated. We also have generated Mesp2-venus knockin mouse in which Mesp2 expression can be visualized in vivo (Fig. 3). The observation clearly showed that Mesp2 protein expression oscillates in the anterior PSM where the next segmental border will be generated.
    By the way, what is the mechanism that operates the clock? We now know that all cells in the PSM have clocks. We can visualize the activity via analyzing Notch activity and gene expression patterns such as Hes7 and Lunatic-fringe. Hes7 is a transcriptional repressor, which can repress its own and L-fng transcription once it is translated to the protein. L-fng is a glycosyltransferase that can modify extracellular domain of Notch receptors and represses Notch activity. This kind of negative feedback mechanism allows clock components oscillate within cells (Fig.4). However, without coupling mechanism, PSM cells never behave in a collective manner, which result in the failure of somite formation. All PSM cells should have mechanism to adjust own clock with others. The identification of the coupling mechanism of segmentation clock in mouse is one of our projects (Project 1). In addition, we are investigating the mechanism of somite segmentation via analyzing the function of transcription factor Mesp2(Project 2 &3).

Project 1: The coupling mechanism of segmentation clock in mice

The coupling mechanism of segmentation clock is understood in zebrafish. However, the mechanism in mice is not known yet. We are currently trying to reveal the mechanism using chimera analyses composed of cells derived from wild-type and gene-knockout mice. In addition, we are planning to use computer simulation which is a strong tool to tackle this kind of complex mechanism.


Fig. 4.The clock mechanism

Project 2:The mechanism to generate segmental border formation

How is the segmental border determined? This question can be changed to how Mesp2 is regulated. Notch signaling is required for Mesp2 expression. But in addition, Tbx6 transcription factor plays a critical role. Tbx6 directly binds to the Mesp2 enhancer and up-regulate the transcription. The expression pattern of Tbx6, especially the anterior border is important since the border completely matches to that of Mesp2 expression domain. Thus the anterior border of Mesp2 expression is defined by Tbx6, which corresponds to the next somite boundary. Furthermore, once Mesp2 is expressed in the Tbx6-dpendent manner, Tbx6 protein expression is suppressed in the Mesp2-expressing cells. As the result, the next anterior border of Tbx6 expression domain is determined.(Fig. 5)。These events are repeated every 2 hours. The detailed molecular mechanism of how Tbx6 protein expression is regulated is still unknown.


Fig. 5 Mesp2 generates segmental border by suppressing Tbx6

Project 3:The mechanism to establish rostral-caudal patterning within a somite

Bones of spine (vertebra) is derived from somitic cells. The anterior-posterior patterning of a single vertebra is prefigured within a somite composed of two distinct compartments (rostral and caudal compartments). The pedicle of neural arch is exclusively derived from caudal half of a somite (Figure 6). Mesp2 and Notch signaling play important roles in the establishment of the rostral-caudal polarity. Mesp2 defines rostral identity of a somite via suppressing Notch signaling pathway. That is why Mesp2-KO mouse have fused vertebra composed of pedicle of neural arch. Actually Mesp2 expression is finally restricted in the rostral half of the presumptive somite. However, the mechanism of how Mesp2 suppresses Notch signaling is still elusive.


Fig. 6.The mechanism to establish Rostral-caudal polarity

Future direction:

Our studies regarding the function of transcription factor Mesp2 receive international evaluation. We are one of the leading labs in the research field of somitogenesis. We are now aiming to reveal molecular mechanisms of phenomena that had been demonstrated via mouse genetics. What are the protein partners of Mesp2 and how do these protein complexes achieve the function? What are the real target genes of Mesp2 and how many genes are targeted and regulated by Mesp2? We need biochemical approach and new technology to address these questions.

 




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