1. Chaper 19 Comparative Genomics and the Evolution of Animal Diversity 2004 生物科学 倪向敏 200431060142

2. outline  Topic 1: Most animals have essentially the same genes  Topic 2: Three ways gene expression is changed during evolution  Topic 3: Experimental manipulations that alter animal morphology  Topic 4: Morphological changes in crustaceans and insects  Topic 5: Genome evolution and human origins

3. Charles Darwin : all animals arose from a common ancestor! Over the course of many millions of years of evolution, a flat worm lived in burrows beneath the ancient oceans spawned the remarkable diversity we now see among modern animals.

4.  There are 25 different animal phyla and most fall into three major groups: lophotrochozoans, ecdysozoans, and deuterostomes. Figure 19-1 summary of phyla

5. Topic 1 : Most animals have essentially the same genes

6. Comparison of the currently available genomes reveals one particularly striking feature : different animals share essentially the same genes. Pufferfish, mice and human genomes comparison: about every human genes has a clear counterpart in the mouse genome ; more than three quarters of human and pufferfish genes can be unambiguously aligned.

7. Figure 19-2 Phylogeny of assembled genomes The relationship among those animals whose genomes have been sequenced to date

8. The genetic conservation seen among vertebrates extends to the humble sea squirt.  It contains half the number of genes present in vertebrates;  Nearly two-thirds of the protein coding genes contain a clear recognizable counterpart in vertebrates;  The increase in gene number seen in vertebrates is due to the duplication of genes present in the sea squirt. (for example, EGF genes )

9. Figure 19-3 phylogenetic tree showing gene duplication of the fibroblast growth factor genes (EFG) Cioca EFGs are shown in orange,whereas vertebrate are shown in orange. Branchless is an EFG found in drosophila.

10. How does gene duplication give rise to biological diversity? Two ways :  Conventional view --- the coding regions of the new duplication genes undergo mutation. These mutant genes encode related proteins with slightly different activities.  Recent view --- duplicated genes contain new regulatory DNA sequences. This allows different copies of the gene to be expressed in different patterns within the developing organism.

11. The high degree of conservation of the genes found in different animals has recently focused the changes in gene expression as a general mechanism in generating evolutionary diversity. how?

12. Topic 2 Three ways gene expression is changed during evolution

13. Pattern determining genes: a class of regulatory genes  Changes of the activities and expression patterns of these genes during evolution seem to cause significant changes in animal morphology.  Distinguishing characteristic---cause correct structures to develop.

14. Three major strategies for altering the activities of pattern determining genes  1.A given pattern determining gene can be expressed in a new pattern. This will cause those genes whose expression it controls to acquire new patterns of expression.  2.The regulatory protein encoded by a pattern determining gene can acquire new functions (for example, a transcriptional activation domain can be converted into a repression domain).  3.Target genes of a given pattern determining gene can acquire new regulatory DNA sequences, and thus come under the control of a different regulatory gene.

15. Figure 19-4 summary of the three strategies for altering the foles of pattern determining genes Altering the expression of the pattern determining gene. Different garget genes are regulated due to changes in enhancer sequences Proteins acquire new functions through mutation.

16. Topic 3 Experimental manipulation that alter animal morphology

17. Abnormal morphologies are obtained through each of the three mechanisms: 1.altering the expression 2.altering the function 3.altering the targets of the pattern determining genes

18. Changes in Pax6 expression create ectopic eyes Pax6  pattern determining gene which controls eye development in most or all animals.  is normally expressed within developing eyes.  misexpressed in the wrong tissues causes the development of extra eyes in those tissues.  expression pattern changes are probably for some of morphological diversity ( positioning of eyes ) among the eyes of different animals.

19. Figure 19-5 misexpression of Pox6 and eye formation in Drosophila

20. Evolutionary changes in regulation of Pax6 expression have been more important for the creation of morphologically diverse eyes than have changes in Pax6 protein function. Pax6 genes from other animals also produce ectopic eyes when misexoressed in drosophila.

21. Fruit flies were engineered to misexpress the squid Pax6 gene. Extra eyes were obtained in the wings and legs.

22. Changes in Antp expression transform antennae into legs Antp:  a second Drosophila pattern determining gene  controls the development of the middle segment of the thorax, the mesothorax.  encodes a homeodomain regulatory protein that is normally expressed in the mesothorax of the developing embryo.  not expressed in the developing head tissues.

23. When misexpressed in the head,Antp causes a striking change in morphology: legs develop instead of antennae. Figure 19-6 a dominant mutation in the Antp gene results in the hemeotic transformation of antennae into legs

24. Importance of protein function: interconversion of ftz and Antp Two related pattern determining genes in Drosophila : the segmentation gene---ftz the homeotic gene---Antp These genes are linked and arose from an ancient duplication event. The two encoded proteins are related and contain very similar DNA-binding domains (homeodomains)

25. The Antp and Ftz proteins recognize distinct DNA-binding sites because they form heterodimers with different “partner” proteins  Antp contains a tetrapeptide sequence motif, YPWM, which mediates interactions with a ubiquitous regulatory protein called Exd (Extradenticle).  Ftz contains s pentapeptide sequence, LRALL, which mediates interactions with a different ubupuitous regulatory protein,FtzF1

26. Figure 19-7 Duplication of ancestral gene leading to Antp and ftz Ftz-FtzF1 dimers recognize DNA sequences that are distinct from those bound by Antp-Exd dimers. Antp and Ftz regulate different target genes.

27. Subtle changes in an enhancer sequence can produce new patterns of gene expression  Changes in the target enhancers that are regulated by pattern determining genes for evolutionary diversity can be nicely illustrated by the Dorsal regulatory gradient in the early fly enbryo.

28. Binding affinities of Dorsal recognition sequences produce distinct patterns of gene expression.  Target enhancers with low-affinity Dorsal binding sites are expressed in the mesoderm, where there are high levels of the Dorsal gradient.  Target enhancers with high-affinity sites are expressed in the neurogenic ectoderm, where there are intermediate and low levels of the gradient.

29. The principle that changes in enhancers can rapidly evolve new patterns of gene expression stems from the experimental manipulation of a 200 bp tissue specific enhancer that is activated only in the mesoderm. The enhancer contains two low-affinity Dorsal binding sites and is activated by high levels of Dorsal gradient in ventral regions (the future mesoderm).

30.  Single nucleotide substitutions that convert each site into an optimal Dorsal binding site cause the modified enhancer to be activated in a broader pattern.  A total of eight nucleotide substitutions (sufficient to create two Twist binding sites) combined with the two nucleotide substitutions that produce high-affinity Dorsal binding sites caused the modified enhancer to direct a broad pattern of gene expression in both the mesoderm and neurogenic.  A few additional nucleotide changes create binding sites for a zinc finger repressor---Snail. The modified enhancer, containing optimal Dorsal sites, Twist activator sites, and Snail repressor sites, is expressed only in the neyrogenic ectoderm where there are low levels of the Dorsal gradient.

31. Figure 19-8 regulation of transgene expression in the early Drosophila embryo

32. Altogether, a series of 2,10, 14 nucleotide substitutions produce a spectrum of Dorsal target enhancers which direct expression in the mesoderm, the mesoderm and neurogenic ectoderm, or just in the neurogenic ectoderm. suggest that enhancers can evolve quickly to create new patterns of gene expression.

33. Ubx  a drosophila pattern determining gene.  It’s analysis illustrates all three principles of evolutionary changes.  New patterns of gene expression are produced by: 1.Changing the Ubx expression pattern 2.Changing the encoded regulatory protein 3.Changing its target enhancer

34. The misexpression of Ubx changes the morphology of the fruit fly Ubx:  encodes a homeodomain regulatory protein that controls the development of the third thoracic segment, the metathorax.  specifically represses the expression of genes that are required for the development of the second thoracic segment, or mesothorax.

35. Antp is one of the genes that Ubx regulated: Ubx represses Antp expression in the metathorax and restricts its expression to the mesothorax of developing embryos.  Mutants that lack the Ubx repressor exhibit an abnormal pattern of Antp expression. The gene is not only expressed within its normal site of action in the developing mesothrax, but it is also misexpressed in the developing metathorax. This misexpression of Antp causes a transformation of the metathorax into a duplicated mesothorax.

36. Figure 19-9 Ubx mutants cause the transformation of the metathorax into a duplicated mesothorax. a. A normal fly contains a pair of prominent wings and a set of halteres. b. A mutant that is homozygous for a weak mutation in the Ubx gene has two pairs of wings and no halteres.

37. The expression of Ubx in the different tissues of the mitathorax depends on regulatory sequences that encompass more than 80 kb of genomic DNA.  A mutants Cbx (Contrabithorax) disrupts this Ubx regulatory DNA without changing the Ubx protein coding region.  Causes Ubx to be misexpressed in the mesothorax, in addition to its normal site of expression in the metathorax.  The mesothorax is transformed into a duplicated copy of the normal metathorax.

38. Figure 19-10 misexpression of Ubx in the mesothorax results in the loss of wings.  The wings are transformed into halteres.

39. Changes in Ubx function modify the morphology of fruit fly The conversion of Ubx into a transcriptional activator causes it to function like Antp and promote the development of the mesothorax. illustrates how changes in the function of a pattern determining regulatory protein can alter morphology.

40. Ubx nromally functions as a repressor.  The Ubx protein contains specific peptide sequences that recruit repression complex.  One such peptide is composed of a stretch of alanine residues---alanine rich repression domains.

41.  The misexpressin of Antp causes all of the head and thoracic segments of the embryo to develop as duplicated mesothoracic segments, all the thoracic segments contain denticle patterns that look like the one normally present only on the mesothorax.  The misexpression of Ubx causes all three thoracic segments to develop denticle patterns typical of the normal first adominal segment.

42.  Fuse the Ubx DNA-binding domain (homeodomain) to the potent activation domain from the viral VP16 protein.  Ubx is converted into an activator.  The misexpression of the Ubx-VP16 fused protein causes all of the segment to develop as mesothoracic segments,not metathoracic segments as seen when the normal Ubx protein is misexpressed in engineered embryos.  The Ubx-VP16 protein produces the same phenotype as that obtained with Antp.

43. Figure 19-11 changing the regulatory activities of the Ubx protein a.Normal embryo b.The misexpression of Ubx c.The misexpression of a Ubx-VP16 fused protein. d.The misexpression of the normal Antp protein.

44. Changes in Ubx target enhancers can alter patterns of gene expression  The Ubx protein contains a homeodomain that mediates sequence-specific DNA binding.  Ubs also contains a tetrapeptide motif [YPWM] that mediates interactions with Exd.  Ubx binds DNA as a Ubx-Exd dimer.

45. Many homeotic regulatory proteins interact with Exd and bind a composite Exs-Hox recognition sequence:  Exd binds to a half-site with the core sequence, TGAT.  Hox proteins such as Ubx binds an adjacent half-site with a different core consensus sequence, A-T-T/G-A/G.

46. The two half-sites are often separated by two nucleotides. determine which Exd-Hox dimer can bind:  Exd-Ubx dimers prefer T-T in the central position.  Exd-Labial dimers prefer G-G central residues. Raise the possibility the target enhancer regulated by one Hox protein can rapidly evolve into a target enhancer for a different Hox protein.

47. Figure 19-12 interconversion of Labial and Ubx biding sites The Hox subunit makes additional contacts with the central two nucleotides (NN). The exact sequence of these residues strongly influences spicificity.

48. Topic 4 Morphological changes in crustaceans and insects

49. Arthropods are remarkably diverse Arthropods embrace five groups: 1.trilobites ( 三叶虫 sadly extinct) 2.hexapods (such as insects) 3.crustaceans (shrimp, lobsters, crabs and so on) 4.myriapods (centipedes and millipedes) 5.chelicerates (horseshoe crabs spiders and scorpions)

50.  The success of the arthropods derives,in part, from their modular architecture.  They are composed of a series of repeating body segments that can be modified in seemly limitless ways.  Some segments carry wings, whereas others have antennae, legs, jaws, of specialized mating devices.  We know more about the evolutionary processes responsible for the diversification of arthropods than for any other group of animals.

51. Changes in Ubx expression explain modifications in Limbs among the crustaceans  Branchiopods : the thoracic segment nearest the head, T1, contains swimming appendages that look like those further back on the thorax (the second through eleventh thoracic segments, T2-T11).  Isopods : contain swimming limbs on the second through eighth thoracic segments, but the limbs on the first thoracic segment are smllar than the others and function as feeding limbs---maxillipeds.

52. Slightly different patterns of Ubx expression in branchiopods and isopods are correlated with the modification of the swimming limbs on the first thoracic segment of isopods.  During the divergence of branchiopods and isopods, the Ubx regulatory sequences changed in isopods.  Ubx expression was eliminated in the first thoracic segment, and restricted to segments,T2-T8.  The head genes can be expressed in the T1 segment due to the loss of the Ubx repressor.

53. Causes maxillipeds to develop in place of normal swimming limbs. Figure 19-13 changing morphologies in two different groups of crustaceans.

54. Why insects lack abdominal limbs  All insects have six legs, two on each of the three thoracic segments.  Other arthropods, have a variable number of limbs.  This evolutionary change---the loss of limbs on the abdomen of insects, is due to functional changes in the Ubx regulatory protein.

55. In insects, Ubx and abd-A repress the expression of Distalless (Dll ), which is required for the development of limbs. In developing Drosophila embryos ：  Ubx is expressed at high levels in the metathorax and anterior abdominal segments;  abd-A expression extends into more posterior abdominal segments.  Together Ubx and abd-A keep Dll off in the first seven abdominal segments.  Ubx does not interfere with the expression of Dll in the metathorax----not interfere with limb development in T3.

56. Why does Ubx repress Dll expression in the abdominal segments of insects, but not crustaceans? The Ubx protein has diverged between insects and crustaceans. This was demonstrated in the following experiment:

57. The misexpression of Ubx throughout all of the tissues of the presumptive thorax in transgenic Drosophila embryos suppresses limb development due to the repression of Dll. Indicate that the Drosophila Ubx protein is functionally distinct from Ubx in crustaceans. The misexpression of the crustacean Ubx protein in transgenic flies does not interfere with Dll gene expression and the formation of thoracic limbs.

58. Figure 19-14 evolutionary changes in Ubx protein function a.The Dll enhancer is normally activated in three pairs of “spots” in Drosophila embryos b.The misexpression of the Drosophila Ubx protein strongly suppressed expression from the Dll enhancer. c.The mixexpression of the Ubx protein form the brineshrimp Antermia causes only a slight supprission of the Dll enhancer.

59. What is the basis for this functional difference between the two Ubx proteins? The crustacean protein has a short motif containing 29 amion acid residues that block repression activity---”antirepression” peptide.  When this sequence is deleted, the crustacean Ubx protein is just as effective as the fly protein at repressing Dll gene expression.  When this peptide is attached to the fly protein,the hybrid protein behaves like the crustacean Ubx protein and no longer represses Dll.

60. Figure 19-15 comparison of Ubx in crustaceans and in insects The C-terminal antirepression peptide blocks the activity of the N-terminal repression domain The C-terminal antirepression peptide was lost through mutation

61. Modification of flight limbs might arise from the evolution of regulatory DNA sequences In Drosophila, Ubx is expressed in the developing halteres.  It functions as a repressor of wing development.  Approximately five to ten target genes are repressed by Ubx.  These genes encode proteins crucial for the growth and patterning for the wings.  In Ubx mutants, these genes are no longer repressed in the halteres, and the halteres develop into a second set of wings.

62. Fruit flies---dipterans : all contain a single pair of wings and a set of halteres. Butterflies---lepidopterans : all contain two pairs of wings. What is the basis for these different wing morphologies?

63. ? Change the Ubx expression pattern so that it is lost in the progenitors of the hindwings in lepidoptera. Permit the developing hindwings to express all of the genes that are normally repressed by Ubx.  There is no obvious change in the Ubx expression pattern in flies and butterflies;  Ubx is expressed at high levels throughout the developing hindwings of butterflies.

64. ? Change in the Ubx protein function. The Ubx protein functions distinctly in flies and butterflies.  The Ubx protein appears to function in the same way in fruit flies and butterflies.  In butterflies, the loss of Ubx in patches of cells in the hindwing causes them to be transformed into forewing structures. Suggest that the butterfly Ubx protein functions as a repressor that suppresses the development of forewings.

65. ? Each of the approximately five to ten target genes that are repressed by Ubx in Drosophils have evolved changes in their regulatory DNAs. They are no longer repressed by Ubx in butterflies.  It is possible that the regulatory DNAs of the wing patterning genes have lost the Ubx binding sites.

66. Figure 19-16 changes in the regulatory DNA of Ubx target genes a.The Ubx repressor is expressed in the halteres of dipterans and hindwings of lepidopterans (orange). b. Different target genes contain Ubx repressor sites in dipterans. These have been lost in lepidopterans. Wingless Drosophila serum fesponse factor

67. Topic 5 : Genome evolution and human origins

68. Human contain surprisingly few genes  The human genome contains only 25,000- 30,000 protein coding genes (there were popular estimates for 100,000 protein coding genes).  Higher vertebrates, such as humans, contain sophisticated mechanisms for gene regulation in order to produce many patterns of gene expression.

69. Organismal complexity is not correlated with gene number, but instead depends on the number of gene expression patterns.  The nematode worm ----nearly 20,000 genes.  The fruit fly---- less than 14,000 genes.  The average fly gene might be regulated by three of four separate enhancers ---- produce about 50,000 total patterns of gene expression.  Each worm gene is regulated by only one or two enhancers ----about 30,000 total patterns of gene expression.

70. The human genome is very similar to that of the mouse and virtually identical to the chimp Mice and humans:  Contain roughly the same number of genes.  80% of these genes possess a clear and unique one- to-one sequence alignment with one another between the two species.  The proteins encoded by these genes are highly conserved and share an average 80% amino acid sequences identity.  Most of the remaining 20% of the genes differ by virtue of lineage-specific gene duplication events.  There are few, if any, “mew” gene in humans that are completely absent in mice.

71. The chimp and human genomes are even more highly conserved. They vary by an average of just 2% sequence divergence----in an average stretch of 100 bp there are only two nucleotide substitutions between a random chimp and human.  Regulatory DNA evolve more rapidly than proteins.  The limited sequence divergence between chimps and human is sufficient to alter the activities of several key regulatory DNAs.

72. The evolutionary origins of human speech Speech is one of the defining features of being human.  We alone possess the capacity for precise communication in the form speech and written language.  How did our distinctive form of language arise in human evolution?

73. Speech depends on the precise coordination of the small muscles in our larynx and mouth. FOXP2  Reduced levels of a regulatory protein— FOXP2 cause severe defects in speech.  The human form of this protein is slightly different from those present in mice and primates.  There are two amino acid residues at positions 303 and 325 that are unique to human: thr to asn (T to N) at position 303 and asn to ser (N to S) at position 325.  Perhaps these changes have altered the function of human FOXP2 protein.

74. Figure 19-17 summary of amino acid changes in the FOXP2 proteins of mice and primates. The numbers indicate nonconservative amino acid substitutions. Figure 19-18 comparison of the FOXP2 gene sequences in human, chimp, and mouse.

75. How FOXP2 fosters speech in humans Perhaps a combination of all three mechanisms:  Changes in the FOXP2 expression pattern  Changes in its amino acid sequences  Changes in FOXP2 target genes might explain its emergence as an important mediator of human speech.

76. Two amino acid residues at positions 303 and 325 are unique to humans.  These changes have altered the function of the human FOXP2 protein.  There is evidence that these changes occur within a repression domain of the protein, thereby raising the possibility that the human FOXP2 fails to regulate target genes that are repressed in mice and chimps.

77. Changes in the FOXP2 regulatory DNA might cause the gene to acquire a new pattern of gene expression in human brain.  In chimps the gene might not be expressed in the appropriate region of the brain at the right time during development.  In humans FOXP2 might be expressed at the right levels in the correct time and place to foster the development of language in the brain of infants. FOXP2 might regulate different sets of target genes in chimps and humans.

78. Consider potential target genes of the FOXP2 regulatory protein.  Some might encode nuerotransmitters or other critical signals that are expressed within the developing larynx.  Perhaps these changes have augmented the levels or timing of gene expression, so that critical signals are active in the larynx during the time when humans are most susceptible to acquiring language as infants.  The corresponding genes might be expressed at lower levels, at later stages, or in the wrong regions, of the developing chimp larynx.

79. Figure 19-19 a scenario for the evolution of speech in humans The human gene is strongly expressed at the critical time in the development of the speech center and activates all three hypothetical target genes in the neocortex. These target genes might encode neurotransmitters important for the formation of the speech center.

80. The future of comparative genomes analysis Now:  There is a glaring limitation in our ability to infer the function of regulatory DNA from simple sequence inspection.  Fewer than 100 regulatory DNAs have been carefully characterized in all animals combined.  This is not a sufficient data set to determine whether regulatory DNAs that mediate similar patterns of gene expression share a common “code”.

81. In the future:  It might be possible to infer both the timing and sites of gene expression by simply scanning the DNA sequences associated with any given gene.  It might also be possible to identify changes in the expression profiles of homologous genes.  The continuing development of new computational methods and the availability of new genome assemblies offer exciting prospects for the use of comparative methods to reveal the mechanisms of evolutionary diversity.

82. The end Thank you !