Development of the Nervous System PDF

7.6.3 Cell types in the cerebellum Interneurons sit in the molecular layer: Basket cells connect individual purkinje cells Stellate cells Input from the brain stem: Mossy fibers form special synapses with the dendrites of the granule cells -> Rosette synapses Climbing fibe...

7.6.3 Cell types in the cerebellum Interneurons sit in the molecular layer: Basket cells connect individual purkinje cells Stellate cells Input from the brain stem: Mossy fibers form special synapses with the dendrites of the granule cells -> Rosette synapses Climbing fibers connect to the purkinje cells Figure 126. Cellular connectivity of the cerebellum 7.7 Summmary: Migration of neuronal precursor cells during brain development Cerebral cortex: Radial migration: Formation of excitatory pyramidal neurons in the cortex At least two major migration modes – somal translocation (early in development) – glial guided locomotion along radial glia (up to 2 cm migration during human cortex development) Tangential migration: Immigration of inhibitory interneurons into the cortex Cerebellum: Radial migration of Purkinje precursor cells Tangential migration of cerebellar granule cell precursors 8 Neural crest cells 8.1 The Neural Crest as a Model System to Study Stem Cell Biology 76  Figure 127. Generation of Neural Crest Cells during Neurulation 8.1.1 Studying epithelial-to-mesenchymal transition (EMT) and migration of Neural Crest Cells Highly migratory cell population in the vertebrate embryo emerging at the dorsal part of the closing neural tube during neurulation The neural crest cells undergo an epithelial to mesenchymal transition (EMT) The migration is defined by the somites The NCC wander off between the somites and the skin or beneath the somites The final destination is in the PNS and in facial structures Figure 128. Migration of neural crest cells 8.1.2 The neural crest 77  Figure 129. The neural crest Neural crest targets: Neural crest cells generate most of the peripheral nervous system, pigment cells in the skin, smooth muscle in the outflow tract of the heart, craniofacial bone and cartilage, etc. Figure 130. Neural Crest Targets 78 8.2 In vivo fate mapping 8.2.1 In vivo cell fate mapping using Cre-recombinase-mediated recombination in mice (left) Tool to recombinate the mouse genome: Cre-recombinase does not exist in mice but is introduced into the mouse genome via transgenic mice Cre-recombinase recognises a sequence flanked by LoxP points in the mouse genome and rearranges the locus The recognised sequence is excised by the cre-recombinase and put into a vector consisting of the excised sequence and one LoxP signal point (right) Use of the Cre-recombinase tool: Cre-recombinase excises the stopp signal between the two LoxP signal points The promoter can now access the gene behind the the originally there stopp signal and the GFP gene can be expressed In the recombinant cells, the GFP gene is now expressed but in non-recombinant cells the GFP gene is still silenced (not expressed) Figure 131. Cre-recombinase tool to recombinate a genome Application example of the cre-recombinase tool: Myth6 is a tissue-specific promoter in the heart After breeding the Myth6-Cre-mouse and the Cre-reporter-strain-mouse, a transgenic mouse recombinant only in the heart tissue cells with the specific promoter Myth6 exists The Myth6 promoter is always active and activates the Cre-recombinase The stopp signal between the LoxP-points (silencing the downstream following GFP) is excised and the GFP gene is expressed -> Heart fluorescents green 79  Figure 132. Application example of the cre-recombinase tool 8.2.2 Expression of Wnt1 and in vivo fate mapping of Wnt1-Cre expressing cells Wnt1 expression: Specifically in the dorsal neural tube and in premigratory neural crest population at all axial levels Figure 133. Im vivo fate mapping of neural crest cells using a transgenic mouse expressing Cre recombinase in the neural crest and the Rosa26R Cre reporter line 8.3 Neural crest cells and their derivates Neural: Glia (Schwann cells satellite glia Sensory nervous system (dorsal root ganglia) 80  Autonomic nervous system (Sympathicus, Parasympathicus) Enteric nervous system Non-neural: Smooth muscle (outflow tract of the heart) Adrenal medulla (Chromaffin cells) Melanocytes Craniofacial bones and cartilage Odontoblasts (producing dentin in teeth) Figure 134. Neural Crest Cells and their Derivatives 8.3.1 Mapping cell derivatives with quail chick chimeras The chicken egg is opened and the a part of the neural crest is swapped out with the corresponding part of a quail embryo The swapped neural crest part can be marked and the migration paths observed 81  Figure 135. Quail chick chimeras 8.4 Fate vs. Potential Figure 136. In vivo NCCs differentiate according to their axial position 8.4.1 Development of multiple cell types Selective effect: Selective effect of a factor in a heterogenous population of lineage-restricted cells Selective elimination or proliferation Instructive effect: Instructive effect of a factor on a homogenous population of multipotent cells Fate specification at expense of all other possible fates 82  Figure 137. Selective efect (left) vs. instructive effect (right) 8.5 The neural crest cell culture system The neural tube is dissected from the embryo and planted on a cell culture dish The cells that leave the neural tube are neural crest cells which form a cell carpet in the culture dish The dissected neural tube is removed from the dish and the left over neural crest cells can be studied The neural crest cells are analysed, if they are a heterogenous population of lineage-restricted cells or a ho- mogenous population of multipotent stem cells Figure 138. Clonal analysis in vitro Labelling the harvested neural crest cells: The harvested neural crest cells are marked with live-labelling factors: – CD271/p75NTR = Low affinity neurotrophin receptor: Surface molecule allowing prospective identification and direct isolation – Sox10: an HMG transcription factor 83  Figure 139. Labelling the harvested neural crest cells 8.5.1 Neural crest stem cells (NCSC) A single cell (primay founder) is replanted and fed with neurotrophic factors to analyse whether it is a lineage- restricted cell or a multipotent stem cell. Clonal analysis revealing multipotency and self-renewal capacity in 90% of the cells The primary clones also contain progenitors like the primary founder cell (-> self-renewal) A single secondary founder is again replanted and the clones contain the same clonal cells like the primary founder cell At least an “in vitro stem cell”, similar to ES cells and others Prospective identification based on expression of CD271/p75NTR (Marker) -> Proof that a stem cell is a stem cell Figure 140. Stemple and Anderson, Cell 1992: Prospective identification based on expression of CD271/p75NTR 8.5.2 Identification of cues regulating NCSC development 84  Figure 141. Based on culture system: Identification of cues regulating NCSC development Wnt1/BMP2 combination: Wnt1/BMP2 combination allows self-renewal and produces new NCSC Wnt1, BMP2 and TGFβ are intructive factors and if applied alone, force differentiation Figure 142. Instructive growth factors regulating early emigrating neural crest cells 8.6 Neural crest cells in vivo 8.6.1 Are the findings obtained in cell culture relevant in vivo? In vivo fate mapping Combination of in vitro and in vivo approaches reveal relevance of TGFβ, Wnt, BMP, etc. signaling pathways for neural crest stem cell self-renewal and fate decisions -> In vitro culture system is relevant for in vivo systems 85  Figure 143. Can the findings found in a culture dish be reproduced in vivo? Tgfbr2 gene inactivation in Neural Crest Stem Cells prevents normal smooth muscle, bone and car- tilage formation: Figure 144. co: control / mt: Tgfbr2 inactivation 8.6.2 Neural crest cells in vivo 86  Figure 145. Homogeneous population of multipotent cells or heterogenous population of fate-restricted cells in vivo? Labelling in vivo neural crest cells in chicken embryos: Opening of the egg and labelling of the desired cell(s) with two methods: – Low titer (low concentration) retroviral infection with labels (multiple cells) – Single cell dye labelling to analyse the progeny of a single neural crest stem cell 1980s: Premigratory and migratory multipotent neural crest cells 2010: Premigratory neural crest cells are lineage-restricted Genetic cell fate mapping using the confetti mouse: Addressing multipotency in vivo Tracing premigratory Wnt1-CreERT The Cre is inducible by hormones (via estrogen-receptor) and will be activ only in a few cells The cells with activated Cre and their progeny can be observed The coloured genes indicate potential fates, which can be freely recombined and lead to different fates 87  Figure 146. Confetti mouse: Tracing of single premigratory NC cells using a multicolor Cre reporter “Confetti” Quantitative analysis of rare color clones at low clonal density: In mice, the vast majority of neural crest cells appear to be multipotent at the stage analyzed, with very few clones contributing to single derivatives. 8.6.3 Cellular identities and developmental trajectories of neural crest cells by scRNAseq New method based on premessengers turning to messengers Despite broad developmental potential in many cells (see Confetti study), there is a quite extensive molecular heterogeneity at the single cell level Neural crest cells differentiate through sequential lineage-restriction events, involving co-expression and com- petition of genes driving alternative fates Neural Crest-Derived Cells with Stem Cell Features Also Persist in Adult Structures – In vivo fate mapping and prospective identification and isolation – Prospectively identified p75/Sox10-positive neural crestderived cells in adult skin display self-renewal capacity and p75/Sox10-positive neural crest-derived cells in adult skin are multipotent 88  Figure 147. Single cell RNA sequencing 9 Synapse formation 9.1 The Synapse Speed: < 1m/s Rate: > 1 kHz Dynamics: 106 -fold 9.1.1 Synapse evolution Figure 148. Convergent synapse evolution 89