Lecture 2 Slides_20240912. KGan.pdf

Reflection questions from Lecture 1 How do cells become different from one another during development? Intrinsic processes: asymmetric cell division → asymmetric distribution of nuclear/cytoplasmic factors → differences in gene expression → different cell fates (...

Reflection questions from Lecture 1 How do cells become different from one another during development? Intrinsic processes: asymmetric cell division → asymmetric distribution of nuclear/cytoplasmic factors → differences in gene expression → different cell fates (ex. Weissman’s theory, Roux’s experiments) Extrinsic processes: How do cells influence the development of neighboring cells?? Signaling cascades: Morphogen gradients: How do genes control cell identity and behavior by regulating protein expression??? Epigenetic control: Transcriptional control: Translational control: Post-translational control:  Specifying cell identity Barresi and Gilbert, Chapter 2 How are cell fates specified? Autonomous Conditional Syncytial How can we map the fates of individual cells during development? Single-cell RNA sequencing 2  A review of cell fate specification and determination Differentiation: cell stops dividing and develops specialized structural elements and distinct functional properties DETERMINED SPECIFIED NOT DETERMINED 3  Autonomous specification Conklin: autonomous specification of muscle cells in tunicate blastomeres Remove B4.1 → larva with no tail muscles Whittaker: Macho mRNA segregates asymmetrically in the cytoplasm to drive tail muscle development How do depletion and ectopic expression of macho affect muscle development? 4  Conditional specification Conditional specification: cell fate depends on its position in the embryo Inducing signals can include Cell-to-cell contacts Secreted factors Physical properties of local environment Recall Driesch’s experiments: conditional specification in the sea urchin embryo Conclusions The potential for a blastomere to adopt any cell fate is greater than its expected fate during normal development Cell-cell interactions are critical for normal development 5  Syncytial specification Syncytial blastoderm in Drosophila Morphogen gradients specify syncytial cell fates Opposing gradients of Bicoid and Caudal transcription factors specify segment identities Nuclei divide without cytoplasmic cleavage Autonomous: transcription factors differentially Membranes eventually form around each nucleus expressed after cellularization How is cell fate determined? Conditional: position relative to neighboring nuclei 6  How can we map the fates of individual cells during development? Single-cell RNA sequencing Define cell identity by determining which genes are being expressed at any given time. Transcriptome: full complement of RNAs produced by a cell Spatial map: cells clustered based on transcriptome Developmental tree: first 12 h of zebrafish similarities and gene expression changes over time embryogenesis 7 Questions??? 3-min break! 8 Mechanisms of differential gene expression Barresi and Gilbert, Chapter 3 Differential gene expression can occur at the levels of gene transcription, pre-mRNA processing, mRNA translation, and protein modification. Transcription: epigenetics, transcription factors Pre-mRNA processing: alternative splicing Translation: ribosomal selectivity, cytoplasmic localization of mRNA, miRNA and RNA interference Proteins: post-translational modifications (later in Ch. 4) 9  Anatomy of the gene Major parts of a eukaryotic gene: human β-globin Non-coding regulatory elements and transcription factors Transcription factors bridge enhancers and promoters to activate gene expression. 10  Enhancers and silencers modulate gene transcription Enhancers bind transcription factors to How can we identify enhancers and silencers? induce tissue-specific gene expression GFP reporter fused to retina- lacZ reporter fused to neuron- specific gene in zebrafish specific L1 gene in mouse ENHANCER SILENCER NRSE: neural restrictive silencer factor 11  Transcription: Epigenetic modifications modulate access to genes Nucleosome and Epigenetic regulation by histone modification chromatin structure Prevents access to promoter and blocks transcription Exposes DNA to RNA pol II and transcription factors to activate transcription How does DNA methylation block transcription? Loss of MeCP2 leads to Rett’s syndrome in females Methyl CpG-binding protein 2 Loss of spoken language Loss of hand skills Repetitive hand movements Difficulty walking 12  Transcription: How do transcription factors regulate gene transcription? 3D model of MITF Transcription factors in each family “Yamanaka factors” induce (helix-loop-helix) share similar DNA-binding domains pluripotent stem cells from differentiated skin cells Contains trans-activating domain: indirectly binds RNA pol II or histone modifiers to modulate transcription Binds co- Binds specific regulators or enhancer other TFs sequence Recruit histone-modifying enzymes Stabilize RNA pol II Coordinate expression of multiple genes by binding to enhancers 13  Pre-mRNA processing: Alternative splicing Differential pre-mRNA processing Drosophila: Dscam gene contains 115 exons and can generate 38,016 splice isoforms! Splicing: cut, rearrange, and ligate exons together Produces wide variety of proteins from same gene Protein isoforms may play similar Dscam: encodes membrane adhesion protein required for roles in different cells or opposite self-avoidance between dendrites from same neuron roles in the same cells Mutations in Dscam homologue may drive neurological defects of Down syndrome in humans 14  mRNA translation: Ribosomal selectivity and cytoplasmic localization of mRNA Mouse: selective activation of translation Drosophila egg: mRNA localization in cytoplasm Nanos mRNA diffuses and is trapped by anchor proteins at posterior pole Hsp83 mRNA is degraded everywhere except at the posterior pole, where it is protected by protein complexes Rpl38: protein in ribosomal large subunit; Bicoid and Oscar mRNAs are expressed in somites that generate vertebrae actively transported by motor Rpl38 deficiency: cannot translate subset of Hox proteins to posterior and genes to specify vertebrae → deformed skeleton anterior poles, respectively 15  mRNA translation: How do microRNAs specifically regulate mRNA transcription and translation? C. elegans: lin-4 gene encodes microRNAs RNA interference (RNAi) complementary to 3’UTR of lin-14 mRNA inhibits gene expression Lin-14: transcription factor Drosha: makes required during first larval individual pre-miRNA phase; not needed afterward hairpins Binding of small RNAs RISC complex: (microRNAs) to repetitive separates dsDNA sequence in 3’UTR of lin-14 strands and aligns with mRNA triggers degradation of 3’UTR of target mRNA transcripts Cleaves mRNA or miRNA structure: “hairpin loop” blocks translation structures trigger protective “RNA interference” mechanism Recognition of target to inhibit transcription and sequence depends on translation of gene strength of miRNA complementarity!16 Questions??? 3-min break! 17  Basic tools of developmental genetics Barresi and Gilbert, Chapter 3 How can we characterize gene expression during development? In situ hybridization ChIP-Seq RNA-Seq How can we test gene function? CRISPR/Cas9 GAL4-UAS system Cre-Lox system 18  In situ hybridization: When and where is a gene expressed in an embryo? Drosophila: localization of odd-skipped Probe hybridization and immunodetection of gene expression by in situ hybridization odd-skipped mRNA Design of RNA probe against odd-skipped mRNA 19  Chromatin immunoprecipitation-sequencing (ChIP-Seq): Where do transcription factors bind? Where are modified nucleosomes located? Isolate chromatin Cross-link proteins Precipitate antibodies out of (nucleosomes or solution with magnetic beads transcription factors) to DNA Separate proteins from DNA and sequence DNA Bind proteins with specific Map sequences to genome antibodies 20  Deep sequencing (RNA-Seq): How does the transcriptome differ in the same tissue at different stages? Chick: comparing gene expression in the Isolate RNA to obtain only genes eye across embryos of different ages that are actively expressed Fragment transcripts and generate cDNA Add specialized adaptors to cDNA ends to enable PCR amplification and immobilization for sequencing 21  CRISPR/Cas9 gene editing How can we test gene function? How can we repair mutations? Design gene-specific guide RNA (gRNA) and introduce into cells with Cas9 nuclease gRNA binds with complementarity to genome and recruits Cas9 to induce a double-stranded break Non-homologous end joining (NHEJ) results in small insertions or deletions (indels) that establish a premature stop codon and loss of protein function 22  GAL4-UAS system How can we activate or repress regulatory genes ectopically in tissues? Gene for yeast GAL4 transcription factor placed GAL4 binding sites placed downstream from enhancer of upstream of Pax6, which “jaw” genes controls eye development ??? 23  Cre-Lox system How can we conditionally eliminate gene expression in specific cell types? Mouse ES cells: knocking out the Hnf4α gene only in liver cells Strategy for generating Hnf4α knock-out mice: Female: WT alleles of Hnf4α replaced by “floxed”Hnf4α alleles at Exon 2 Male: Cre-recombinase active only in liver cells Female x male → Liver cells in progeny lack functional Hnf4α 24 Questions??? 25