Chapter 16

1. Chapter 16 The Genetic Basis of Development 4 and 6 April, 2005 zygote  adult

3. Overview • Instructions in the genome establish the developmental fate of cells in multicellular organisms. • Developmental pathways consist of sequences of various regulatory steps. • The zygote is totipotent, giving rise to all body cells. • Gradients of maternally-derived regulatory proteins establish polarity of the body axis and control transcriptional activation of zygotic genes. • Transcriptional regulation and cell signaling mediate development in animals and plants. • The same set of genes appears to regulate early development in all animals.

4. Development • In multicellular organisms, life begins as a single cell. • With few exceptions, somatic cells contain the same genetic information as the zygote. • In development, cells commit to specific fates and differentially express subsets of genes. • Cells identify and respond to their position in developmental fields. • Daughter cells may differ with respect to regulatory instructions and developmental fate.

5. Building the embryo • Developmental decisions • made at specific times during development • many are binary, e.g., male or female, germ line or somatic. • most are irreversible • many involve groups of cells rather than single cells • In animals decisions are made to • establish anterior-posterior and dorsal-ventral axes • subdivide anterior-posterior axis into segments • subdivide dorsal-ventral axis into germ layers • produce various tissues and organs • Most decisions involve changes in transcription

6. Sex determination • XX-XY chromosomal systems for sex determination have evolved many times • Different molecular pathways for sex determination in different groups of animals • Drosophila • each cell lineage makes sexual decision • ratio of X chromosomes to autosomes determines sex • cascade of differential mRNA splicing • Mammals • TDF gene on Y chromosome determines maleness • endocrine hormonal system

9. Sxl toggle • Ratio of NUM bHLH proteins to DEM bHLH proteins measures X:A ratio by competing for dimer formation • DNA binding domain of NUM proteins recognizes Sxl early promoter • twice as much NUM protein in females with two X chromosomes as males with one X results in more NUM-NUM homodimers • Sufficient NUM-NUM homodimers activate Sxl early promoter resulting in SXL protein that alternatively splices larger Sxl transcript from late promoter • sets up autoregulatory loop in flies with X:A ratio of 1.0 • in flies with X:A ratio of 0.5, insufficient NUM-NUM homodimers results in no SXL protein and late transcript is normally processed (yields nonfunctional protein)

11. Sxl downstream target • SXL protein activates downstream shunt that leads to female development • SXL protein binds to primary transcript of tra (transformer) resulting in spliced transcript that produces TRA protein • TRA protein in turn is RNA-binding protein that produces female-specific splicing of dsx (doublesex) transcript • DSX-F transcription factor represses male-specific gene expression resulting in female development • In absence of SXL, there is no functional TRA protein, and dsx is spliced to produce DSX-M transcription factor which represses female-specific genes, leading to male development

13. Sex determination in mammals • Presence of Y chromosome determines maleness • SRY gene in humans encodes transcription factor (testis-determining factor) • expression of SRY in developing gonad causes it to develop into testis • testis secretes testosterone resulting in male development • In XX individuals, absence of SRY protein and subsequent absence of testosterone results in default female shunt pathway

15. Role of cytoskeleton in development • Consists of highly organized rods and fibers • microfilaments (actin) • intermediate filaments • microtubules • Such structures are polar, with distinct “+” and “–” ends • Serve as highway system for intracellular transport • Asymmetry of cytoskeletal elements plays fundamental roles • location of mitotic cleavage plane • control of cell shape • directed transport of molecules

16. Origin of germ line • In animals, germ line is set aside from soma in early development • only germ cells can undergo meiosis • somatic cells form body of organism • Asymmetric distribution of cytoplasmic particles (e.g., P granules of Caenorhabditis elegans) by cytoskeleton • cells receiving particles develop into germ line • particles anchored to actin in some organisms, to microtubules in others

18. Drosophila anterior-posterior axis • Determined by gradients of BCD (product of bicoid) and HB-M (product of hunchback) • mRNA maternally deposited in egg • BCD mRNA tethered to “–” ends of microtubules via 3’ UTR • HB-M protein gradient depends on NOS protein • nos mRNA tethered to “+” end of microtubule via 3’ UTR • NOS protein gradient blocks translation of hb-m mRNA, resulting in HB-M gradient • Resulting opposite gradients of BCD and Nos determine axis

22. Drosophila dorsal-ventral axis • Determined by gradient of transcription factor DL (encoded by dorsal) • gradient established by interaction of spz and Toll gene products deposited in oogenesis and released during embryogenesis • SPZ-TOLL complex triggers signal transduction pathway in cells that phosphorylates inactive DL • Phosphorylated DL migrates to nucleus, activating genes for ventral fates

25. Positional information • Localization of mRNAs within cell establishes positional information in cases where developmental fields begin as a single cell • Formation of concentration gradients of extracellular diffusible molecules establishes positional information in multicellular developmental fields • works by signal transduction • diffusible molecules are known as morphogens

27. Complex pattern: Drosophila • Successive interpretation of established, changing, and new gradients • Largely due to changes in transcription • Genes targeted by gradients of maternal A-P and D-V transcription factors are cardinal genes • respond to these factors at enhancers and silencers • similar genes in other animals

29. Drosophila development (1) • Early syncitial development • zygotic nucleus divides 9 times with no cell division • some nuclei migrate to posterior pole to give rise to germ line • 4 more mitotic divisions without cell division • Nuclei migrate to surface of egg cytoplasm • membrane forms around them (cellularization) • begin responding to positional information in A-P and D-V transcription factor gradients.

31. Drosophila development (2) • At 10 hours, 14 segments • 3 head • 3 thoracic • 8 abdominal • At 12 hours, organogenesis begins • At 15 hours, exoskeleton begins to form • At 24 hours, larva hatches

33. Drosophila development (3) • Developmental fate determined through transcription-factor interactions • A-P cardinal genes = gap genes • Kruppel and knirps (mutants have gap in normal segmentation) • promoters have differential sensitivity to BCD and/or HB-M • establishes different developmental fields along embryo, roughly defining segments • Bifurcation of development: targets of gap gene encoded transcription factors • one branch to establish correct number of segments • one branch to assign proper identity to each segment

34. Drosophila development (4) • Segment number • gap gene products activate pair-rule genes • several different pair-rule genes • expression produces repeating pattern of seven stripes, each offset • pair-rule products act combinatorially to regulate transcription of segment-polarity genes • expressed in offset pattern of 14 stripes • Segment identity • gap gene products target cluster of homeotic gene complexes • encode homeodomain transcription factors • mutations alter developmental fate of segment • e.g., Bithorax (posterior thorax and abdomen) and Antennapedia (head and anterior thorax)

40. Pattern formation • Transcriptional response to gradients (asymmetrical distribution) of transcription factors • Memory of cell fate • intracellular and intercellular positive-feedback loops • e.g., homeodomain protein binds to enhancer elements of its own gene, ensuring continued transcription • Cell-cell interactions • inductive interaction commits groups of cells to same developmental fate • lateral inhibition results in neighboring cells assuming secondary fate

43. Generalizations • Asymmetry of maternal gene products establishes positional information used for early development • Successive rounds of expression of genes encoding transcription factors establish axes and body part identity • Positive feedback loops maintain differentiated state • Components of one developmental pathway are also found in many others • Differences in types and concentrations of transcription factors result in different outputs

44. Developmental parallels • Early animal development follows fundamentally similar pattern • Remarkable similarity among homeotic genes • one HOM-C cluster in insects • four HOX clusters in mammals • paralogous to insect cluster • expressed in segmental fashion in early development • Knockout and genome studies suggest animal development uses same regulatory pathways

47. Development in plants • Plants have different organ systems than animals and plant cells can not migrate • Plants do not separate soma from germ-line until flower development • Plants too have hormones and signaling pathways • Arabidopsis thaliana is model system • Transcriptional regulators control fate of four whorls (layers) giving rise to flower • process similar action of homeotic genes in animal development

48. Assignment: Concept map, Solved Problems 1 and 2, All Basic and Challenging Problems.