Neuronal Determination and Differentiation

Cell Differentiation A human body contains more than 210 major types of differentiated cells Cell determination commits a cell to a particular developmental pathway -Can only be “seen” by experiment -Cells are moved to a different location in the embryo -If they develop according to their new position, they are not determined

Donor Recipient Before Overt Differentiation After Overt Normal No donor Recipient Before Overt Differentiation After Overt Normal Not Determined (early development) Determined (later development) Tail cells are transplanted to head Tail cells develop into head cells in head into tail cells in head Tail Head Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Cell Differentiation Cells initiate developmental changes by using transcriptional factors to change patterns of gene expression Cells become committed to follow a particular developmental pathway in one of two ways: 1) via differential inheritance of cytoplasmic determinants 2) via cell-cell interactions

Cell Differentiation Cytoplasmic determinants -Female parent provides egg with macho-1 mRNA -Encodes a transcription factor that can activate expression of muscle- specific genes

Cell Differentiation Induction is the change in the fate of a cell due to interaction with an adjacent cell If cells of a frog embryo are separated: -One pole (“animal pole”) forms ectoderm -Other pole (“vegetal pole”) forms endoderm -No mesoderm is formed If the two pole cells are placed side-by-side, some animal-pole cells form the mesoderm

All cells contain the full genome (which cells do not All cells contain the full genome (which cells do not?) or the full set of genetic information for the whole organism. Cellular differentiation in vertebrates, therefore, is not a loss of DNA (as in some insects and nematodes), but rather, a development of selective gene expression  specialization. How do we know this? Early cloning studies in which nuclei from cells from later developing tissues were implanted into enucleated eggs  normal, complete embryo. In the latter part of the 19th century, it was believed that neurons and glia arise from distinct pressures. In fact, all cells of the nervous system arise from the same neuroepithelium.

Points to portend about cell migration (next lecture): What is the 1° migration route? What is the purpose of 2° migration? When do they begin to differentiate to their final structure? Which ones are expected to differentiate first?

In the vertebrate, significant neuronal differentiation occurs once the neurons are post-mitotic. Do neurons divide at birth? How about at age 2? For the most part, we don’t get new ones. In the adult, ongoing neurogenesis is evident only in the hippocampus and olfactory bulb (see end of this set of slides).

Two major types of signals for differentiation Extrinsic cues Intrinsic cues Signals inherited from precursor (present in the cytoplasm or nucleus). Uses transcriptional activators or repressors. May be timed and automatic (may be influenced by extrinsic cues). Signals from the environment. Distance (hormones). Neighboring cells (paracrine) – can be secreted or membrane-bound. Signaling molecules  surface receptors  signal transduction  activates transcription factors certain genes are expressed to confer the phenotype of that particular cell (e.g., shape, nt type, how it fires)

Ways to study Neuronal Determination What is determination? Cell fate… “What will I be when I grow up?” Transplantation – way to find out if, at certain developmental time point, cells’ fate is fixed or is still flexible. Genetic Mutations – how is the normal gene product important? - spontaneous mutant (reeler) - transgenic (knockout) - mosaic (pt mutation, part normal)

3. Cell/tissue culture - Isolate or create a controlled system -cell ablation – way to understand a cell’s fate when a neighboring cell is removed (what kind of cue is this?)

Specific Example of Intrinsic/extrinsic cues: proneuronal and neurogenic genes Invertebrate system: - proneuronal – neuroprogenitors (neuroblast) are instructed to help maintain the structural integrity of the intercellular space (without this gene, much apoptosis (PCD) would occur), rather than epidermablasts. - neurogenic – neuroprogenitors restructure the # of other neural progenitors (extrinsic signals to keep certain cells as epidermablasts)

2. Vertebrate system - Local (extrinsic) signals from the organizer have already separated neuroectoderm from future epithelium. - Proneural genes are used to separate post-mitotic neurons (which will do what?) from other neuroepithelial cells that continue to divide. -Required for the determination of certain populations of neural cells. -Neurogenic genes promote an epithelial phenotype and inhibit formation of post-mitotic neurons.

Differentiation of Neurons and Glia Are also capable of turning other uncommitted cells to neural precursors. How are specific cell types controlled in a vertebrate system? e.g., glial differentiation in vertebrate optic nerve: expression of the 3 glial cell types is controlled by a combination of intrinsic and extrinsic signals. PDGF and NT-3 (growth factors) are secreted by 1 type of cell (type I astrocytes), which allow another cell type to continue dividing (w/o these growth factors, it will stop dividing and immediately differentiate). This cell is then able to use an “internal clock” (# of cell divisions) prior to its differentiation.

Example from insect: - switch from neuroblast to glial cell is controlled by a transcription factor, engrailed, a DNA binding protein.

Determination of Individual Cell Fate Example of extrinsic cues controlling 2 specific cell types in the PNS. - Sympatho-adrenal progenitors  adrenergic neurons vs. endocrine chromaffin cells, which differ both morphologically and biochemically. Glucocorticoids: ligand-receptor complex translocates to nucleus and then directs actions of txn factors, which decr neuronal genes and incr chromaffin genes. (note that txn factors can decr, as well as activate)

Transmitter switching by target-derived factors. A) All sympathetic neurons start as noradrenergics. Some innervate the sweat glands and switch their transmitter phenotype as the sweat gland matures and stop tyrosine hydroxylase and start choline acetyltransferase synthesis. B) The adrenergic-to-cholinergic switch can be prevented by replacing sweat gland-rich targets with tissue usually receiving adrenergic innervation. Conversely, an adrenergic-to-cholinergic switch can be accomplished by transplanting foot pad tissue onto hairy skin, which is usually innervated by adrenergic sympathetic neurons. C) Factors such as LIF and CNTF, found in target tissues can influence neurotransmitter choice in cultured sympathetic neurons causing cells that would differentiate as adrenergic neurons to become cholinergic.

bFGF/NGF: bFGF secreted by progenitor cells promotes neuronal development and also induces expression of NGF receptor – making cell responsive to NGF. Cell-cell signaling in the insect compound eye. Ommatidia: photoreceptor units consisting of many cell types; i.e., photoreceptor cones. Progenitors start out completely uncommitted; cell-cell interactions determine their fate.

Differentiation occurs as a ‘wave’ over the eye – regulated by txn factors signalled using the Ras cascade (Ras is ‘molecular switch’ for photoreceptor vs. cone cells). SUP (‘seven-up’) is a nuclear receptor protein, which specifies photoreceptor cells. Activation of ras pathway is necessary for it to function: Ras  generic photoreceptor  subset of specific outer photoreceptors (occurs using sup)

Original signal activates the Ras pathway via an EGF-type receptor. Target-derived signal for (final) neuronal differentiation. Final choice of fate for a neuron may come only after the cell has established contact with its synaptic target. Example of a population of neural crest cells all start out adrenergic (producing NE) Those reaching their axons out to smooth muscle remain adrenergic (becoming sympathetic neurons)

Those making contact with sweat glands switch their nt production and become cholinergic. E.g., this experiment has shed some light: Co-culturing young neurons with sweat gland tissue could induce. Transplanting in vivo could cause switch. Factor involved has not yet been fully clarified.

Differentiation Neurons become fixed post mitotic and specialized They develop processes (axons and dendrites) They develop NT-making ability They develop electrical conduction

Schematic diagram of an idealized embryo in cross section showing pathways of neural crest migration in trunk and derivatives formed. Neural crest cells migrate along two primary pathways: dorsally under the skin, ventrally through the sclerotome. Dorsal migrating cells form pigment cells, ventrally migrating cells form dorsal root and sympathetic ganglia, Schwann cells, and cells of the adrenal medulla. Drawn by Mark Selleck.

Trunk neural crest cells migrate in a segmental fashion. A) Schematic diagram demonstrating that neural crest cells migrate through the sclerotomal portion of the somites, but only through the rostral half of the sclerotome. B) In longitudinal section, neural crest cells (green) can be seen migrating selectively through the rostral half of each somitic sclerotome (S). From Bronner-Fraser (1986).

Differentiation of cranial neural crest cells: TGF-ß Regulates Expression of Transcription Factors to Determine the Fate of Cranial Neural Crest Cells CNC cells give rise to an array of tissue types: odontoblasts, chondroblasts, osteoblasts, neural tissues, such as sensory neurons and cranial nerve ganglia etc. Both ectoderm and endoderm of the branchial arch provide signaling instructions for the fate specification of these progenitor cells. Chai et al., 2003

Neuroblast differentiation: Series of GF and transcriptional regulators affecting neurogenesis of neural crest progenitors. Neural crest cells can be identified by the expression of FoxD3 and SOX10. Progenitor cells differentiate into sympathetic, parasympathetic, enteric, or sensory neurons dependent upon instructive signals encountered at or near the time of egress from the neural tube. Extrinsic cues encountered during migration or at sites where neural crest-derived cells differentiate influence patterns of gene expression. From Howard, 2004

Specification and differentiation of peripheral autonomic neurons are dependent upon the interplay between cell extrinsic and cell intrinsic factors. Initial instructive cues from the neural tube influence neural crest cells to respond to BMPs. Induction of Phox2b and MASH1 is followed by the induction of HAND2 and Phox2a resulting in expression of pan-neuronal (SCG10, NF) and cell type-specific (TH, DBH, ChAT, VAChT) genes. M.J. Howard, 2004

Neuronal & Glial Lineages are derived from common progenitors A) 2 identified neuroglioblasts in the Drosophila neuroblast map (see Fig 7A). B) Separation of neuronal and glial sublineages in progenitor 6-4. The glial regulatory protein, Gmc, is expressed in 6-4. When this cell divides into two equally sized daughter cells, 6-4 G and 6-4 N, the Inscuteable complex and Miranda segregate Gmc into 6-4 G, which thereby becomes specified as glioblast. C, D) The MNB neuroblast produces both glial cells and neurons. The engrailed gene, which encodes a homeodomain transcription factor, is required for glial sublineage. When en function is reduced by injecting antisense oligonucleotides, MNB forms only neurons (D).

Neuronal lineage determined by intrinsic factors: Mouse Numb is inherited asymmetrically Asymetrical division of cells. Dividing pair of daughter precursor cells uniformly stained for a proneural gene. Only one daughter cells inherits the numb protein (green). Double labeling for proneuronal and numb gene (yellow). Asymmetrical distribution in neuronal progenitor cells from cortex, neural crest, and spinal cord.

Neurons are born: Neurogenesis Neurons are born in the ventricular zones close to the brain ventricles Neurons are born mostly prior to birth Birth dating studies can determine the time of the last cell division [3H]thymidine or BrdU labeling to determine “birthdate” of the neurons Glia cells proliferate throughout life

Cortical neurons are born consecutively Neurogenetic timetable for the neocortex, based on long-survival [3H]thymidine auto-radiography in the rat. SA Bayer & J Altman, 1993

Development of the cerebral cortex. The ventricular zone (VZ) contains the progenitors of neurons and glia. The first neurons to be generated establish the preplate (PP); their axons, as well as ingrowing axons from the thalamus, establish the intermediate zone (IZ). Neurons of cortical layers II–VI establish the cortical plate (CP), which splits the preplate into the marginal zone (MZ), or future layer I, and the subplate (SP), a transient population of neurons.At the end, six cortical layers are visible overlying the white matter (WM) and the subplate has largely disappeared. Neural precursors in the subventricular zone (SVZ) continue to generate neurons that migrate rostrally into the olfactory bulb, even during postnatal life.

Laminar fate determination in the cerebral cortex. Morphogenesis of the mammalian cerebral cortex. Neural precursors are born in the ventricular layer and migrate away from the ventricular surface, following tracks provided by radial glial cells. The first born cells are the Cajal-Retzius neurons (left). Later born neurons accumulate in a dense matrix of cells, the cortical plate (middle). In this plate, neurons are ordered by birth date in such a way that older neurons (magenta) remain in deep layers, and younger neurons (blue) migrate through the deep layers to attain a superficial position (right). B) If ventricular cells from young donors (which would become deep cells) are transplanted into an old host, they adapt to their new environment and develop as superficial neurons (arrow). C) In converse heterochronic transplantation (old donor to young host), transplanted ventricular cells maintain their laminar fate and become superficial neurons. D) Layer 4 neurons transplanted into older brains switch their fate so that it is appropriate for the upper layer neurons. E) When layer 4 neurons are transplanted into younger hosts, they end up in layers 4 and 5, but not layer 6.

Is there neurogenesis in the adult Brain? Yes there is, in the Dentate gyrus Olfactory bulb

Neurogenesis in adult rat hippocampus One day after BdDU injection 4 weeks after BrDU injection Neuronal marker NeuN plus BrDU labeled cells F. Gage, Salk, San Diego