1 embryonic development in the fruit flyJohn Noto BIO441 Lecture 24 April 2017

2 Development Development refers to interaction of the genome with the cytoplasm and external environment to produce a programmed sequence of typically irreversible events. Differentiation Differentiation refers to the formation of cell types, tissues, and organs through specific gene regulation. A single cell with one genotype produces a variety of specialized tissues and organs. Development and differentiation can be studied at many levels: Morphology Biochemistry Genetics

3 Development Development refers to interaction of the genome with the cytoplasm and external environment to produce a programmed sequence of typically irreversible events. Differentiation Differentiation refers to the formation of cell types, tissues, and organs through specific gene regulation. A single cell with one genotype produces a variety of specialized tissues and organs. Development and differentiation can be studied at many levels: Morphology Biochemistry Genetics

4 Development Development refers to interaction of the genome with the cytoplasm and external environment to produce a programmed sequence of typically irreversible events. Differentiation Differentiation refers to the formation of cell types, tissues, and organs through specific gene regulation. A single cell with one genotype produces a variety of specialized tissues and organs. Development and differentiation can be studied at many levels: Morphology Biochemistry Genetics

5 Flies are a great model organism

6 Flies are a great model organism

7 Flies are a great model organism

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11 Developmental stages of Drosophila(10-12 days) Egg  Larva (3 instars) Pupa Adult

12 EMS Mutation screeningWieschaus, Lewis and Nusslein-Volhard Nobel Prize, genetics basis of development

13 EMS Mutation

14 Recombination

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16 Fertilization and subsequent development

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27 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

28 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

29 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

30 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

31 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

32 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

33 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

34 Embryonic development in Drosophila:Development begins with fertilization. Prior to fertilization, molecular gradients exist within the eggs. Polar cytoplasm occurs at the posterior end---example of maternal effect. 2 nuclei fuse after fertilization to form a zygote. 9 mitotic divisions occur without cell division, and after 7 divisions, some nuclei migrate to the polar cytoplasm (posterior) creating germ-line precursors. Other nuclei migrate to the cell surface and form blastoderm precursor. 4 more mitotic divisions occur and all nuclei are separated by cell membranes.

35 Embryonic development in Drosophila.

36 Embryonic development in Drosophila.

37 Embryonic development in Drosophila.

38 Embryonic development in Drosophila.

39 Embryonic development in Drosophila.

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41 ZEISS Lightsheet Z.1 - Imaging of Drosophila embryo for cell trackingZEISS Lightsheet Z.1 - Drosophila cell tracking using a color gradient https://www.youtube.com/watch?v=FChS4KU5jDM

42 Cytoskeletal proteins create cytoplasmic islands around nucleiDNA Actin microfilaments microtubules

43 Subsequent development depends on two processes:Anterior-posterior and dorsal-ventral molecular gradients exist in the egg---mRNAs and proteins placed in egg by mother confer maternal effect. Formation of (1) parasegments and (2)embryonic segments, which give rise to (3) adult segments. Adult segmentation reflect Embryo segmentation

44 Subsequent development depends on two processes:Anterior-posterior and dorsal-ventral molecular gradients exist in the egg---mRNAs and proteins placed in egg by mother confer maternal effect. Formation of (1) parasegments and (2)embryonic segments, which give rise to (3) adult segments. Adult segmentation reflect Embryo segmentation

45 Three major classes of genes control development and differentation*Mutations identified by presence lethal or abnormal structures during development. Maternal effect genes Segmentation genes Homeotic genes

46 1. Maternal effect genes Expressed by the mother during egg production; they control polarity of the egg and the thus embryo. bicoid gene Regulates formation of anterior structures (mutants possess posterior structures at each end). Gene is transcribed during egg production, and expressed after fertilization. nanos gene Regulates abdomen formation (mRNAs collect in posterior of the egg). torso gene Transcription and translation occur during egg production. Occurs throughout the eggs, but is only active at the poles.

47 1. Maternal effect genes Expressed by the mother during egg production; they control polarity of the egg and the thus embryo. bicoid gene Regulates formation of anterior structures (mutants possess posterior structures at each end). Gene is transcribed during egg production, and expressed after fertilization. nanos gene Regulates abdomen formation (mRNAs collect in posterior of the egg). torso gene Transcription and translation occur during egg production. Occurs throughout the eggs, but is only active at the poles.

48 1. Maternal effect genes Expressed by the mother during egg production; they control polarity of the egg and the thus embryo. bicoid gene Regulates formation of anterior structures (mutants possess posterior structures at each end). Gene is transcribed during egg production, and expressed after fertilization. nanos gene Regulates abdomen formation (mRNAs collect in posterior of the egg). torso gene Transcription and translation occur during egg production. Occurs throughout the eggs, but is only active at the poles.

49 1. Maternal effect genes Expressed by the mother during egg production; they control polarity of the egg and the thus embryo. bicoid gene Regulates formation of anterior structures (mutants possess posterior structures at each end). Gene is transcribed during egg production, and expressed after fertilization. nanos gene Regulates abdomen formation (mRNAs collect in posterior of the egg). torso gene Transcription and translation occur during egg production. Occurs throughout the eggs, but is only active at the poles.

50 Distribution of bicoid mRNA and protein in the eggA = Anterior P = Posterior

51 Distribution of bicoid mRNA and protein in the eggA = Anterior P = Posterior

52 Distribution of bicoid mRNA and protein in the eggA = Anterior P = Posterior

53 Bicoid protein

54 Bicoid protein mRNA Protein

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57 2. Segmentation genes: Determine the segments of the embryo and adult, and thus divide the embryo into regions that correspond to the adult segmentation patterns. Gap genes Subdivide the embryo along the anterior-posterior axis. Mutation results in the deletion of several adjacent segments. Pair rule genes Divide the the embryo into regions, each containing parasegments. Mutations cause deletions of the same part of a pattern in every other segment. Segment polarity genes Determine regions that become segments of larvae and adults Mutants possess parts of segments replaced by mirror images of adjacent half segments.

58 2. Segmentation genes: Determine the segments of the embryo and adult, and thus divide the embryo into regions that correspond to the adult segmentation patterns. Gap genes Subdivide the embryo along the anterior-posterior axis. Mutation results in the deletion of several adjacent segments. Pair rule genes Divide the the embryo into regions, each containing parasegments. Mutations cause deletions of the same part of a pattern in every other segment. Segment polarity genes Determine regions that become segments of larvae and adults Mutants possess parts of segments replaced by mirror images of adjacent half segments.

59 2. Segmentation genes: Determine the segments of the embryo and adult, and thus divide the embryo into regions that correspond to the adult segmentation patterns. Gap genes Subdivide the embryo along the anterior-posterior axis. Mutation results in the deletion of several adjacent segments. Pair rule genes Divide the the embryo into regions, each containing parasegments. Mutations cause deletions of the same part of a pattern in every other segment. Segment polarity genes Determine regions that become segments of larvae and adults Mutants possess parts of segments replaced by mirror images of adjacent half segments.

60 2. Segmentation genes: Determine the segments of the embryo and adult, and thus divide the embryo into regions that correspond to the adult segmentation patterns. Gap genes Subdivide the embryo along the anterior-posterior axis. Mutation results in the deletion of several adjacent segments. Pair rule genes Divide the the embryo into regions, each containing parasegments. Mutations cause deletions of the same part of a pattern in every other segment. Segment polarity genes Determine regions that become segments of larvae and adults Mutants possess parts of segments replaced by mirror images of adjacent half segments.

61 2. Segmentation genes: Determine the segments of the embryo and adult, and thus divide the embryo into regions that correspond to the adult segmentation patterns. Gap genes Subdivide the embryo along the anterior-posterior axis. Mutation results in the deletion of several adjacent segments. Pair rule genes Divide the the embryo into regions, each containing parasegments. Mutations cause deletions of the same part of a pattern in every other segment. Segment polarity genes Determine regions that become segments of larvae and adults Mutants possess parts of segments replaced by mirror images of adjacent half segments.

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63 Functions for segmentation genes defined by mutations.

64 3. Homeotic genes: Homeotic genes specify the body part to develop at each segment. Adult body parts develop from undifferentiated larval tissues called imaginal discs. Homeotic mutants develop a different body part at a particular segment (imaginal disc) than the usual body part. Different homeotic gene groups share similar sequences of ~180 bp called homeoboxes that code proteins. Homeoboxes regulate development and produce proteins that bind upstream of the gene units. Homeotic gene complexes are abbreviated Hox. Hox genes also specify body plans in vertebrates and plants.

65 Homeotic genes: HOX genes

66 Fig. 19.21, Locations of homologous imaginal discs in larva and adult.

67 Fig. 19.21, Locations of homologous imaginal discs in larva and adult.

68 Examples of homeotic Drosophila mutant with the bithorax mutationWhat is wrong with one of these flies?

69 Antennapedia and aristapedia mutants

70 antennapedia

71 antennapedia

72 Aristapedia

73 Ectopic expression: Dpp>eyeless

74 Fig , Organization of bithorax homeotic genes in a 300kb region of the Drosophila genome. T = thoracic A = abdominal

75 Fig Homologous Hox gene clusters occur in Drosophila and the mouse.

76 How do development biologists study differential expression of genes during development and differentiation? Immunofluorescene assays that bind to specific mRNAs and proteins.

77 How do development biologists study differential expression of genes during development and differentiation? Quantitative real-time RT-PCR of cDNA from mRNA transcripts.

78 How do development biologists study differential expression of genes during development and differentiation? Quantitative real-time RT-PCR of cDNA from mRNA transcripts.

79 RNA-Seq

80 Ribosome Profiling – sequencing of ribosome-bound mRNAs

81 How do development biologists study differential expression of genes during development and differentiation? Gene knockout using transformation or transduction, or other gene silencing techniques like RNAi.

82 RISC = RNA-induced silencing complex

83 CRISPR/Cas9 DNA editing tools---snip DNA and replaceHow do development biologists study differential expression of genes during development and differentiation? CRISPR/Cas9 DNA editing tools---snip DNA and replace “clustered regularly interspaced short palindromic repeats” present in bacteria 1. CRISPR tell Cas9 where to snip the DNA 2. Cas9 recognizes sequences about 20 bp long 3. Guide RNA to match target sequence