10/2/2015. Chapter 4. Determination and Differentiation. Neuroanatomical Diversity

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1 Chapter 4 Determination and Differentiation Neuroanatomical Diversity 1

2 Neurochemical diversity: another important aspect of neuronal fate Neurotransmitters and their receptors Excitatory Glutamate Acetylcholine (nicotinic AChR) Norepinephrine Inhibitory GABA Glycine Modulatory Acetylcholine (muscarinic or nicotinic AChR) Serotonin Peptides e.g. oxytocin Determination & Differentiation Concepts The process of fate specification is stepwise and progressively restrictive Several different mechanisms appear to contribute to nerve cell determination & differentiation Neurons become specified by INTRINSIC means (e.g. by virtue of cell lineage or built-in temporal mechanisms) by EXTRINSIC means (e.g. local chemical factors in their environment or signals from other neurons) by an interaction of intrinsic & extrinsic influences Transmembrane receptors and transcription factors are often involved in transducing signals that influence cell fate. 2

3 Experimental Approaches Gene and protein expression studies in situ hybridization (ISH) or qrt-pcr using probes for nucleotide sequences e.g. pax6 immunohistochemistry (IHC) or Western blot using antibodies to proteins Mutational and transgenic approaches Forward genetics- random mutagenesis, screening Reverse genetics- knockout, overexpression Transplantation studies in space (heterotopic) in time (heterochronic) In vitro studies IHC cone protein in retina IHC cerebellum GFAP and Calbindin ISH FGF8 ISH cortical layer markers 3

4 Heterotopic Transplantation: test potential fate Fig 4.1 In vitro manipulations of cell fate Fig 4.1 4

5 Invertebrate Models Drosophila melanogaster Invariant Lineages: Transcriptional Hierarchies Gonad Fig 4.2 C. elegans lineage map for nerve cell fate 5

6 Fate specification through transcription factor cascades Marty Chalfie -/- -/- -/- Fig 4.3 Positional and Temporal Identity of Drosophila Neuroblasts Nbs Hb, Egl, Cas Positional Information Fig 4.5 Fig 4.6 6

7 Positional and Temporal Identity of Drosophila Neuroblasts Knockout or misexpression of positional identity genes } GMCs Each transcription factor represses the previous and activates the next one. Thus competence to produce each cell type is temporally restricted. Corrected Fig 4.6 Asymmetric division produces asymmetric fates e.g. Drosophila nerve cord Fig

8 Notch-Delta signaling promotes asymmetric fate through lateral inhibition Fig Asymmetric division of a sensory organ precursor (SOP) Red- chromatin Green- Numb (Notch inhibitor) Fig

9 Control of Asymmetric Division Par is found at apical pole of all epithelial cells Par and Insc/Pins orient the mitotic spindle to prepare for asymmetric division. Numb (in red) inhibits the Notch pathway in the GMC that inherits it, promoting determination of its different fate. The Neuroblast goes on to divide again. Homologs of the same genes function similarly in mammals Fig. 4.7 Asymmetric inheritance and lateral inhibition in fly mechanosensory receptor lineage delamination Numb promotes asymmetric fates Notch promotes self-renewal of progenitors and prevents differentiation Numb inhibits Notch Thus Numb blocks the fate that Notch would have promoted Evidence: Notch -/- has two SP IIb cells and no socket or shaft cells; Numb -/- cannot suppress Notch so it has two SP IIa cells (and no neuron, hence numb ) Similarly, overexpression of Notch (thus overcoming Numb action) also leads to 2 SP IIa cells and no neuron. Fig

10 Genetic control of eye development through inductive cellular interactions in Drosophila Morphogenetic furrow blue= ato Notch again! Inductive interactions Fig 4.10 Photoreceptor Mosaic Normal Sevenless Fig

11 Sevenless Transduction Pathway Boss-Sev binding triggers signaling pathway leading to R7 fate Fig 4.12 Vertebrate Models 11

12 Chick-quail chimera method of Nicole LeDouarin Neural crest cell fates are highly subject to local influences. What are they? Progenitor cell Lineage tracing reveals diversity of daughter fates Fig. 1.7 Fig

13 In vitro determination by signaling molecules Fig Specification of neurotransmitter fate in neural crest (sympathoadrenal cells) BMPs Phox2b, Mash1 TH NA Fig

14 Chick-Quail Chimera approach to study of neural crest cell fate Test of commitment to fate Fate depends on location Fig Neurotransmitter fate in neural crest can be altered by the target Older pup TH switch to ChAT Noradrenaline acetylcholine Fig

15 3 stages: 1. Preplate formation 1. Cajal-Retzius cells 2. Subplate cells 2. Cortical plate splits the preplate 3. Formation of 6 layers in an inside-out order Cortical Histogenesis Fig 3.13 Intrinsic and extrinsic influences on laminar fate in cerebral cortex Fig

16 When is laminar fate determined? Fig 4.21 Feedback produces mixed-fate clones in retina +/+ Ath5 -/- Negative feedback on RGC number. Through Shh? Limits on Ath5 response? Notch? Progenitors produce mixed clones Fig Sequence of generation of cell types 16

17 Fate can be influenced by birthdate/age and local signals Fig Age-dependent restriction of potential Potential to produce progenitor vs. fated cell can vary even in same environment due to asymmetric division or very local signals Retinal progenitor cell Fig

18 Cell fate specification through molecular gradients Fig 4.27 Motor Neuron Fate: Shh vs. BMP on D-V axis induce Class II HD genes and inhibit Class I HD genes. RA induces Class I HD genes, setting up cross-repression boundaries. RA, FGF gradients on AP axis, regulate hox genes through cross-repression, creating sharp boundaries A-P fate D-V fate Fig

19 Shh/BMP countergradients instruct D-V positional identity, first in progenitors, then in daughter cells Fig 4.29 Positional Identity in Spinal Cord Motor Neurons Normal Before axonogenesis, fate is flexible After axonogenesis, it isn t Fig

20 Movement coordination requires sensory and motor match Fig 4.32 Control of sensory to motor matching Force ventral sensory axon to innervate dorsal muscle, then it switches its motor neuron partner. Muscle controls projections of inputs! Loss of muscle loss of labels Muscles induce TF factor expression in their partners Fig

21 2014 Exam 1 Results 2013 Exam 1 Results EXAM UG mean: 71 G mean: 81 # Students Score, % 21

22 Glial Specification Fig

23 Genetic Regulation of Fate 23