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Neurogenesis and cell fate specification: the role of transcriptional regulatory networks Joe Corbo Washington University School of Medicine ([email protected]) Unicellular organism Unicellular organism Multicellular organism Proliferation and differentiation Fertilized egg Diverse cell types Cellular differentiation on an epigenetic landscape Fertilized egg Differentiated cell types Why do mammals have so few cell types? - A cell type is defined by the complement of genes it expresses. - ~25,000 genes in the mammalian genome. - Let’s assume that each gene can be either ON or OFF. - A cell type consists of a specific combination of ON and OFF genes. - Thus, the genome permits 225,000 possible cell types. - But mammals have only several hundred cell types. Why? Cell type #1 Cell type #2 Cell type #3 The number of cell types is constrained by the structure of transcriptional regulatory networks cis-regulatory element coding sequence Today’s lecture Part 1: Neurogenesis and cell fate specification Part 2: Transcriptional regulatory networks Photoreceptors are the entry point of our visual system The retina has seven basic cell classes Rod Cone Horizontal cell Bipolar cell Amacrine cell Ganglion cell How to define a cell type? -molecules -morphology -connectivity (Mueller glia) not depicted The retina contains as many as 80 different cell types Data for mouse retina Cell classes cell types Rods Cones Horizontal cells Bipolar cells Amacrine Cells Ganlion cells Mueller glia total # of cell types: 1 2 1 15 30 30 1 80 retina Generation of neuronal diversity in the developing retina pupil ONL INL GCL Embryonic retina Adult retina light sensory information Proliferation and differentiation Retinal progenitor Seven retinal cell classes ONL INL GCL Embryonic retina Adult retina light sensory information The cell cycle in the developing retina Interkinetic nuclear migration Three types of cell division: Symmetric mitotic Asymmetric Symmetric post-mitotic Proliferation in the developing retina Q fraction: proportion of cell division progeny exiting the cell cycle (“Quitting fraction”) P fraction: proportion of cell division progeny continuing To proliferate Proliferation in the developing brain Q fraction: proportion of cell division progeny exiting the cell cycle (“Quitting fraction”) P fraction: proportion of cell division progeny continuing To proliferate Retinal cell types are born in overlapping waves Cell number Rod Bipolar Amacrine Gang. Cone Müller glial P11 P9 P7 P5 P3 P1 E18 E16 E14 E12 E10 Horiz. Birth date Adapted from Young, 1985. Cell lineage in the retina Three types of cell division: Symmetric mitotic Asymmetric Example cell lineage: Symmetric post-mitotic Final output: 2 ganglion cells 1 horizontal cell 5 rods 1 bipolar cell The competence model of retinal development Competence model: progenitors pass through a series of competence states, during each of which the progenitors are competent to produce a subset of retinal cell types. Environmental factors: (1) Small molecules (2) Secreted proteins (3) Cell surface molecules Progenitor Etc. Competence • Time Differentiated cell types Today’s lecture Part 1: Neurogenesis and cell fate specification Part 2: Transcriptional regulatory networks What is a transcription factor? A transcription factor is a protein with two parts: (1) A sequence-specific DNA-binding domain (2) An activation or repression domain Activation domain Transcription factor DNA-binding domain DNA What is a transcription factor? A transcription factor is a protein with two parts: (1) A sequence-specific DNA-binding domain (2) An activation or repression domain Activation domain Transcription factor DNA-binding domain DNA CTAATCCC A transcription factor can bind a family of related sequences CRX: CTAATCCC CAAATCCC CTAATCGC CTAAGCCC CAAATCCC CTAAGCGC etc. A transcription factor can bind a family of related sequences CRX: CTAATCCC CAAATCCC CTAATCGC CTAAGCCC CAAATCCC CTAAGCGC etc. ~1,000 TFs in human genome and they fall into various families http://www.bioguo.org/AnimalTFDB/images/pic_family.jpg How does transcriptional regulation work? brain cis-regulatory elements (CREs) are in blue retina coding sequence limbs = transcriptional activators 300-600 bp Transcription factors control: -spatial pattern -timing -levels = transcriptional repressor Transcriptional regulatory networks cis-regulatory element coding sequence Photoreceptors are the entry point of our visual system Fertilized egg Photoreceptor precursor cell rod cone “The complex system of interactions underlying the epigenetic landscape” photoreceptor Crx Nrl Nr2e3 photoreceptor Gene networks in photoreceptor development: Otx2 Crx probe • • • Otx2 is a homeodomain-containing transcription factor (TF) One of the first TFs to be expressed in developing photoreceptors When mutated, Crx expression is lost and photoreceptors fail to differentiate Wt Wt Otx2-/- Otx2-/Otx2 Crx Furukawa et al., Nat. Neurosci. 6: 1255-1263 Gene networks in photoreceptor development: Crx Otx2 • • • • Crx is a homeodomain-containing transcription factor (TF) similar to Otx2 One of the first TFs to be expressed in developing rods and cones When Crx is mutated photoreceptors fail to differentiate normally; causes several forms of blindness in humans Expression of both rod-specific and conespecific genes is affected Crx Rod-specific genes Cone-specific genes Furukawa et al., Nat. Genet. 23: 466-470 Gene networks in photoreceptor development: Nrl • • • Otx2 Nrl is a leucine zipper transcription factor Nrl is expressed only in rods, not in cones It is required for activation of rod genes and repression of cone genes Crx Nrl Rod-specific genes Cone-specific genes Furukawa et al., Nat. Genet. 23: 466-470 Wild-type rod ON OFF Nrl mutant “rod” OFF ON Gene networks in photoreceptor development: Nrl regulation • Otx2 Nrl is directly regulated by at least three upstream transcription factors: Otx2, Crx and Rorb Rorb b Crx Nrl Rod-specific genes Cone-specific genes Furukawa et al., Nat. Genet. 23: 466-470 Gene networks in photoreceptor development: Nr2e3 • • • • • Otx2 Nr2e3 a member of the nuclear hormone receptor superfamily Nr2e3 is only expressed in rods, not cones Nrl activates expression of Nr2e3 Nr2e3 represses cone genes in rods It also activates rod genes Rorb b Crx Nrl Nr2e3 probe Wt Nrl-/Pnr Nr2e3 Rod-specific genes Cone-specific genes Wild-type rod ON OFF Nr2e3 mutant “rod” ON ON Transcriptional networks as the driving force behind the generation of neuronal diversity Otx2 Rorb b Crx Nrl Retinal progenitor Pnr Nr2e3 Retinal Cell types Rod-specific genes Cone-specific genes Differentiated cell types occupy defined position in the ‘state space’ Of the cell’s transcriptional regulatory network Embryonic stem cell Photoreceptor precursor cell rod cone Today’s lecture Part 1: Neurogenesis and cell fate specification Part 2: Transcriptional regulatory networks [email protected] http://pathology.wustl.edu/~corbolab/