Published: 28 February 2024 Cellular and molecular control of vertebrate somitogenesis Yuchuan Miao & Olivier Pourquié Nature Reviews Molecular Cell Biology volume 25, pages517–533 (2024)

https://www.nature.com/articles/s41580-024-00709-z

 

下の動画の説明がわかりやすかった。
Somite Formation & 90min clock Crash Biology チャンネル登録者

 

FGFの体節形成に対する抑制的な役割

FGF (Fibroblast Growth Factor) does play a role in suppressing new somite formation, particularly in the posterior presomitic mesoderm (PSM). High levels of FGF in the posterior PSM maintain cells in an unsegmented, undifferentiated state. This suppression helps create the gradient required for the orderly, sequential formation of somites as cells move from the posterior to the anterior region of the PSM.

If FGF signaling is experimentally suppressed or reduced:

1. Premature Somite Formation: Cells that would normally be held in an unsegmented state in the posterior PSM can undergo premature segmentation. This can lead to the formation of somites before the cells reach the proper position in the anterior PSM, where segmentation is normally coordinated.
2. Disruption of Rostrocaudal Polarity: Lowered FGF levels might interfere with the normal rostrocaudal patterning within somites. FGF signaling is also involved in establishing the correct polarity within each somite, so its suppression can cause defects in somite boundary formation and polarity.
3. Alteration of Determination Front Position: The “determination front” — the zone where cells transition from unsegmented to segmented states — is influenced by the balance of FGF and RA gradients. Suppressing FGF shifts this determination front posteriorly, resulting in somites forming prematurely, closer to the posterior PSM.

Experimental studies have shown that reducing FGF signaling can indeed lead to the formation of smaller, irregularly patterned somites, as cells segment earlier than intended. This highlights FGF’s role in controlling the timing and spatial pattern of somite formation, ensuring that somites form in an orderly, regulated manner as cells progress through the PSM.

(ChatGPT)

FGFシグナリング、WntシグナリングによるTbx6遺伝子発現の促進

FGF and Wnt signaling are critical for activating and maintaining Tbx6 gene expression in the posterior presomitic mesoderm (PSM) during somitogenesis. Here’s how these pathways regulate Tbx6:

1. Activation and Maintenance of Tbx6 Expression: High levels of FGF and Wnt signaling in the posterior PSM provide a supportive environment that promotes Tbx6 expression. These signals maintain Tbx6 at levels necessary for keeping cells in an undifferentiated, mesenchymal state, preparing them for future segmentation as they move toward the anterior.
2. Regulation Through Gradients: FGF and Wnt form gradients that are highest in the posterior PSM and gradually decrease toward the anterior PSM. As cells “move” or are positioned closer to the anterior, they experience decreasing levels of FGF and Wnt, leading to the downregulation of Tbx6. This shift in signaling cues is essential for transitioning cells out of the Tbx6-expressing, unsegmented state, allowing them to prepare for segmentation and somite boundary formation.
3. Indirect Activation: While FGF and Wnt signaling are not transcription factors that bind directly to the Tbx6 promoter, they activate Tbx6 expression through downstream effectors and pathways within the cell. These downstream factors likely include various transcriptional regulators that respond to FGF and Wnt signals, which in turn promote Tbx6 transcription in the posterior PSM.

In summary, FGF and Wnt signaling indirectly activate and maintain Tbx6 expression in the posterior PSM, playing an essential role in somitogenesis by supporting Tbx6‘s role in maintaining the unsegmented, mesenchymal state of PSM cells.

(ChatGPT)

Tbx6の体節形成における抑制的な役割

In somitogenesis, Tbx6 is a key transcription factor that plays a crucial role in maintaining the undifferentiated, mesenchymal state of cells in the presomitic mesoderm (PSM) and promoting their commitment to the paraxial mesoderm lineage. This role is essential for the orderly formation of somites along the anterior-posterior axis. Here’s a detailed look at Tbx6’s role in somitogenesis:

1. Specifying Paraxial Mesoderm Lineage: Tbx6 helps specify cells in the PSM as paraxial mesoderm, which gives rise to somites. In the absence of Tbx6, PSM cells may adopt alternative fates, such as forming neural tissue instead of somites. This lineage specification function is critical in ensuring that the correct tissue types emerge in the right positions during development.
2. Maintaining Cells in an Undifferentiated State: Tbx6 expression is high in the posterior PSM, where it keeps cells in an undifferentiated and mesenchymal state, preventing them from undergoing premature segmentation. This maintenance of an undifferentiated state is essential for the proper timing of somite formation. When cells move toward the anterior PSM (注）, Tbx6 expression decreases, which allows them to transition toward epithelialization and segmentation.
3. Coordination with FGF and Wnt Signaling: Tbx6 expression is influenced by FGF and Wnt signaling gradients, which are high in the posterior PSM and gradually decrease toward the anterior. These signaling pathways help maintain Tbx6 expression in the posterior PSM, where cells are held in a proliferative, unsegmented state. As cells approach the anterior, decreasing FGF and Wnt signals lead to downregulation of Tbx6, allowing cells to exit the proliferative state and begin the process of segmentation.
4. Regulation of Somite Boundary Formation: Although Tbx6 itself does not directly trigger the mesenchymal-epithelial transition (MET) needed for somite boundary formation, it creates the conditions necessary for cells to respond to other cues, such as Mesp2, that initiate boundary formation. As cells exit the Tbx6-expressing region, they become competent to form boundaries and undergo MET, marking the anterior edge of a new somite.
5. Interaction with Mesp2 for Rostrocaudal Polarity: Tbx6 indirectly influences the establishment of rostrocaudal (head-to-tail) polarity within somites by interacting with pathways that activate Mesp2 expression in the anterior of each forming somite. Mesp2 expression is critical for defining the anterior-posterior polarity, which is essential for the correct alignment and differentiation of somites. Proper timing of Tbx6 downregulation is necessary for Mesp2 to function appropriately in defining this polarity.

In summary, Tbx6 is essential for maintaining the mesenchymal state and paraxial mesoderm identity of cells in the PSM, coordinating with signaling pathways to control the timing and positioning of somite formation. By controlling when and where cells become competent to segment, Tbx6 ensures the sequential and ordered formation of somites, a fundamental aspect of vertebrate body segmentation.

(ChatGPT)

（注）上の説明でmoveという言葉が使われていたので、てっきり細胞移動が起こると説明しているのかと思い、？？でしたが、よくよく聞いてみたら物理的な移動を意味するmoveではないとのことでした。

In this context, “move toward the anterior PSM” does not imply physical movement of individual cells. Instead, it refers to the positional progression of cells along the anterior-posterior axis within the presomitic mesoderm (PSM) due to developmental processes.

More accurately, what happens is this:

• Cells in the PSM are gradually positioned closer to the anterior as new cells are added posteriorly due to tail bud growth and axial elongation. This process creates a progressive shift without requiring individual cell migration.
• As cells reach the anterior PSM (or the “determination front”), their exposure to signaling gradients changes (with decreased FGF/Wnt and increased RA), which influences gene expression patterns, such as the downregulation of Tbx6.

Thus, a clearer phrasing might be: “As cells are positioned closer to the anterior PSM…”

(ChatGPT)

英語のmoveという言葉には、物理的な移動だけでなく状況の変化を意味する用法もあるというわけです。

In English, the word “move” can indeed have both literal and figurative meanings, depending on the context:

1. Literal Meaning (Physical Movement): “Move” often implies physical movement of an object from one place to another. For example, “The car moved down the street” means the car physically traveled along the road.
2. Figurative or Conceptual Meaning (Change Without Physical Movement): “Move” can also describe changes that are not physical. For instance:
   • Position in a sequence or process: “The project moved to the next phase” implies progression in steps rather than physical movement.
   • Change in state or condition: “He moved to a new job role” indicates a change in status or position without physical relocation.
   • Shift in time or focus: “We moved the deadline” or “The story moves to a new topic” means a change in timing or subject matter, not a physical shift.

In scientific or technical language, “move” is often used figuratively to describe progression, transition, or reconfiguration within a defined space or system, even if no physical movement occurs. For instance, in biology, when we say cells “move toward differentiation,” it may mean they progress through developmental stages without physically migrating.

(ChatGPT)

 

レチノイン酸の役割

以下,ChatGPT 4oの回答ですが、RAはMesp2遺伝子の発現を直接、活性化するわけではないようです。FGFが抑制していたものを解き放つという、間接的な形での作用だとのこと。

In somitogenesis, retinoic acid (RA) plays a key role in setting up the anterior-posterior (A-P) gradient within the presomitic mesoderm (PSM). This gradient interacts with other signaling gradients, particularly those of fibroblast growth factor (FGF) and Wnt, to establish a regulatory environment that is crucial for segmental patterning.

RA does not directly activate the expression of Mesp2, a transcription factor essential for somite boundary formation and rostrocaudal polarity. Instead, RA indirectly influences Mesp2 expression by opposing the posteriorizing effects of FGF and Wnt signaling in the PSM. High levels of FGF and Wnt signaling in the posterior PSM inhibit Mesp2 expression, whereas RA, which is synthesized in the anterior region, helps to repress FGF and Wnt as cells progress anteriorly. This repression allows the anterior PSM cells to express Mesp2 in a specific region, known as the “determination front,” where conditions are favorable for somite segmentation.

Thus, RA’s role in somitogenesis is to help balance the signaling environment in the PSM by counteracting FGF and Wnt, creating a permissive environment for Mesp2 expression at the right place and time, rather than acting as a direct activator of Mesp2.

レチノイン酸のFGFシグナリングやWntシグナリングに対する抑制的な作用

レチノイン酸とFGF/Wntは拮抗した作用をもつわけですが、具体的に何を抑制するのでしょうか。FGFやWntの遺伝子発現そのものを抑制するわけではなく、例えばFGFシグナリングの要素であるマップキナーゼを脱リン酸化する酵素の遺伝子発現を促進することでFGFシグナリングを弱めたりするのだそうです。またFGF受容体の遺伝子発現を抑制するという報告もあるそう（以下のChatGPTの説明を参照）。

Retinoic acid (RA) opposes FGF and Wnt signaling in the presomitic mesoderm (PSM) through several mechanisms, but it does not primarily act by directly suppressing the transcription of FGF or Wnt genes. Instead, RA counteracts FGF/Wnt signaling by influencing downstream signaling pathways and components in the following ways:

1. Inhibition of FGF and Wnt Signaling Pathways:
   • RA can indirectly interfere with FGF and Wnt signaling by modulating the expression of downstream targets or components within these pathways.
   • For example, RA has been shown to induce the expression of MAP kinase phosphatases (such as Dusp6), which inactivate components of the FGF signaling cascade, thereby reducing FGF signaling activity. This reduces the phosphorylation and activity of key FGF signaling molecules, effectively dampening FGF’s influence in the anterior PSM.
2. Antagonistic Gradient Formation:
   • RA and FGF/Wnt establish opposing gradients within the PSM. RA is synthesized in the anterior PSM and diffuses toward the posterior, whereas FGF and Wnt are expressed more strongly in the posterior and decrease toward the anterior.
   • This spatial gradient positioning allows RA to gradually counterbalance the effects of FGF and Wnt signaling as cells move anteriorly. Rather than directly suppressing FGF or Wnt gene transcription, RA establishes a microenvironment in the anterior PSM where FGF/Wnt signaling becomes progressively less dominant.
3. Indirect Regulation of Oscillatory and Segmental Gene Expression:
   • RA promotes the expression of genes required for segmentation and differentiation, such as Mesp2, which is necessary for somite boundary formation. This indirectly opposes the role of FGF and Wnt in maintaining cells in an oscillatory, undifferentiated state.
   • By promoting segmental gene expression, RA effectively counteracts FGF/Wnt-driven oscillatory gene activity, allowing cells to exit the oscillatory phase as they approach the determination front.
4. Suppression of FGF Receptor Expression:
   • In some studies, RA has been shown to downregulate the expression of FGF receptors (e.g., Fgfr1), which decreases the responsiveness of cells to FGF signals as they move toward the anterior PSM. This reduction in receptor expression decreases FGF pathway activity without directly impacting FGF ligand expression.
5. Regulation of Cell Cycle and Differentiation States:
   • RA promotes cell cycle exit and differentiation, which opposes the proliferative effects of FGF and Wnt signaling. By pushing cells toward a differentiated, segmentation-competent state in the anterior PSM, RA helps counteract the posteriorizing effects of FGF and Wnt, which maintain cells in a proliferative, mesenchymal, and unsegmented state.

Summary

In summary, RA opposes FGF and Wnt signaling mainly through indirect mechanisms rather than directly suppressing FGF or Wnt gene transcription. It modulates the signaling environment by affecting downstream signaling components, receptor expression, and promoting differentiation. These actions ensure that the determination front is established at the correct position within the PSM, allowing for the orderly transition from oscillatory to segmented states necessary for somite formation.

(ChatGPT 4o)

FGFやWntと周期的に変動する遺伝子との関係

In somitogenesis, FGF (Fibroblast Growth Factor) and Wnt signaling interact with Notch signaling to regulate the timing, positioning, and progression of somite formation. Here’s how FGF and Wnt influence Notch signaling within this developmental process:

1. Establishing the Posterior-to-Anterior Gradient

• FGF and Wnt form a gradient across the presomitic mesoderm (PSM), with high concentrations in the posterior PSM (closer to the tail end) and decreasing toward the anterior PSM.
• This gradient creates a “wavefront” that determines where cells are kept in an undifferentiated, oscillatory state versus where they are able to stop oscillating and begin somite formation.

2. Maintaining the Oscillatory State of Notch Signaling in the Posterior PSM

• High FGF and Wnt levels in the posterior PSM promote and maintain oscillatory gene expression, including that of Lfng (lunatic fringe), Hes7, and Delta in the Notch pathway.
• These oscillations are crucial for the “segmentation clock” that drives periodic Notch signaling activity. FGF and Wnt signaling help sustain this clock in the posterior region by keeping cells in a proliferative, undifferentiated state.

3. Influencing the Determination Front

• As cells progress anteriorly in the PSM, they encounter decreasing levels of FGF and Wnt and increasing levels of retinoic acid (RA).
• At a certain threshold—called the determination front—the low levels of FGF and Wnt allow cells to exit the oscillatory Notch signaling state and stabilize their gene expression patterns, leading to somite boundary formation.
• This interaction sets the position at which the oscillatory clock stops, allowing Notch signaling to stabilize and initiate differentiation.

4. Regulating Somite Boundary Formation

• Notch signaling is essential for establishing the boundaries of somites, and the cessation of oscillatory Notch activity is key to determining where boundaries form.
• By controlling the oscillatory vs. stabilized states of Notch signaling, FGF and Wnt ensure that somite boundaries are positioned correctly along the anterior-posterior axis.

5. Temporal and Spatial Coordination of Somite Formation

• Wnt signaling also works alongside FGF to influence the periodicity of the segmentation clock by modulating the expression of clock genes like Hes7 and Lfng.
• By maintaining cells in an oscillatory state until they reach the determination front, FGF and Wnt ensure that Notch signaling follows a precise temporal pattern that matches the physical positioning along the body axis, contributing to the regular, sequential formation of somites.

Summary

In somitogenesis, FGF and Wnt signaling maintain Notch signaling in an oscillatory state in the posterior PSM, essential for the segmentation clock. As cells move toward the anterior, lower FGF/Wnt levels allow them to exit this oscillatory state at the determination front, where Notch signaling stabilizes to form somite boundaries. This regulation ensures precise timing and spatial organization in somite formation.

(ChatGPT)

周期的に変動する遺伝子

周期的に変動する遺伝子がいくつかありますが、どれが一番の発信源といえるのでしょうか。Deltの遺伝子発現には周期性がありますが、Notchにはないようです。ノッチシグナリングとしては周期性があるという言い方は可能です。

In the oscillatory system of somitogenesis, Notch signaling is generally considered the most upstream component that initiates and regulates the segmentation clock, of which HES genes (like Hes1 and Hes7) are key downstream oscillatory elements.

Reasons Why Notch is Upstream of HES

1. Activation of HES Genes by Notch Signaling:
   • Notch signaling is activated when Delta ligands bind to Notch receptors on neighboring cells. This interaction triggers the Notch intracellular domain (NICD) to be released and translocate to the nucleus, where it activates the transcription of downstream target genes, including the HES family (e.g., Hes1, Hes7).
   • Thus, Notch signaling initiates the oscillatory expression of HES genes in the presomitic mesoderm (PSM).
2. Synchronization Role of Notch:
   • Notch signaling is also critical for synchronizing oscillations between neighboring cells in the PSM, ensuring coordinated oscillatory gene expression across cells that will form a somite boundary. This intercellular communication mediated by Notch and Delta helps maintain the rhythmic, synchronized oscillations of HES genes across the tissue.
3. Regulation by FGF and Wnt Gradients:
   • Notch activity, influenced by upstream FGF and Wnt gradients, helps establish the oscillatory environment in the posterior PSM. These gradients set up the context for Notch signaling to initiate the oscillations, positioning Notch as a primary regulator in the segmentation clock.
4. Feedback Mechanism with HES Genes:
   • HES proteins, once expressed, participate in a negative feedback loop to repress their own transcription and the transcription of other oscillatory genes. However, without initial Notch signaling, this feedback loop in HES genes would not be activated, making Notch the upstream trigger.

Summary

In somitogenesis, Notch signaling is the most upstream component of the oscillatory system, activating and coordinating the expression of HES genes and other oscillatory genes within the segmentation clock. HES genes then function in feedback loops as downstream effectors, creating the cyclic gene expression patterns essential for somite formation.

Notchは周期的に変動しないことに関して

In somitogenesis, Notch gene expression itself does not oscillate like Delta; rather, Notch signaling activity oscillates due to the oscillatory expression of its ligands (like Delta) and downstream effectors (such as HES genes).

Here’s how Notch signaling oscillations occur without Notch gene expression itself being oscillatory:

1. Constant Notch Receptor Expression:
   • The expression of Notch receptors (such as Notch1 in mice) remains relatively stable in the presomitic mesoderm (PSM) and does not oscillate. Instead, the Notch receptor is always present on the cell surface, ready to respond to Delta ligand binding.
2. Oscillatory Delta Ligand Expression:
   • Delta ligands (e.g., Dll1 in mice) do oscillate in their expression, driven by the segmentation clock. When Delta levels rise on the surface of one cell, it binds to Notch receptors on neighboring cells, activating Notch signaling in those cells in an oscillatory manner.
   • This oscillatory expression of Delta ligand initiates periodic activation of Notch signaling in adjacent cells, creating a synchronized oscillation of Notch pathway activity across the PSM.
3. Oscillatory Downstream Targets (HES Genes):
   • The activation of Notch by oscillating Delta triggers the expression of downstream targets like HES genes (e.g., Hes1, Hes7), which also oscillate. HES proteins provide negative feedback that helps control the oscillatory cycle of Delta and other segmentation clock genes.
4. Intercellular Synchronization:
   • The stable presence of Notch receptors and the oscillatory expression of Delta enable the synchronized activation of Notch signaling across neighboring cells. This intercellular communication is critical for maintaining coordinated oscillations in the PSM, which ultimately determine the timing and location of somite boundaries.

Summary

While Notch gene expression itself does not oscillate in somitogenesis, the Notch signaling pathway oscillates due to the periodic expression of Delta ligands and HES gene feedback loops. This oscillatory signaling is essential for the segmentation clock and synchronized somite formation across the presomitic mesoderm.

Notch-Deltaシグナリングの役割

下の動画はノッチシグナリングレポーターによるノッチシグナリングの可視化。レポーターの設計の原理は下の論文を参照。

Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA binding protein CSL [CBF1/RBPjκ/Su(H)/Lag1] to activate gene expression.　luciferase complementation imaging (LCI) reporters for NICD-CSL association　https://pubmed.ncbi.nlm.nih.gov/21775282/

Oscillatory Notch signalling during somite boundary formation, imaged using SPIM The Company of Biologists チャンネル登録者数 5930人

 

同様にChatGPTの回答です。

In somitogenesis, the Notch-Delta signaling pathway is essential for coordinating the timing and spatial organization of somite formation. This pathway functions as part of a molecular “segmentation clock,” which ensures that somites form in a regular, sequential manner along the anterior-posterior (A-P) axis of the developing embryo.

Key roles of Notch-Delta signaling in somitogenesis include:

1. Oscillatory Gene Expression: Notch-Delta signaling drives oscillations of specific “clock genes” in the presomitic mesoderm (PSM), such as Hairy1, Hes7, and others, which cycle in a periodic manner. This oscillation occurs with a set frequency, creating a wavefront of gene expression that moves through the PSM. This “segmentation clock” is crucial for determining the periodicity of somite formation.
2. Synchronization of Cells: Notch-Delta signaling helps synchronize the oscillatory behavior of neighboring cells in the PSM. Through intercellular communication, Delta ligands from one cell bind to Notch receptors on adjacent cells, coordinating their segmentation clocks. This synchronization ensures that cells within a future somite boundary express genes likeMesp2 together, enabling the precise formation of somites.
3. Boundary Formation and Rostrocaudal Polarity: Notch signaling is critical in specifying somite boundaries and establishing rostrocaudal (head-to-tail) polarity within each somite. At the determination front, where somites bud off from the PSM, Notch signaling interacts with factors like Mesp2 to trigger the epithelialization process that creates the somite boundary. The periodic activation of Notch also helps establish differential gene expression within each somite, giving them a distinct anterior and posterior polarity.
4. Coordination with FGF and RA Gradients: Notch-Delta signaling is integrated with FGF and RA gradients in the PSM, which help establish the anterior-posterior axis. Notch oscillations occur within a field shaped by these gradients, defining the region where somites form (the “determination front”). As the PSM cells move out of the FGF-rich posterior region toward the RA-rich anterior region, Notch signaling cues become crucial in determining where and when a somite boundary will form.

In summary, Notch-Delta signaling coordinates the temporal and spatial patterning of somite formation by driving oscillatory gene expression, synchronizing cell behaviors, defining boundaries, and interacting with other signaling pathways. This fine-tuned regulation is critical for forming the segmented structure of the vertebrate body.

(ChatGPT)

In somitogenesis, Delta (a ligand in the Notch signaling pathway) exhibits oscillatory gene expression primarily through its regulation by the segmentation clock, which is driven by negative feedback loops involving oscillatory genes like Hes family members (e.g., Hes1 and Hes7). Here’s a breakdown of the mechanism behind Delta’s oscillatory expression:

1. Notch-Delta Signaling and Feedback Loop:
   • Delta ligands (e.g., Dll1 in mice) are part of the Notch signaling pathway, which is crucial for coordinating the segmentation clock across neighboring cells in the presomitic mesoderm (PSM).
   • When Delta binds to Notch receptors on neighboring cells, it activates the Notch pathway, leading to the expression of downstream genes, including Hes genes. This interaction synchronizes oscillatory gene expression between neighboring cells, which is essential for organized somite formation.
2. Hes Genes as Transcriptional Repressors:
   • Hes genes, activated by Notch signaling, function as transcriptional repressors and play a key role in generating oscillations. For example, Hes7 represses its own transcription as well as that of Delta by binding to their promoters, creating a negative feedback loop.
   • After Hes proteins are synthesized, they inhibit further transcription of their own gene and Delta. As Hes proteins degrade, repression is lifted, allowing Delta and Hes transcription to restart, resulting in a cycle of oscillatory expression.
3. Negative Feedback with a Time Delay:
   • This oscillatory behavior relies on a time delay in the negative feedback loop, which is a function of mRNA and protein synthesis, degradation rates, and cellular delays in transcriptional repression.
   • The degradation of Hes proteins and mRNA is relatively rapid, and once they are sufficiently degraded, repression is released, allowing a new cycle of Delta and Hes expression to begin. This delay-dependent negative feedback is critical for generating stable oscillations.
4. Synchronization Across Cells via Notch-Delta Interactions:
   • Notch-Delta signaling between neighboring cells helps synchronize their oscillatory patterns. Delta expression in one cell activates Notch in an adjacent cell, which in turn influences Hes gene expression in that neighboring cell, creating coordinated oscillations.
   • This intercellular coupling ensures that Delta expression oscillates synchronously across cells in the PSM, which is essential for creating uniform, periodic somite boundaries.
5. Interaction with FGF and Wnt Gradients:
   • Delta oscillations also interact with the FGF and Wnt signaling gradients across the PSM. These gradients are high in the posterior PSM and decrease toward the anterior, modulating the pace and location of Delta oscillations.
   • As cells move toward the anterior PSM, where FGF and Wnt levels are lower, Delta oscillations slow and eventually stabilize, marking the transition point where somite boundaries begin to form.

Summary

Delta’s oscillatory expression in somitogenesis is driven by a negative feedback loop involving Hes genes, with a time delay that produces cyclical repression and derepression. This oscillation is synchronized across cells by Notch-Delta signaling between neighboring cells, and modulated by the FGF and Wnt gradients to ensure coordinated somite formation.

(ChatGPT)

Lunatic fringe (Lfng)

Lunatic fringe (often abbreviated as Lfng) is a gene that encodes an enzyme known as a glycosyltransferase, which modifies the Notch receptor, influencing how it responds to Delta and other Notch ligands. It plays an important role in regulating Notch signaling oscillations during somitogenesis. Here’s a breakdown of lunatic fringe’s function, its name, and how it fits into the Notch signaling pathway.

1. What Lunatic Fringe Does

• Role in Notch Signaling: Lunatic fringe modifies the Notch receptor by adding sugars to it (glycosylation). This modification changes the sensitivity of Notch to its ligands, including Delta-like (Dll1) and Jagged ligands.
• Oscillatory Expression: Like Delta and HES genes, Lfng expression oscillates in the presomitic mesoderm (PSM) as part of the segmentation clock. These oscillations help regulate the timing of Notch activation, contributing to the rhythmic formation of somites.
• Function in Somite Formation: By modulating Notch signaling, lunatic fringe helps maintain the periodic activation of Notch in a way that coordinates somite segmentation across neighboring cells.

2. Literal Meaning of the Name “Lunatic Fringe”

• The name “lunatic fringe” is somewhat playful, inspired by the idea of an “edge” or “fringe” that behaves in a periodic or “lunatic” (crazy, unpredictable) manner.
• Fringe refers to the family of fringe proteins (other members include manic fringe and radical fringe), which all influence Notch signaling by modifying the Notch receptor’s response to its ligands.
• Lunatic implies something erratic or periodic, likely a nod to the cyclic or “oscillatory” behavior seen in Notch signaling and segmentation, somewhat like the moon’s periodic phases (the root “luna” refers to the moon).

Summary

Lunatic fringe is a glycosyltransferase enzyme that modifies the Notch receptor, affecting how it responds to ligands like Delta. Its oscillatory expression is crucial for somite formation in somitogenesis. While it is neither Delta nor HES, it plays a critical role in controlling the oscillations of Notch signaling that drive the segmentation clock. The name “lunatic fringe” reflects the periodic or “fringe” nature of its action in the oscillatory system of somitogenesis.

(ChatGPT)

下の動画はマウス胚で体節が形成されるごとに遺伝子発現が変動するLunatic fringeという遺伝子。

Imaging Clock Oscillations in the Mouse Embryo Cell Press チャンネル登録者数 2.58万人

Wave of gene expression in an embryo European Molecular Biology Laboratory (EMBL) チャンネル登録者数 1.34万人
In vivo imaging of gene activity oscillations — Two-photon time-lapse imaging of using the LuVeLu transgenic line (reporting lunatic fringe gene activity); side view on the paraxial mesoderm, merged YFP and brightfield channel. Real-time recordings reveal oscillatory patterns of gene activity (Venus channel), sweeping from posterior to anterior mesoderm as periodic waves. Simultaneously, segment formation occurs where waves come to halt. Credit: Nature

Hairy

HES1はHairy and Enhancer of Split 1の略で、Hairy遺伝子ファミリーの一部です。 HES1は、哺乳類のHairyホモログであり、Notchシグナル伝達経路によって誘導される転写因子です。もともとHairyはショウジョウバエ（Drosophila）で発見され、発生中の体節形成や遺伝子発現の制御に関与しています。哺乳類では、HES1やHES7などが体節形成や神経発生の際に遺伝子発現の振動を担い、体の発生において重要な役割を果たしています。 このため、HES1は「哺乳類のHairy遺伝子の一種」として位置づけられ、振動的な発現パターンを示す遺伝子として、発生学的な研究で頻繁に参照される遺伝子の1つです。

Avian hairy Gene Expression Identifies a Molecular Clock Linked to Vertebrate Segmentation and Somitogenesis Isabel Palmeirim1 ∙ Domingos Henrique2,§ ∙ David Ish-Horowicz2 ∙ Olivier Pourquié CELL Volume 91, Issue 5p639-648November 28, 1997 https://www.cell.com/fulltext/S0092-8674%2800%2980451-1

 

 

新しく形成される体節の「境界」を決めるシグナル

ChatGPTの回答。

The specification of the caudal (posterior) boundary of a newly formed somite is tightly regulated by several interacting signaling pathways and genes. Key signals and regulatory elements involved include:

1. Notch Signaling: Notch signaling is crucial for determining the rostrocaudal (head-to-tail) polarity within each somite. In particular, Mesp2, a downstream target of Notch, is expressed in the anterior portion of the forming somite. This differential expression contributes to defining the boundary between the rostral (anterior) and caudal (posterior) compartments of each somite. Notch activity helps restrict Mesp2 expression, thereby influencing the formation of the boundary at the caudal end.
2. FGF and Wnt Gradients: Fibroblast Growth Factor (FGF) and Wnt signaling gradients are high in the posterior presomitic mesoderm (PSM) and gradually decrease toward the anterior. As cells move anteriorly within the PSM, they leave the influence of high FGF and Wnt levels, which allows for changes in gene expression critical for somite boundary formation. These gradients work together with Notch signaling to ensure that cells in the anterior PSM can form somites with distinct anterior and posterior regions.
3. RA (Retinoic Acid) Gradient: Retinoic acid, produced in the anterior PSM, creates a gradient opposing that of FGF and Wnt. This gradient helps create a permissive environment in the anterior PSM for somite boundary formation. The balance between RA and FGF/Wnt gradients helps set up the “determination front” where somites form, with the anterior boundary of the new somite influenced by RA and the posterior boundary still affected by FGF and Wnt signaling.
4. Tbx6 and Ripply Genes: Tbx6, a transcription factor expressed in the posterior PSM, helps maintain the unsegmented state of cells in this region. As cells reach the determination front, Tbx6 expression is downregulated in a process partly mediated by the Ripply genes, which are also regulated by Mesp2. This downregulation allows for the differentiation of the anterior and posterior compartments of the somite, specifying the caudal boundary of the newly formed somite.
5. Ephrin/Eph Signaling: Once the somite boundary has been established, Ephrin/Eph signaling becomes active at the newly formed boundary, reinforcing segmentation and boundary stabilization. Ephrin ligands are typically expressed in the posterior cells, while Eph receptors are in the anterior cells of the next forming somite. This interaction helps create a clear caudal boundary for each somite.

Together, these signals and gene interactions establish the caudal boundary of the somite, ensure correct rostrocaudal polarity, and contribute to the distinct segmentation pattern necessary for organized vertebrate body plan development.

新しい体節ができるときにその新しい体節の2つの境界すなわち吻側の境界と尾側の境界は同時に形成されるのかどうかについて

下のChatGPTの回答によれば、尾側の境界が先にできて、そのあとで吻側の境界ができるのだそうです。意外でした。自分は逆かと思いました。

When a new somite forms, the establishment of its anterior and posterior boundaries is a successive process rather than a simultaneous one.

Here’s a breakdown of how these boundaries form:

1. Posterior Boundary Formation: The posterior boundary of the new somite is defined first. This boundary is established as cells in the presomitic mesoderm (PSM) reach the “determination front,” where they encounter conditions favorable for somite segmentation. At this front, signals such as reduced FGF and Wnt levels (alongside elevated RA levels in the anterior PSM) allow these cells to undergo the segmentation process. Notch signaling also plays a crucial role here, helping to define the initial boundary.
2. Anterior Boundary Formation: After the posterior boundary is defined, the anterior boundary of the forming somite is established. This is typically marked by the differential expression of genes like Mesp2, which is initially restricted to the rostral (anterior) half of the somite. This expression leads to the establishment of the anterior boundary, defining the complete somite with distinct anterior and posterior halves.

This stepwise process ensures that each somite forms with the correct orientation and rostrocaudal polarity. By establishing boundaries sequentially, the somitogenesis machinery ensures that each somite is properly aligned with its neighboring segments, creating the precise, segmented structure essential for vertebrate body patterning.

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間葉-上皮 転換

In somitogenesis, the Mesenchymal-Epithelial Transition (MET), which is essential for somite boundary formation, involves the gene Paraxis as well as other key players. Here’s how these genes contribute to the MET process:

1. Paraxis: Paraxis is a basic helix-loop-helix (bHLH) transcription factor that is crucial for the epithelialization of somites. Paraxis promotes the transition of mesenchymal cells in the presomitic mesoderm (PSM) to a more epithelial, organized structure within each somite. In Paraxis mutants, somites fail to undergo proper epithelialization, resulting in disorganized, mesenchymal-like structures rather than well-defined somites.
2. E-cadherin and N-cadherin: Cadherins are adhesion molecules essential for establishing and maintaining the epithelial structure of cells. During MET, cells in the forming somite upregulate cadherins (especially N-cadherin), promoting cell-cell adhesion and the formation of a segmented epithelial structure.
3. Fibronectin and Integrins: These extracellular matrix (ECM) components and receptors help stabilize the epithelial structure within somites. Fibronectin provides structural support in the ECM, and integrins on the cell surface interact with fibronectin to promote cell adhesion and polarity, reinforcing the MET process.
4. Notch Signaling and Mesp2: Notch signaling, via Mesp2, also influences the MET process. Mesp2 expression in the anterior portion of each forming somite contributes to boundary formation and epithelialization, working in concert with Paraxis and cadherins to establish the segmented, epithelial state.

Together, these genes and molecular pathways drive the mesenchymal-epithelial transition in somitogenesis, enabling the formation of organized, epithelialized somites essential for the segmented structure of the vertebrate body plan.

(ChatGPT)

上の説明では体節の前半分という話がでてきましたが、後ろ半分に関してはどうなのでしょうか。

In the posterior portion of each forming somite, the mesenchymal-epithelial transition (MET) and establishment of somite boundaries are regulated by specific genes that contribute to the segmentation and polarization of somites. Key genes essential for MET and somite formation in the posterior region include:

1. Mesp2: Mesp2 is a crucial transcription factor that plays a role in defining the anterior-posterior polarity within each somite. In the anterior portion of a forming somite, Mesp2 expression helps initiate MET and boundary formation. This, in turn, indirectly defines the posterior boundary by establishing a sharp contrast with the posterior region, which has lower Mesp2 expression.
2. Notch Signaling Pathway: The Notch pathway is integral to maintaining the segmentation clock and ensuring that the posterior PSM cells enter a synchronized cycle of differentiation and segmentation. Notch signaling also helps specify the rostrocaudal polarity within each somite. In particular, genes such as Dll1 (Delta-like 1) and Lfng (Lunatic fringe) within the Notch pathway show oscillatory expression patterns that help set the timing and boundary for somite formation. Notch activity is crucial for the posterior portion to remain distinct from the anterior and to respond appropriately during the MET.
3. Uncx4.1 (Uncx): Uncx4.1 is expressed specifically in the posterior half of each somite after it forms. This transcription factor helps maintain the identity and structure of the posterior compartment and is essential for establishing the rostrocaudal (head-to-tail) polarity within the somite. It represses anterior markers and maintains the distinct identity of the posterior portion.
4. Tbx6: Tbx6, expressed in the posterior PSM, helps keep cells in an undifferentiated state until they reach the determination front, where FGF and Wnt levels are reduced. Once cells are positioned within the forming somite, Tbx6 downregulation is required for epithelialization to proceed in the anterior, which indirectly helps establish the posterior identity in cells that did not fully downregulate Tbx6 initially.
5. Ephrin/Eph Signaling: Ephrin ligands (often expressed in the posterior region) and Eph receptors (expressed in the anterior of the next somite) create a boundary of cell-cell repulsion between somites. This signaling enforces a clear distinction between the posterior of one somite and the anterior of the next. Ephrin/Eph interactions are essential for the stabilization and integrity of the posterior boundary.

In summary, genes like Mesp2, Uncx4.1, and components of the Notch, Ephrin/Eph, and FGF signaling pathways collectively ensure that the posterior portion of each somite retains its unique identity and undergoes the MET process in a way that preserves somite polarity and structure. These interactions allow the proper segmentation of the vertebrate body plan during somitogenesis.

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周期的に遺伝子発現レベルが変動する遺伝子の制御

The regulation of oscillatory genes like hairy (known as Hes genes in vertebrates, such as Hes7 in mice) in somitogenesis is governed by a network of signaling pathways and genes that collectively form the segmentation clock. This clock drives the periodic expression of oscillatory genes, which is essential for the timing of somite formation. Key regulators of these oscillatory genes include:

1. Notch Signaling Pathway: Notch signaling plays a central role in regulating oscillatory gene expression in the presomitic mesoderm (PSM). The activation of Notch receptors by ligands such as Delta-like 1 (Dll1) triggers a cascade that induces the transcription of Hes7, a gene that oscillates in a periodic manner. Hes7 itself then functions as a transcriptional repressor, leading to negative feedback that contributes to its oscillatory expression pattern.
2. FGF (Fibroblast Growth Factor) Signaling: FGF signaling, which is high in the posterior PSM, is also involved in the regulation of oscillatory genes. FGF maintains cells in an undifferentiated state in the posterior PSM and influences the periodicity of oscillatory genes. It works in conjunction with Wnt signaling to sustain the oscillatory behavior in the posterior PSM, and as cells progress anteriorly (where FGF levels decrease), the oscillations slow and eventually stop, signaling the onset of somite boundary formation.
3. Wnt Signaling Pathway: Similar to FGF, Wnt signaling is active in the posterior PSM and forms a gradient that decreases toward the anterior. The Wnt pathway contributes to the maintenance and synchronization of oscillatory gene expression by interacting with Notch and FGF signaling pathways. This pathway also influences genes like Axin2, which oscillates in the PSM and is involved in the regulation of the segmentation clock.
4. Negative Feedback Loops: Oscillatory genes, such as Hes7, are regulated by negative feedback mechanisms that are crucial for their periodic expression. Hes7 protein represses its own transcription by binding to its promoter, leading to a delay in re-expression after degradation. This negative feedback loop creates the oscillatory dynamics, with cycles of expression and repression in a tightly regulated timeframe.
5. Intercellular Coupling and Synchronization: The synchronization of oscillations between neighboring cells in the PSM is mediated by Notch signaling. Notch allows cells to communicate and coordinate their oscillatory patterns with one another, ensuring a cohesive wave of gene expression that moves across the PSM. This coupling is essential for creating a uniform segmentation pattern along the anterior-posterior axis.
6. Clock Genes (such as Lfng): Other clock-related genes, like Lfng (Lunatic fringe), also oscillate in synchrony with Hes7. Lfng modulates Notch receptor sensitivity, adding an additional layer of feedback regulation that contributes to the segmentation clock.

In summary, the oscillatory expression of genes like Hes7 (hairy) is regulated by a complex interplay of Notch, FGF, and Wnt signaling pathways, supported by negative feedback loops and intercellular coupling. These regulatory interactions form the segmentation clock that controls the rhythmic, timed formation of somites in somitogenesis.

(ChatGPT 4o)

Mesp2によるEph遺伝子発現

Eph gene expression is indeed driven by Mesp2 during somitogenesis, specifically in the context of establishing somite boundaries and the rostrocaudal (head-to-tail) polarity within each forming somite. Here’s how this relationship works:

1. Activation of EphA4 by Mesp2: Mesp2, which is expressed in the anterior portion of each forming somite, directly induces the expression of EphA4, a member of the Eph receptor family. This expression is crucial for defining the boundary between the anterior and posterior portions of the somite and establishing proper segmentation.
2. Role in Boundary Formation and Polarity: EphA4, once expressed in the anterior half of a somite under the influence of Mesp2, interacts with ephrin ligands (e.g., ephrin-B2) expressed in the posterior half of the neighboring somite. This Eph-ephrin interaction creates a repulsive boundary that physically separates adjacent somites, ensuring they form as distinct segments.
3. Regulation of Rostrocaudal Polarity: By driving EphA4 expression in the anterior portion of each somite, Mesp2 helps establish distinct anterior and posterior identities within each somite. This polarity is essential for correct vertebral patterning and alignment of tissues derived from somites.

In summary, Mesp2 directly activates EphA4 expression, which plays a central role in somite boundary formation and establishing rostrocaudal polarity through interactions with ephrin ligands in adjacent segments. This regulatory pathway ensures that somites form with distinct, polarized boundaries essential for the segmented organization of the vertebrate body plan.

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Eph受容体はMesp2により遺伝子発現が誘導されるという上の説明でしたがephrinリガンドに関しては、そのような制御機構はないようです。また別の仕組みがephrinリガンドの遺伝子発現を制御しているとのこと（下の説明）。

Mesp2 does not directly drive ephrin gene expression. Instead, Mesp2 primarily drives the expression of EphA4 in the anterior portion of each forming somite, while ephrin (specifically ephrin-B2) expression is regulated in the posterior region of the somite through other mechanisms.

Here’s how the relationship between Mesp2, Eph receptors, and ephrins functions in somite boundary formation:

1. Mesp2-Driven EphA4 Expression: Mesp2 induces the expression of EphA4 specifically in the anterior half of the forming somite. This localized EphA4 expression is crucial for the anterior-posterior patterning and segmentation process.
2. Ephrin-B2 Expression in the Posterior: Ephrin-B2 is expressed in the posterior half of the adjacent somite, not directly due to Mesp2 but rather through regulatory mechanisms that establish complementary expression patterns of Eph receptors and ephrins across somite boundaries. The ephrin-B2 gene is expressed in response to other signaling inputs that are distinct from those regulating Mesp2.
3. Eph-Ephrin Interaction and Boundary Formation: The interaction between EphA4 (induced by Mesp2 in the anterior half of a somite) and ephrin-B2 (in the posterior half of the adjacent somite) creates a repulsive boundary between somites. This boundary formation does not require Mesp2 to directly regulate ephrin expression; rather, it relies on the complementary expression and interaction of Eph receptors and ephrins across the somite boundary.

In summary, Mesp2 specifically drives the expression of EphA4 but not ephrin genes. The complementary expression of ephrin-B2 in the posterior region of adjacent somites is regulated by other factors, allowing for the necessary Eph-ephrin interaction that leads to somite boundary formation and segmentation.

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Eph/Ephrinの発現の局在

一つの体節とその直後の体節との境界がどうやってきまるのかというと、「反発シグナル」であるEph（リガンド）/ephrin（受容体）シグナリングが活躍しているそうです。Ephリガンドは体節の尾側の境界になる細胞で発現します。ephrin受容体はその体節の尾側の体節の吻側境界となる細胞に発現します。

Patterning and mechanics of somite boundaries in zebrafish embryos Save Related Papers Chat with paper May 2020Seminars in Cell and Developmental Biology 107(8–10) DOI:10.1016/j.semcdb.2020.04.014 LicenseCC BY-NC-ND 4.0　https://www.researchgate.net/figure/Repulsive-cues-A-Eph-ephrin-molecules-are-expressed-in-stripes-in-the-anterior-PSM_fig2_341505871

Clock-and-wavefront仮説でキリンの首の長さは説明できるのか

The “clock-and-wavefront” hypothesis provides a model to explain the periodic formation and size of somites along the body axis, including how changes in this mechanism can lead to variations in neck length across species, such as the long neck of a giraffe. This model combines two main elements: a segmentation clock (oscillatory genes like Hes) and a wavefront (signaling gradients) that together regulate the number and size of somites, which ultimately determine vertebral length and number. Here’s how this applies to somite length and neck length in species with elongated necks like the giraffe:

1. Segmentation Clock and Somite Number:
   • In the clock-and-wavefront model, the segmentation clock operates via oscillations of genes like Hes7 in the presomitic mesoderm (PSM), which determine the periodicity of somite formation. The frequency of these oscillations sets the timing for each somite’s formation: a faster clock results in smaller somites, while a slower clock results in larger somites.
   • In giraffes, it is not just the somite size but also the number of cervical somites that contributes to their long necks. Giraffes have a greater number of cervical vertebrae due to changes in clock periodicity or regulatory mechanisms that extend the duration of somite formation specifically in the neck region.
2. Wavefront Position and Somite Size:
   • The wavefront in this model is governed by opposing gradients of FGF/Wnt (high in the posterior PSM) and retinoic acid (RA) (high in the anterior PSM). These gradients set a threshold (the wavefront) where cells transition from an oscillatory state to a stable, segmented state, allowing them to form a somite.
   • The speed at which the wavefront moves anteriorly relative to the segmentation clock determines the length of each somite. In species with longer necks, slower wavefront progression or a slower segmentation clock could lead to more numerous and/or larger somites, thus lengthening the neck region.
3. Genetic and Evolutionary Adaptations in Giraffes:
   • In giraffes, genetic modifications likely adjust the clock and wavefront to generate more and/or slightly larger cervical somites compared to shorter-necked animals. Evolutionary adaptations may involve changes in the expression or function of segmentation genes (Hes7, Lfng) or wavefront-controlling signals (FGF/Wnt and RA pathways) specifically in the neck region, resulting in an extended period of somite formation.
   • This extended segmentation in the cervical region leads to a greater number of vertebrae in the neck, thereby lengthening it. This mechanism can work similarly in other animals with elongated body segments, such as snakes, where a higher number of smaller somites along the entire body axis results in a long, flexible body.
4. Somite Length and Scaling:
   • The size of each somite is largely determined by the interplay between the oscillation period of the segmentation clock and the speed of the wavefront. Slower oscillations and/or a more gradual wavefront progression allow each somite to form at a larger size. Conversely, faster oscillations and a rapid wavefront progression create smaller somites.
   • In giraffes, modifications in these parameters during embryonic development result in relatively longer somites in the cervical region, though the exact cellular and molecular adjustments in giraffes are still an area of research.

In summary, the clock-and-wavefront hypothesis explains the length of the giraffe’s neck through modifications in the timing of the segmentation clock, the movement of the wavefront, and the regional regulation of somite number and size. Together, these factors lead to a greater number and/or larger size of cervical somites, contributing to the giraffe’s unique neck length.

(ChatGPT 4o)

Determination frontとは

The “determination front” in somitogenesis is generated by the interaction and balance between opposing gradients of retinoic acid (RA) and FGF/Wnt signaling within the presomitic mesoderm (PSM). This front serves as a threshold boundary where cells transition from an oscillatory state to a stable, segmented state, leading to the formation of somites. Here’s how the determination front is generated and how cells interpret the gradient ratios:

1. Generation of the Determination Front

The determination front is established by:

• Opposing Gradients:
  • FGF and Wnt signaling are highly active in the posterior PSM, where they promote cell proliferation and maintain cells in an undifferentiated, mesenchymal, and oscillatory state.
  • Retinoic acid (RA), produced in the anterior PSM, creates a gradient that opposes FGF and Wnt, promoting differentiation and segmentation.
• Anterior-Posterior Positioning:
  • Cells in the PSM are sequentially positioned along the anterior-posterior axis as the embryo elongates, so as cells move closer to the anterior, they encounter gradually decreasing levels of FGF and Wnt and increasing levels of RA.
  • The determination front forms where these gradients reach a critical balance. This is where the FGF/Wnt signals are low enough, and RA is high enough, to trigger the transition from oscillatory to stabilized gene expression, allowing segmentation to occur.

2. Interpretation of the RA and FGF/Wnt Gradient Ratios

Cells in the PSM interpret the ratio of RA to FGF/Wnt, which serves as a positional cue:

• Threshold Mechanism:
  • Cells use the relative levels of FGF/Wnt and RA to determine whether they are within the oscillatory region (posterior) or the segmentation-competent region (anterior).
  • When FGF/Wnt signals are high relative to RA, cells maintain the oscillatory gene expression pattern (segmentation clock).
  • When RA levels become higher than FGF/Wnt at the determination front, the oscillations stop, and cells enter a segmentation-competent state. This ratio effectively serves as a “threshold” that cells interpret to decide when to commit to somite formation.
• Effect on Oscillatory Gene Expression:
  • High FGF and Wnt signaling maintains the expression of oscillatory genes like Hes7, which are essential for the segmentation clock’s periodic cycles.
  • As cells move anteriorly and the RA/FGF-Wnt ratio increases, oscillatory gene expression is gradually suppressed. This stabilization in gene expression coincides with the expression of segmentation-specific genes like Mesp2 at the determination front, which marks the start of somite boundary formation.
• Gradual Transition from Oscillatory to Stable State:
  • The transition from an oscillatory to stable gene expression state happens progressively as cells move across the determination front.
  • This ratio of RA to FGF/Wnt ensures that segmentation occurs at the correct position, as cells “sense” this gradient ratio and stop oscillating only when they reach the front.

Summary

The determination front is generated by the opposing RA and FGF/Wnt gradients, which establish a positional threshold in the PSM. Cells interpret the ratio of RA to FGF/Wnt as a signal to either maintain oscillatory gene expression (when FGF/Wnt is high) or transition to a stable state for somite formation (when RA is high). This ratio serves as a positional cue, enabling cells to transition to segmentation precisely at the right position along the axis, ensuring orderly somite formation.

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論文

1. Published: 01 July 2008 Control of segment number in vertebrate embryos Nature volume 454, pages335–339 (2008)　https://www.nature.com/articles/nature07020#MOESM224