The role of Notch Signaling Pathway in Xenopus laevis Gastrulation and Neurulation

After fertilization, the egg first divides into two halves forming the 2-cell stage, then the second cleavage creates a 4-cell stage, the third cleavage creates an 8-cell stage, and cell divisions continue until a hollow ball of cells is formed, called the blastula. Then gastrulation begins. Gastrulation marks the onset of changes in cell behaviors that begin to shape an individual (Contakos et al, 2005). The process of gastrulation results in the formation of the three germ layers: ectoderm, mesoderm and endoderm. These germ layers are responsible for organogenesis. However, the signaling pathway that controls nerulation has not been thoroughly studied in Xenopus laevis. Over 100 years ago, the Notch signaling pathway is discovered in Drosophila melanogaster. This pathway controls the segregation of the three germ layers and determines the cell fate (lateral inhibition) of tissues arising from the germ layers in Drosophila, and it’s believed that there must be a homologue of Notch signaling pathway in vertebrates. So, what role does the Notch signaling pathway play in X. laevis gastrulation and nerulation? Recently, there are studies that focus on investigating the role of Notch signaling pathway in Xenopus laevis.

The three Germ Layers (Purves et al, 1998)

Gastrulation (Purves et al, 1998)

A video clip of frog development

1. Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis

  • One experiment was done in 2010 by Revinski et al, investigating whether Delta-Notch signaling is involved in the segregation of the three germ layers by studying the expression of neural, endodermal and mesodermal markers (sox2, sox17alpha, and bra respectively) at gastrula and early neurula stages in embryos where Notch signaling was manipulated. The Notch signaling pathway is activated when a Notch ligand (Delta) binds and interacts with the receptor Notch on the surface of the neighboring cell which triggers the cleavage of Notch receptor intracellular domain NotchICD. NotchICD then enters the nucleus and activate transcription of target genes lead to suppression of the neuronal fate in the cells surrounding the neuronal precursor (Lai, 2004; Fortini, 2009). Notch signaling pathway is inhibited by a member of CSL protein Su(H) (Supressor of Hairless). In this experiment, the pathway is activated by injecting notchICDmRNA, and inhibited by injecting su(H)1DBMmRNA, delta-1STU into the embryos. The control embryos are injected with standard control morpholinos.

Figure 1: Effects of NotchICD on neural and paraxial mesodermal markers at the neural plate stage (Revinski et al, 2010).

– First, the Notch signaling pathway is turn on by injecting 1 ng of NotchICD into the embryos and monitoring the expression of sox2 (marker for neural ectoderm) and the myogenic related factors myoD and myf5 (Figure 1). NotchICD-injected embryos show that there are more sox2 expression (purple staining) compare to the control. Also, myoD and myf5 expression are decreased on the injected site. When looking at the tranverse section (D, E, H, I), we see that not only the neural plate is widened in the medio-lateral axis, but it is also thicker on the injected side (compare white bars in Figs. 1E, H, I and the thickness of the sox2 domain between the injected- and the noninjected sides in Figs. 1F′, G, K). The result suggests that Notch signaling pathway may be favoring neural development at the expense of paraxial mesoderm.

Fig. 2. Effects of NotchICD on markers of the three germ layers at gastrula stage (Revinski et al, 2010).

– In figure 2, the embryos were injected with 1 ng of notchICD in 2 or 4 cell stages and fixed at neural plate stage. Comparing A – B and D – E, there is a decreasing in expression of bra (marker for mesodermal). Also, in G and I, we observe the change in shape of the blastospore on the injected site. We could come to a conclusion that Notch activation alters the expression of markers of the three germ layers and impairs morphogenetic movements during gastrulation (figure 2).

  • Because gain-of-function experiment sometimes causes abnormal activities of gene expression, the author also performed a loss-of-function experiment to see whether this pathway involved in segregation of the three germ layers during gastrulation. There are different ways that loss-of-function experiment could be done. One is to inject su(H)1DBM mRNA to impair CSL dependent, canonical Notch activity (Wettstein et al., 1997), another way is using delta-1STU mRNA to block signaling by the ligand Delta-1 (Chitnis et al., 1995), or using an antisense morpholino oligonucleotide against Notch (Notch Mo) to produce a general blockade of the Notch pathway (López et al., 2003).

Fig. 3. NotchICD alters the expression domain of myf5 and impairs morphogenetic movements during gastrulation. nis: non injected site. is: injected site (Revinski et al, 2010).

– When blocking CSL-dependent Notch signaling at early gastrulation, we observe an increase in myf5 and decrease in sox2 expression at injection sites (figure 3 A – I), which shows an expansion of paraxial mesodermal while the neural ectoderm is reduced (figure 3).

Fig. 4. Effects of blocking Notch signaling on neural and paraxial mesodermal markers at the late gastrula and neural plate stage (Revinski et al, 2010).

– When delta-1 signaling was impaired, it results in an expansion of myf5 and myoD as well as a reduction of sox2, it also means that the mesoderm is expanded while the neural plate is reduced (figure 4A – F’).  In general blockade of Notch pathway, embryos were injected with Notch-Mo, ISH reveals an increasing in myoD expression and decreasing in sox2 expression (figure 4 G – L)

  • Overall, these experiments suggest that Notch signaling is involved in the building of the embryonic dorsal midline in X. laevis, favoring floor plate against notochord development.

Proposed model for role of Notch signaling during the subdivision of mesoderm and endoderm germ layers during gastrulation (Contakos et al, 2005).

– According to the model above, a normal levels of Notch signaling allow for appropriate amounts of endoderm and mesoderm tissue formation (A). Ectopic activation of Notch signaling during gastrulation results in anincrease of endodermal cell types and a decrease in mesodermal cell types(B).  The opposite effect is observed when Notch signaling is suppressed, where mesoderm cell types increase at the expense of endodermal tissues (C) (Contakos et al, 2005).

  • In summary, we observe a consistence expansion of the bra domain and an animal shift of the neural border, while the suprablastoral endoderm was either expand if Delta-1 signaling was block or reduced after a general knock-down of Notch (figure 5A). And it also demonstrates that Delta-Notch signaling is involved in the segregation of the three germ layers during gastrulation. Figure 5B shows a possible pathway, activated Notch moves the limit of involution toward the vegetal pole (type A decision) we get neural plate, while blocking Notch pathway moves the limit toward the animal pole (type B decision) we get notochord development.

Fig. 5. Schematic model summarizing the phenotypes after manipulating Delta-Notch signaling. nis: non-injected site, is: injected site (Revinski et al, 2010).

2. Notch signaling acts during neurulation to promote secondary specification of the medial floor plate:

Model of Xenopus dorsal midline development:

Neurulation (Purves et al, 1998).

  • Notch signaling promotes floor plate and hypochord fates over notochord. It regulates dorsal midline cell fate decisions, promoting FP and hypochord over the default notochord. The floor plate (FP) is a specialized population of cells in the ventral portion of the vertebrate neural tube, morphologically and molecularly distinct from other regions of the central nervous system.  FP cells do not give rise to neurons and adopt a wedge-shaped morphology. The FP expresses Shh and FoxA2 and has signaling activities crucial to neural development. Notch signaling plays an integral role in cell fate decisions in the dorsal midline of Xenopus laevis, similar to that observed in zebrafish and chick.
  • There had been models that proposed the mechanism of dorsal midline specification in Xenopus, such as the “induction” model, based on classical embryology experiments in chick and mutant phenotypes in mouse, states that Shh secreted by the notochord induces the formation of the FP in the overlying naïve neurectoderm (Artinger and Boomer-Fraser, 1993; Yamada et al, 1991), and the “allocation” model states that all midline structures—notochord, FP, and dorsal endoderm—arise at the same time from the organizer in chick indicated that the FP was pre-determined prior to notochord signals (Catala et al, 1996, 1995; Teilet et al, 1998). These models have been supported by experiment done on zebrafish and chicken, but not on Xenopus. The results of this new study show that Notch signaling promotes formation of both floor plate and hypochord at the expense of notochord, Shh expression is uncoupled from floor plate formation in Xenopus and Notch signaling acts during neurulation to promote secondary specification of the medial floor plate (Peyrot et al, 2011). This new model is called the “revised allocation” mechanism for FP development.

Fig. 6. Notch signaling promotes formation of floor plate and hypochord and represses notochord (Peyrot et al, 2011).

– SDBM is DNA binding mutant of Suppressor-of-Hairless (which is the same as su(H)1DBM in the last paper) used to turn off Notch signaling pathway. NICD is the Notch intracellular domain (which is same as notchICD) used to turn on Notch signaling pathway. Netrin, F-spodin, and FoxA2 are FP markers, while AxPC is notochord marker, and VEGF is hypochord marker. Among those, Netrin is the marker that specifies secondary MFP. Blocking the Notch pathway by injection of mRNA encoding Su(H) DNA binding mutant leads to decreasing of Netrin, F-spodin, and FoxA2 (figure 6 B, E, H, K, N); and result in embryos with greatly reduced or absent mature FP. Activation of Notch pathway by injection of Notch intracellular domain mRNA leads to increasing of Netrin, F-spodin, and FoxA2; and result in loss of differentiated notochord (figure 6 C, F, I, L, O). This experiment confirms that notch signaling pathway is required for the formation of the FP and represses notochord development in Xenopus, also, it’s required for hypochord formation.

  • Another question that was asked was that whether shh expression has anything to do with FP formation. Peyrot’s experiment shows that perturbation of Notch signaling has various effects on shh expression. They studied shh expression in neurula and tadpole stage of embryos upon blockade and activation of Notch signaling pathway. The result showed that a slight reduction in the FP domain of shh was observed in 31% of the embryos, but 69% embryos showed normal expression. Also activation of Notch signaling pathway led to increase expression through the midline but normal expression at FP in 27% embryos, and in 67% embryos shh expression was decreased. There is no correlation between the variable expression of shh and the variable expression of NICD protein. FP cells can either express shh or not, but all express NICD protein (Peyrot et al 2011). And the conclusion is shh is uncoupled from FP formation.

These experiments have shown that Notch signaling pathway plays an important role in segregation of the three germ layers and also acts during neurulation to promote secondary specification of the medial FP. One of the strengths of these papers is that they did not only perform gain-of-function experiments, they also performed loss-of-function experiments in order to illustrate the role of Notch signaling. Another good point is that they performed the experiments at different stages of development, such as early, mid, and late gastrulation, so they could distinguish the role of Notch Signaling at different stages. Although the role of Notch Signaling pathway has been revealed, but we still dont’t know what are the effectors of Notch Signaling and what molecules regulate choices between notochord and primary MFP. More extensive studies needed to be done in order to fully understand the pathway.


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