1. How did differences in morphologies between animal phyla evolve if Hox gene expression patterns are so uniform across species?  Changes in Hox-protein responsive elements of downstream genes  Changes in Hox gene that gives the protein new properties  Changes in Hox gene expression within a region of the body  Changes in Hox gene expression between regions of the body  Changes in the number of Hox gene

2. Why a butterfly has four wings but a fly has only two Carroll 1995 Nature 375: 58-61 DipteraLepidopteraColeoptera Evolution of Ubx-regulated hindwing patterns Four-winged ancestor From Carroll et al 2001 Fig 5.13, Gilbert Fig 23.3 Changes in Hox-protein responsive elements of downstream genes

3. Testing Hypothesis 2: Butteflies have hindwings because Ubx is not expressed in T3 imaginal disc (Warren et al 1994, Carroll et al 1995) Ubx is expressed in haltere imaginal disc in T3 in flies & Ubx is expressed in hindwing imaginal disc in T3 in butterflies Fig 4 Warren et al 1994 Nature 372: 458-461 Conclusion: presence of hindwings in butterflies is not due to absence of Ubx expression. The difference must lie in the response of the downstream targets of Ubx

4. How did differences in morphologies between animal phyla evolve if Hox gene expression patterns are so uniform across species?  Changes in Hox-protein responsive elements of downstream genes  Changes in Hox gene that gives the protein new properties  Changes in Hox gene expression within a region of the body  Changes in Hox gene expression between regions of the body  Changes in the number of Hox gene

5. Changes in Hox gene expression within a region of the body Why caterpillars have abdominal prolegs but maggots do not Warren et al 1994 Nature 372: 458-461 Adult Flies and Butteflies: 3 pairs of legs that arise from imaginal discs T1, T2 & T3 of larva Fly larvae: maggots Moth and butterfly larvae: rudimentary abdominal legs called prolegs

6. A new gene: Distal-less (Dll) and the formation of legs These distal-less expressing imaginal discs give rise to the jaws and legs. No limbs ever form on the abdomen of adults…BUT, butterfly and moth larvae have rudimentary abdominal prolegs…. Is this due to differences in expression patterns of Dll? vestigal/scalloped Distal-less eyeless Distal-less: gene that is important for determining proximal-distal axis of appendages In Insects, Distal-less is expressed in cephalic and thoracic limb forming imaginal discs, but is excluded from abdomen by AbdA and Ubx homeodomain proteins

7. Expression of distal-less in a buckeye butterfly (Precis coenia) cf Drosophila Fig 23.4 Expression of distal-less in the larvae of the buckeye butterfly. Expression of Distal-less is different to Drosophila, in that there is clear distal-less expression in A3-A6. From Gilbert, pg 757 Like Drosophila Initially Distal-less is expressed in embryo in head and thoracic regions that give rise to the imaginal discs…however, Unlike Drosophila As development proceeds, Distal-less expression extends to abdominal segments A3-A6 Is Distal-less (Dll) regulated differently in dipterans compared to lepidopterans Warren et al 1994 Nature 372: 458-461

8. Is Distal-less (Dll) regulated differently in dipterans compared to lepidopterans 2 options: 1. Dll genes of buckeye butterfly are not repressed by Ubx and AbdA 2. Expression of Ubx and AbdA is different in abdominal regions of Precis A: Early Precis caterpillar, thoracic segments and jaws express Dll. Some abdominal segments begin to have holes in AbdA/Ubx expression (white arrows) B: Later-stage Precis caterpillar: Dll is Expressed in holes where there is no AbdA/Ubx expression Green: AbdA/Ubx protein Red: Dll protein Thus, due to downregulation of AbdA & Ubx Dll is expressed in developing embryo, and prolegs form in larvae of buckeye moth. Warren et al 1994 Nature 372: 458-461

9. How did differences in morphologies between animal phyla evolve if Hox gene expression patterns are so uniform across species?  Changes in Hox-protein responsive elements of downstream genes  Changes in Hox gene that gives the protein new properties  Changes in Hox gene expression within a region of the body  Changes in Hox gene expression between regions of the body  Changes in the number of Hox gene

10. Changes in Hox gene that gives the protein new properties Can different morphologies be explained by differences in Hox gene coding regions? Case study: How well conserved is Ubx in ancient athropods? Comparison of Ubx isolated from a Oncophora (OUbx) to Drosophila Ubx (DUbx) (Grenier et al 2000, PNAS 97: 704-709) Common ancestor Athropoda Tardigrada Onycophora (velvet worm)

11. Fossils from Middle Cambrian Burgess shale that resemble Onycophora (velvet worm) Onycophora: ancient arthropods Orthologue of Ubx in onycophora identified (OUbx) DNA binding domain highly conserved Are OUbx and DUbx functionally interchangeable?...test by ectopic expression

12. Comparison of Drosophila Ubx (DUbx) to Oncophora Ubx (OUbx) (Grenier et al 2000, PNAS 97: 704-709), Ectopic expression of DUbx and OUbx in Drosophila Assayed Phenotype of forewings on T2 T3A1A2 DUbx and OUbx can both induce haltere formation instead of forewing on T2 when ectopically expressed in Drosophila Conservation of Ubx function BUT….not all effects of ectopic expression were similar…… Fig2 Grenier & Carroll 2000 PNAS Ectopic expression of DUbx OUbx Adult forewing on T2 transformed towards a haltere Forewing and haltere in WT fly

13. Comparison of Drosophila Ubx (DUbx) to Oncophora Ubx (OUbx) (Grenier et al 2000, PNAS 97: 704-709), Ectopic expression of DUbx and OUbx in Drosophila Assayed Phenotype of T3, A1, A2 (patterns of denticle belts) Phenotype of T3, A1 and A2 in WT larvae Look at pattern of hairs in denticle belts of WT Drosophila larvae T3A1A2 rows of hairs: 2 or 3 4 6 trapezoidal pattern DUbxOUbx Denticle belts in T3 of larvae with ectopic expression of Ectopic expression of DUbx converts T3 into segment with A1 identify Ectopic expression of OUbx does not change T3 segment identify Fig2 Galant & Carroll Nature 2002

14. Comparison of Oncophora Ubx (OUbx) to Drosophila Ubx (DUbx)… (Grenier et al 2000, PNAS 97: 704-709), Difference between DUbx and OUbx due to i)Divergence of sequences along length of coding region ? ii)Presence or absence of discrete functional motifs ? Ectopic expression of DUbx and OUbx in Drosophila Assayed Expression of reporter gene under control of Dll promoter (Dll304-lacZ) In WT embryos, Dll promoter is active in leg imaginal discs on T1, T2 and T3 When DUbx is ectopically expressed, Dll expression is repressed When OUbx is ectopically expressed, Dll expression is not repressed Fig2 Galant & Carroll Nature 2002

15. Comparison of Ubx form and function in other taxa (Galant & Carroll, 2002, Nature 415: 910-913), Difference between DUbx and OUbx due to i)Divergence of sequences along length of coding region ii)Presence or absence of discrete functional motifs Galant and Caroll (2002) Cloned Ubx from phylogenetically intermediate taxa Red flour beetle: Tribolium castaneum (TrUbx) Butterfly: Junonia coenia Gilbert Fig 23.6 Tribolium castaneum Junonia coenia Conserved DNA binding domain, but divergent polyalanine and QAQA domains

16. Is there a functional difference between insect and Onycophoran Ubx orthologues : Galant & Carroll, 2002, Nature 415: 910-913 Ectopic expression of DUbx, TcUbx and OUbx in Drosophila Assayed 1.Phenotype of T3, A1, A2 (patterns of denticle belts) 2.Expression of reporter gene under control of Dll promoter (Dll304-Dll) Fig 2: Galant and Carroll 2002 Nature 415: 910-913 Ectopic expression of WT A2A1T3 DUbxTcUbxOUbxO/QA 1. 2, DUbx TcUbx OUbxO/QA WT Ectopic expression of Patterns of hair In denticle belts Dll304-lacZ activity in thoracic segments DUbx and TcUbx transform segment identify & represses Dll expression OUbx does not: Is this due to conserved QA-polyA region in C-terminus? T3

17. Is there a functional difference between insect and Onycophoran Ubx orthologues : Galant & Carroll, 2002, Nature 415: 910-913 Constructed chimaeric proteins of DUbx and OUbx Including C-terminal 24 amino acids from DUbx fused to OUbx (O/QA) Assay phenotype of ectopic expression of O/QA chimaera 1.Phenotype of T3, A1, A2 (patterns of denticle belts) 2.Expression of reporter gene under control of Dll promoter (Dll304-Dll) Fig 2: Galant and Carroll 2002 Nature 415: 910-913 Ectopic expression of WT A2A1T3 DUbxTcUbxOUbxO/QA 1. 2, DUbx TcUbx OUbxO/QA WT Ectopic expression of Patterns of hair In denticle belts Dll304-lacZ activity in thoracic segments Conclusion: Conserved insect QA-polyA, C-terminal domain is a repressor domain which is sufficient to convert Ubx into transcriptional repressor of Dll T3

18. Fig 23.6 Gilbert: changes in the Ubx protein associated with the insect clade in the evolution of arthropods. Only the insect Ubx protein is able to repress Distal-less Ronshaugen et al, 2002 Galant and Carroll, 2002 The 3’poly-alanine domain is conserved in the insect lineage This polyalanine region acts a repressor of Distal-less Thus insects have evolved the ability to suppress Distal-less Leading to the suppression of legs in the abodomen of insects Comparison of Ubx orthologues in other species

19. The presence of the poly-alanine domain in the insect lineage explains why insects have only six legs Crustacean –like arthroprod ancestors had multiple limbs Six-legged insects diverged from arthropods about 400 million years ago In insects, Ubx and AbdA are expressed in abdominal segments, where they suppress Limb development by suppressing distal-less expression In a branchiopod crustacean, Ubx/AbdA are expressed in abdominal segments but do NOT suppress limb development Fig 1a Ronshaugen et al 2002 shrimp

20. How did differences in morphologies between animal phyla evolve if Hox gene expression patterns are so uniform across species?  Changes in Hox-protein responsive elements of downstream genes  Changes in Hox gene that gives the protein new properties  Changes in Hox gene expression within a region of the body  Changes in Hox gene expression between regions of the body  Changes in the number of Hox gene