Markers in vertebrate neurogenesis

Submitted by: Nisha Yadav B17M4B34 Jyoti Mangla B17M4B35

Overview
Embryologists have long used morphological characteristics, and more recently marker genes, to identify neural tissue and to test the neural-inducing activity of specific cell populations and signalling molecules.

These markers are also used to assess the function(s) of neural genes themselves. Progression from neural induction to terminal differentiation of neurons is a multistep process, and each step involves the activation and/or repression of genes that can be used as molecular markers for these different events.

Introduction
Cells acquire a neural cell fate in the vertebrate embryo in response to signals provided by the organizer region, its precursor cells and some of its derivatives.

The generation of neurons (neurogenesis) involves many steps mediated by multiple signalling pathways that collude to sculpt the gene expression profiles of specific subtypes of neuron.

Neural development begins with neural induction. This defines the neural plate, which consists of neural precursors that express pan-neural genes. These

proliferative cells ultimately give rise to individual neurons, which are postmitotic
cells with distinct cell identities.

Marker genes can be used to identify key steps in neural development

Neural inducers & specifications

Organizer signals induce the expression of neural-specific genes and the acquisition of pseudo-stratified epithelial morphology in competent ectoderm cells. Three signalling pathways have been implicated in this neural induction step: 1. repression of bone morphogenetic protein (BMP) signalling, 2. Activation of the fibroblast growth factor (FGF) 3. Wnt pathways

 BMP signalling more generally regulates dorsoventral patterning of the embryo, such signals promote (ventral) epidermal differentiation, whereas BMP antagonists for example the Wnt pathway, which reduces BMP signalling, promote (dorsal) neural cell fates.
1

FGF signalling has been shown to initiate and to be required for neural induction ,acting in part by suppressing BMP4 transcription.

FGF signals are also required for the generation of the posterior nervous system in the frog.

Main Marker Genes

Several markers, including NCAM, Sox1, Sox2, Sox3, SoxD, L5 and ERNI, have been used along with a variety of region-specific neural markers, as well as genes that are expressed in the mesoderm and epidermis. a good number of marker genes should be used together to define early neural tissue, and they must be used in combination with markers of other tissues.

Anteroposterior regional character

neural tissue is induced with anterior character Neural induction elicited by BMP antagonists generates neural tissue with anterior character, then further posteriorizing signals, including FGFs, Wnts and retinoic acid, induce midbrain, hindbrain and spinal cord. neural plate has anterior identity and needs further signals that are provided by both axial and paraxial mesoderm to acquire posterior characteristics

Neurogenesis when and where

Domains in which neurogenesis takes place are often referred to as neurogenic regions, and are prefigured by the expression of genes with proneural activity, which serves to promote the formation of neurons. Many of these proneural genes are homologous to the achaete-scute genes of Drosophila.

 A good example of how the pattern of neuronal differentiation is controlled 1 is found within this region of the frog neural plate, where the position of neurogenic regions is regulated by signals from the midline (Shh and retinoic acid) .
These signalling molecules control the expression of the Gli and Zic2 genes, the activating and repressive activities of which help to restrict the expression of the proneural gene X-ngnr-1 to three longitudinal stripes, in which neuronal differentiation can take place. A regulated pattern of neuronal differentiation is also seen in the spinal cord, where the first neurons appear in an anterior to posterior sequence. This follows the downregulation of the basic helixloophelix (bHLH) gene cash4 and the homeobox containing gene Sax1 which therefore identify a unique region of the neural plate where neurogenesis is not progressing.

From neural precursor to neuron

The transition from a proliferative neural precursor cell to a postmitotic neuron is a highly regulated step, which, in many instances, has been shown to involve a cascade of transcription factors that is triggered by proneural genes (for example, X-ngnr-1 induces NeuroD in the frog neural plate).

The events underlying this progression are easily visualized in the neural tube,where they are spatially confined to distinct cell layers; 1. the ventricular layer where neural precursors (which may include stem cells) divide, 2. the intermediate layer where the first postmitotic cells can be identified, and 3. the mantle layer where neuronal differentiation takes place

This spatial organization facilitates the interpretation of the significance of marker genes, as does the use of cell-cycle markers such as 5-bromodeoxyuridine (BrdU), proliferating-cell nuclear antigen (PCNA), phosphorylated histone H3 (pH3), and cyclin-dependent kinases (CDKs) and their regulators, which distinguish neural precursors from postmitotic neurons. For example, Sox1 is expressed in the ventricular layer and is downregulated prior to neuronal differentiation, ath3/NeuroM, NeuroD and NKL (neuronal Kruppel-like) are expressed outside this layer and are present transiently in postmitotic cells, and SCG10 is expressed in differentiating neurons in the mantle layer.

Role of Notch Signalling

Proneural genes, such as X-ngnr-1, promote neuronal differentiation, but in doing so also trigger the process of lateral inhibition; as a cell becomes a neuron it activates Notch signalling in neighbouring cells, thereby inhibiting their differentiation. This mechanism ensures that not all cells in the neuroepithelium differentiate simultaneously.

Cells poised to become neurons express the Notch ligands Delta or Serrate and expression of these genes in single cells marks a pivotal and conserved point in the neurogenesis pathway. As a consequence of Notch signalling (delivered by a Delta/Serrateexpressing cell), neighbouring cells express repressors of neuronal differentiation that belong to the enhancer of split-hairy family of transcription factors; for example,Hes1 and Hes5

Neuronal identity
The identity of a neuron depends on its position and time of birth within the neural tube. The molecular control of neuronal subtype specification has been investigated in detail within the ventral spinal cord, where neurons of a specific subtype derive from a particular dorsoventral population of proliferating neural precursors that is referred to as a progenitor domain. Each domain is characterized by the expression of a combination of homeodomain transcription factors (Dbx1, Dbx2, Irx3, Nkx2.2, Nkx6.1, Pax6 and Pax7), which is responsible for the specification of a particular neuronal subtype. Interestingly, some of these patterning genes also seem to regulate neurogenesis; Pax6 is required for the normal expression of the proneural gene Ngn2,whereas, in the frog, Xiro3 (the homologue of Irx3) has been shown to repress neuronal differentiation, as assessed by expression of the general neuronal marker N-tubulin. Different proneural genes are also expressed in distinct progenitor domains within the neural tube, and have recently been shown to regulate not only neurogenesis, but also neuronal subtype specification (for example, Ngn1, Ngn2 and Math1). So, neurons are specified by combinations of transcription factors, which might initially have been identified as proneural or patterning genes. This underscores the observation that it is not possible to separate the production of neurons from the acquisition of neuronal identity, and complicates the interpretation of the expression of these genes as markers of cell subtype or cell differentiation state.

The future
The creation of accessible microarrays that display cDNA pools from different cell types will significantly improve our understanding of cell identity, and will provide quantitative gene expression profiles for cells of interest Using GFP(green fluorescent protein) allows us to examine the dynamics of gene expression in real time, and places still further emphasis on understanding gene function in very specific cellular contexts. It remains to be seen whether these precise gene hierarchies and interactions, which control cell type within the spatially organized embryo, can and need to be replicated in stem cells in vitro to generate stable and specific neuronal phenotypes.