How does Notch signalling mediate lateral inhibition? How is this mechanism thought to regulate timing in neurogenesis

How does Notch signalling mediate lateral inhibition? How is this mechanism thought to regulate timing in neurogenesis?

In my essay I will be focusing on how Notch signals in the nervous system and how the ligand Delta mediates Notch signalling. In addition, I will also be explaining how various genes also play a role in lateral inhibition via Notch signalling. Furthermore I will explain how this mechanism regulates neurogenesis.

Specification of cells at distinct times and places plays an important role for the production of cellular diversity in the vertebrate nervous system. Molecular signals that influence the generation of distinct cell types are spatially and temporally controlled. So therefore the neural pattern formation needs coordination of signals that provide temporal and spatial coordination. Lateral inhibition is one mechanism by which patterns of different cell types are produced and is a type of intercellular interaction by which a cell adopting the primary fate can inhibit its immediate neighbours from doing the same and therefore they adopt the secondary fate.

Figure 1: This figure shows the process of lateral inhibition where nerve cell inhibits neighbouring epithelial cells from adopting the epithelial fate through Delta-Notch mediated interactions.

The stimulated Notch pathway stops the differentiation of cells and preserves them in a proliferative state. This is known as ‘standard lateral inhibition’. It has been noted that in some cells, activation of the Notch pathway inhibits the neuronal fate and actively influences another fate and this is known as ‘instructive lateral inhibition’. Notch signalling plays a role in the development of multicellular organisms by maintaining the self-renewal potential of some tissues and inducing the differentiation of other tissues. Notch signalling can control stem cell or precursor populations in an undifferentiated state and promote binary cell fate decisions via lateral inhibition. Notch receptors contain a Lin Notch repeat region on the extracellular side and on the intracellular side, there exists a subtransmembrane domain with a subsequent domain of six ankyrin repeats and a domain enriched in proline, glutamine, serine and threonine (Beatus et al, 1998).

Delta expressing cells can stimulate Notch in surrounding cells which directs them into an alternative developmental pathway. In an equivalence group of cells (scattered cells in a field of undifferentiated cells), one cell acquires a unique fate and communicates to neighbouring cells that they should acquire a different fate. All cells within an equivalence group are in a particular state and contain similar proportions of Notch and Delta. Intrinsic and extrinsic signals results in a new cell state and disrupts this balance by raising the quantities of Delta in one or a few cells. As a result Notch signalling is stimulated, through successive proteolytic cleavages and initiates lateral inhibition.

Experiments conducted on drosophila show that embryos that lack Delta/Notch function have excess neural cells at the expense of the epidermis(Campos-Ortega et al,1995).Notch is a large, single transmembrane domain receptor protein where ligands such as serrate as well as Delta act on(Cornell et al,2005).

The ligand Delta, a transmembrane protein which has a number of EGF repeats on the extracellular side binds Notch receptor and cleavage of the intracellular domain of Notch occurs by an extracellular enzyme. This produces a Notch intracellular domain (NICD/Notch 1C) where it is further processed by presenilin, ubiquitinylated and is transported into the nucleus. It then binds to RBPJK, a CSL transcription factor and plays a role in upregulating the bHLH gene, Hes1.This gene then in turn suppresses MASH1 expression and various other bHLH proneural genes which keep the cells in an undifferentiated state. These proneural genes are part of the family of the achaete-scute complex (A-Sc) and the products of the A-Sc genes are basic helix-loop-helix (bHLH) transcription factors. These factors have an upstream role where they induce the expression of genes contained within the proneural cluster. When cells become committed to a neural fate, the level of A-Sc proteins rises and the surrounding neighbours within the cluster decreases proving that these factors also have a downstream role. The increased expression of A-Sc genes in the nascent neural cell is transient which also reflects the Delta expression (Lewis et al, 1996). A-Sc is expressed in drosophila and MASH is expressed in mammals and so A-Sc/MASH positively controls Delta expression (Beatus et al, 1998).Cells which express Delta will promote stronger expression of E (spl) in surrounding cells via the Notch pathway. These neighbouring cells with raised levels of E (spl) /Hes will inhibit A-Sc/MASH expression and also of Delta. But after a certain time these cells impair their ability to signal and convert into receiving signals.

The signalling cells which started off having high levels of Delta in contrast to neighbouring cells will receive fewer stimuli of their Notch receptors due to low levels of Delta on neighbouring cells. This results in low levels of E (spl) /Hes which raises A-Sc/MASH activity and Delta expression.

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Figure 2: This diagram shows that in embryos cultured for 24 hours in control (A, C, E) demonstrate expression of cHes5 genes in neural tube, in the anterior and posterior domains of the otic cup. When they are exposed to DAPT, an inhibitor of Notch signalling and a gamma secretase inhibitor, the expression of cHes5 genes are no longer observed (B, D, and F). This demonstrates that these genes are vital for specifying the neuroblastic fate through Delta-dependent lateral inhibition (Daudet et al, 2007).

Delta binds specifically to the EGF repeats in the extracellular domain of the Notch receptor. A protease (e.g. tumour necrosis factor-α (TNF-α) converting enzyme) cleaves Notch specifically at the S2 site and removes the bulk of the extracellular domain. Furthermore cleavage of the membrane tethered intracellular domain at site S3 occurs by the presenilin complex. This occurs either in the plasma membrane or after endocytosis which releases the NICD.NICD then translocates to the nucleus where it associates with CSL to form a binary complex. This recruits coactivators to form the transcription activating complex. NICD is controlled by endocytosis and so NICD becomes monoubiquitylated and targets the receptor for lysosomal degradation. NICD gets destroyed in a proteasome-dependent process.

NICD has nuclear localisation signals and includes a domain that mediates binding to the suppressor of hairless (Su (H)) which is a product of a neurogenic gene. It has been demonstrated that in drosophila S2 cultured cells that stimulation of Notch by Delta results in a movement of Su (H) protein into the nucleus in the absence of intracellular processing of Notch. Su (H) in drosophila and RBP-JK in mammals are vital molecules in relaying the Notch signal and acts downstream specifically of Notch. Su (H) binds to regulatory regions of the E (spl)-C (Enhancer of split complex) and causes transcription and upregulation of E (spl)-C genes. Studies of homologous genes in mammalian cells have confirmed that the action of Su (H) relies on the presence of Notch 1C bound to it. The E (spl)-C encodes for repressive bHLH proteins and in association with the co-repressor Groucho, it suppresses expression of the A-Sc genes. These genes encode for activator bHLH proteins that results in Delta expression. The overall effect is that stimulation of Notch limits Delta expression via a reduction in A-Sc expression. Delta dimers regulate the production of Notch dimers in signal-receiving cells. Deltex is a gene that encodes a positive regulator of the Notch signalling pathway and it is known that monomeric Notch interacts physically with deltex bound to the NICD ANK (Ankyrin repeat motif) repeat array (Muskavitch et al, 1994).The stimulation of the receptor and release of Notch-bound Deltex is a result of Delta-mediated dimerization of Notch. In signal receiving cells this results in ANK repeat-dependent binding of Su (H) to NICD.

XASH-3 is a bHLH transcription factor and it is seen that it influences a neuronal fate and the expression of the inhibitory ligand X-Delta-1.This stimulates X-Notch-1 on surrounding cells and lowers the activity of XASH-3 and therefore limits X-Delta-1 expression in these cells(Chitnis et al,1996).X-Notch-1 and X-Delta-1 are the xenopus homologs of Notch and Delta. The interaction between XASH-3 and X-Delta-1 demonstrate that the salt and pepper pattern of ectopic NECS (cells expressing N-tubulin) created when injecting lower doses of XASH-3 is a result of lateral inhibition.

Figure 3 : This figure shows that XASH-3 expressed ectopically and given in low doses induces ectopic NECs in a salt and pepper pattern as shown by the dark purple line which represents the N-tubulin RNA marker(Chitnis et al,1996).

When Notch is stimulated on a cell, the activity of the proneural genes are reduced possibly by stimulating genes contained within the E(spl) complex(Jennings et al,1995).The suppression of proneural gene activity by Notch not only suppresses cells from following a neuronal fate but Delta expression is also reduced. The ectopic XASH-3 expression could have a possible proneural activity by promoting the expression of neural bHLH transcription factors (Chitnis et al, 1996). BHLH transcription factors function as heterodimers and are negatively regulated when they bind to HLH proteins (proteins that do not contain DNA binding domains) to generate inactive dimers. Therefore the ectopic XASH-3 expression could influence the activity of other proneural genes by interacting with negative regulatory molecules that normally suppress their function and hence generate ectopic neurons.

The inhibitory action of Notch on the proneural activity of XASH-3 is regulated by two ways. The first is that the activity of neurogenic genes promotes the post-transcriptional modification of XASH-3.The second mechanism is that neurogenic genes act on downstream targets of XASH-3.

If XASH-3 and Neuro-D (another type of bHLH transcription factor) stimulate distinct downstream targets to influence neurogenesis and if these targets are differentially susceptible to suppression by neurogenic genes it would explain why XASH-3 is more sensitive to Notch inhibition. This would be precisely the case if XASH-3 induced neurogenesis by stimulating Neuro-D and other downstream bHLH transcription factors. The activity of neurogenic genes may repress bHLH transcription factors but it cannot suppress their ability to express neuron specific genes. So overall XASH-3 and Neuro-D responds to lateral inhibition in distinct ways and demonstrate that these two bHLH transcription factors stimulate downstream target genes in different ways (Chitnis et al, 1996).

Scattered cells found in the proliferative zones express Delta but evidence shows that in chick cells Delta 1 is not expressed in the s-phase of the cell cycle. This demonstrates that Delta 1 expression is limited to only post mitotic neurons (Myat et al, 1996).

The cells in the ventricular zone are inhibited by differentiation by Delta action on the surrounding neurons. This leads to their expression of Hes genes which are target sites of Notch which therefore suppresses the expression of proneural and neurogenic genes. Evidence has been shown that throughout the dorsoventral extent of the trunk, the neural tube expresses Notch 1a, Notch 1b and Notch 5(Appel et al, 2001).Differentiating neurons do not express Notch 1a and Notch 1b but neural precursors do. Notch 1a is also shown to be expressed in post mitotic neurons as shown by expression of Hu proteins.

Figure 4: This is a figure of a day-4 embryo showing nascent dorsal root ganglion (DRG).Here Notch 1 is only expressed in the periphery of the DRG as shown by the purple/blue region. There are no differentiating Hu-positive neurons in the DRG core. (Wakamatsu et al, 2000).

Observations have demonstrated that a subset of proliferative neural precursors and cells specified for neuronal development express delta A, delta B and Delta D throughout the trunk neural tube.

Figure 5 :Notch 1a(B) and Delta 1b(N) is clearly expressed in zebrafish trunk neural tube and that these are Hu-positive cells which shows that Notch 1a and Delta 1b is clearly expressed in post mitotic neurons(Appel et al,2001).

Ablating CaP and VaP primary motor neurons showed that these ablated cells are replaced with the CaP and VaP motor neurons again (Appel et al, 2001).

This evidence shows that primary motor neurons suppress surrounding cells laterally from adopting the neuroblastic fate (Doe et al, 1985).The identities of primary motor neurons are suitable for their positions in the neural tube. In delta A mutant embryos neural precursor cells differentiate prematurely and develop into neurons(Appel et al,2001).Many early specified primary motor neurons, fewer later specified secondary motor neurons and radial glia are generated which demonstrates that inhibition of Delta-Notch signalling causes this. The reason for this is that the neural precursors leave early from the cell cycle which cause the depletion of the pool of precursor cells and is later replaced by post-mitotic neurons. Hence the Delta-Notch signalling controls specification of a suitable population of primary motor neurons but does not control specification of motor neuronal identity.

Notch family members are expressed widely in zebrafish ectoderm and premigratory neural crest at open neural plate stages. Precursors of the Rohon-Beard spinal sensory neurons (RBs) are part of the Delta family and these are early neural crest derivatives that cannot migrate into the periphery.Misexpression of dominant negative Delta 1 causes interruption of Notch signalling which results in a large number of RBs at the expense of premigratory neural crest cells. This is precisely what happens in the peripheral nervous system in the fly in lateral inhibition during neurogenesis where Notch signalling inhibits the neuroblastic fate and allows the epidermoblast fate. Therefore in zebrafish, cells within the premigratory trunk neural crest are all set to form RBs but Delta expression within these cells activates the neighbouring cells to follow a different fate via the Notch pathway (Cornell et al, 2000).

Figure 6 :Embryos homozygous for a missense mutation of Delta A contain a huger proportion of post-mitotic neurons(huc expression-blue) and fewer proliferative cells(brdU incorporation-brown) demonstrating that neural precursor cells leave the cell cycle early and mature as neurons(Appel et al,2001).

Ngn1 is part of the bHLH transcription factor family involved in specifying neuronal differentiation and this neurogenin is found within RBs and neurons and that suppression of ngn1 causes the decrease in RBs. Activation of Notch signalling decreases ngn1 expression in RBs.Experiments done on disrupting ngn1 function using morpholino antisense oligonucleotides found that premigratory trunk neural crest cells was restored in embryos in the absence of Notch function(Cornell et al, 2002 ).This is an example of a standard Notch mediated lateral inhibition where Notch signalling inhibits a neurogenic fate. Experiments show that injecting RNA coding for full-length Delta-1 protein, suppresses the generation of neurons. A gene that is normally found only in nascent neurons and is forced to be expressed in all cells suppresses cells from becoming nascent neurons. This is an example of lateral inhibition and so when all cells are forced to express a particular gene, they all suppress one another. But in normal conditions, isolated cells that contain the gene, suppress their neighbours and at the same time these neighbours do not deliver inhibition to the isolated cells.

Injection of a RNA that codes for a Delta-1 protein with a truncated intracellular domain results in overproduction of neurons. Injection of a RNA encoding an activated Notch, suppresses neuronal generation. Furthermore, the activated Notch prevents the neural fate and stops expression of the endogenous Delta-1(Lewis et al, 1996).This gives evidence that lateral inhibition operates in a feedback system. A cell expressing Delta-1 more strongly than its surrounding cells will suppress them from adopting the neuronal fate. As the cells express Delta more strongly than its neighbours, the neighbours stimulate the Su (H) →E(Spl) pathway more strongly and hence generate less Delta ligand. The outcome is that most cells are now generating the Notch receptor and only one cell is producing the Delta ligand.This maintains the neighbouring cells in a Delta-free state. In vertebrates, neurons are produced in three different ways:

Within the central neuroepithelium that generates the neural plate and neural tube.
By migration from the neural crest.
By delamination from the cranial placodes.

In the central neuroepithelium, scattered postmitotic cells are the first neurons to appear in a variety of different regions and these neurons are closely associated with undifferentiated proliferative neuroepithelial cells. This pattern of neurons reflects the pattern seen in the neural plate of amphibians and fish where the generation of a set of primary neurons is separated by a time lag from the generation of subsequent sets of neurons. Primary neurogenesis occurs in cells that express Notch-1 and other Notch homologues. In xenopus and chick, the Delta homologue, Delta-1 seems only to be expressed in nascent neurons that have stopped dividing and this demonstrates that Delta-Notch signalling works to determine which cells adopt a neuronal fate.

Figure 7: Here injections of X-Delta-1 suppresses generation of all classes of islet1-positive primary neurons (sky-blue region) and inhibit primary neurogenesis (Haddon et al, 1998).

Premigratory and migratory zebrafish trunk neural crest cells interact with each other and stops them from adopting the same neural fate. There are two groups of neural crest cells that migrate on the medial pathway. One is the ventrolateral group which migrate early and produces neurons of the dorsal root ganglion (DRG) and non-neuronal derivatives. The other is the dorsolateral group which migrate much later and only produce non-neuronal derivatives. It appears that neurogenesis is determined before the cells migrate because experiments show that early migrating cells have the potential to produce neurons when they are transplanted so that they migrate along with later migrating cells (Raible et al, 1996).

Evidence shows that when removing early migrating cells before they migrate, later migrating cells accumulate in the empty space and start to generate neurons in the DRG .However, later migrating cells have the ability to produce neurons if early migrating cells are removed after they had migrated. This shows that within neural crest cells, there exists a contact mediated anti-neurogenic signal. In the embryonic CNS of drosophila, neuroblasts are produced from the neuroectoderm in continuous waves (Lewis et al, 1996).In each wave of neurogenesis, the proneural and neurogenic genes regulate the proportion of cells that are singled out for this neuroblastic fate. Each neuroblast will then perform repeated asymmetric divisions to produce neurons and/or glial cells. Lateral inhibition is also known to control the generation of otic neuroblasts and this mechanism functions in the anterior part of the otic epithelium to reduce the cells to adopt the neuroblastic fate. When signalling is absent, all cells in that area adopt the neural fate and a small number stay epithelial (Daudet et al, 2007).

Nascent retinal neurons which express Delta-1 suppress neighbouring progenitors from leaving the cell cycle and initiating neuronal differentiation. So lateral inhibition not only delays but prevents cells from dividing and differentiating in the retina to produce post-mitotic neurons and regulates the number of progenitor cells(Henrique et al,1997).In the absence of lateral inhibition, the number of early neurons rises and a reduction of progenitors occurs and the process of neurogenesis would stop early as a result .Lateral inhibition would permit neurogenesis to process over several days and neurons generated at distinct times in this period adopt distinct characters, which reflects alterations in the intrinsic competence of the progenitor cells and in the surrounding environment where neurogenesis is happening(Henrique et al,1997).

During later neurogenesis, nascent neurons are expressed and Notch-1 is present in uncommitted dividing progenitors and hence these findings show that Delta-Notch signalling regulates continuing sequential generation of neurons.

In the neural retina, an isolated cell that is forced to express Delta-1 differentiates as a neuron but groups of cells with high levels of Delta-1 remain as progenitor cells. This points out the relevance that the behaviour of a cell relies on the exposure of a Delta signal from surrounding cells (Henrique et al, 1997).

Therefore lateral inhibition regulated by Delta-Notch signalling and delivered by nascent neurons to surrounding progenitors is probably likely to have a general and a conserved function in vertebrate neurogenesis.The mechanism helps to regulate entry of progenitor cells into the neuronal differentiation pathway and stops them differentiating early. Hence neurogenesis continuously generates neurons and helps production of neuronal diversity.

References

Appel B Givan L.A Eisen J.S (2001) Delta-Notch signalling and lateral inhibition in zebrafish spinal cord development.BMC Developmental Biology. 1:1-11.
Beatus P Lendahl U (1998) Notch and neurogenesis. Journal of Neuroscience Research 54:125-136.
Campos-Ortega JA (1995) Genetic mechanisms of early neurogenesis in Drosophila melanogastor.Mol Neurobiol 10:75-89.
Chitnis A Kintner C (1996) Sensitivity of proneural genes to lateral inhibition affects the pattern of primary neurons in Xenopus embryos. Development 122: 2295-2301.
Cornell RA Eisen JS (2005) Notch in the pathway: The roles of Notch signalling in neural crest development. Seminars in cell and developmental Biology 16:663-672.
Cornell RA Eisen JS (2002) Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function. Development 129:2639-2648.
Cornell RA Eisen JS (2000) Delta signalling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate. Development 127:2873-2882.
Daudet N Ariza-McNaughton L Lewis J(2007) Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear. Development 134:2369-2378.
Doe CQ Goodman CS (1985) Early events in insect neurogenesis.II.The role of cell interactions and cell lineage in the determination of neuronal precursor cells Dev Biol 111:206-219.
Haddon C Smithers L Schneider-Maunoury S Coche T Henrique D Lewis (1998) Multiple delta genes and lateral inhibition in zebrafish primary neurogenesis. Development 125:359-370.
Henrique D Hirsinger E Adam J Le Roux I Pourquie O Ish-Horowicz D Lewis J (1997) Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina. Current Biology 7:661-670.
Jennings B deCelis J (1995) Role of Notch and the achaete-scute complex in the expression of Enhancer of split bHLH proteins. Development 121:3745-3752.
Lewis J(1996) Neurogenic genes and vertebrate neurogenesis. Current Opinion in Neurobiology 6: 3-10.
Muskavitch MA.T (1994) Delta-Notch signalling and Drosophila cell fate choice. Developmental Biology 166:415-430.
Myat A Henrique D Ish-Horowicz D Lewis J (1996) A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. Developmental Biology 174:233-247.
Raible DW Eisen JS (1996) Regulative interactions in zebrafish neural crest. Development 122:501-7.
Wakamatsu Y Maynard TM Weston JA (2000) Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis. Development 127:2811-2821.
Lecture resources on Fate determination by Dafe Uwangho.
http://scienceblogs.com/pharyngula/2006/12/notch.php
http://genome.ib.sci.yamaguchi-u.ac.jp/~gon/notch/no-image/lateral_diagram.gif

How does Notch signalling mediate lateral inhibition? How is this mechanism thought to regulate timing in neurogenesis

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