XIII Developmental Genetics XIII Developmental Genetics A Overview

XIII. Developmental Genetics A. Overview: - Multicellular organisms have different cell types, but all

XIII. Developmental Genetics A. Overview: B. Genetic Control of Body Plan: - Drosophila has

B. Genetic Control of Body Plan: - 1. There are two genomes involved: -

And the most dramatic examples of homology are in the hox genes, as well.

Ey (eyeless): a selector gene in Drosophila. Turn it ON and get eyes in

Homologous to the Pax 6 gene in vertebrates, that affects eye and brain development.

In fact, the homology is so good that knock-out flies lacking the eyeless gene

XIII. Developmental Genetics A. Overview: - This requires two more levels of genetic regulation:

B. Co-ordination of cell differentiation with other cells Notch genes code for a transmembrane

B. Co-ordination of cell differentiation with other cells When bound, NICD is cleaved and

B. Co-ordination of cell differentiation with other cells How such signals coordinate gene expression

Organogenesis of the vulva in C. elegans There are two cells; one will become

Organogenesis of the vulva in C. elegans The anchor cell now interacts with 6

Organogenesis in flowers Arabidopsis thaliana Thale kress Mustard family

Organogenesis in flowers ‘A’ genes are usually on in first and second whorls. When

Organogenesis in flowers S = sepal, p = petal, st = stamens, c =

Homeobox Genes: - have a 180 bp sequence, which codes for 60 aa’s in

KAMAKURA, M. 2011. Royalactin induces queen differentiation in honeybees. Nature. 473: 478 -483.

Nutritional Control of Reproductive Status in Honeybees via DNA Methylation, R. Kucharski*, et al.

2. Silence methylation in embryos, get a queen phenotype with larger ovaries. (suggests that

Spannhof, et al. 2011. EMBO 12: 238 -243. Histone deacetylase inhibitor activity in royal

But it helps 5 -Aza turn on genes No demethylation by itself 5 -Aza

10 -HDA is structurally similar to a known Histone deacetylase inhibitor (HDACi)…. (don’t let

From Nov. 1944 – May 1945: - Hard winter in northern Europe - Germany

60 years later, children starved early in development had lower methylation of IGF -2

So, perhaps changes incurred during a lifetime can cause a heritable change to the

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XIII. Developmental Genetics

XIII. Developmental Genetics A. Overview: - Multicellular organisms have different cell types, but all cells are descended from a single zygote and have the same DNA.

XIII. Developmental Genetics A. Overview: - Multicellular organisms have different cell types, but all cells are descended from a single zygote and have the same DNA. - So, cell specialization is really a very special type of gene regulation; the regulation of genes that influence the developmental fate of a cell. - This requires two more levels of genetic regulation: - regulation of cell specialization/differentiation - co-ordination of this specialization with other cells/tissues - One of the most remarkable revelations in this area of genetics is that all animals (from flies to worms to mammals, and even radially symmetrical coral) share the same genes that co-ordinate the basic body plan and its polarity (anterior-posterior axes and body regions)…. But of course, why not?

XIII. Developmental Genetics A. Overview: B. Genetic Control of Body Plan: - Drosophila has been the model organism for body plan development, and resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets). - 1. There are two genomes involved: the maternal genome produces maternal proteins that form a gradient in the egg and influence the initial development of the embryo. The zygote’s genome produces proteins that react to these gradient in two ways: - segmentation genes (gap, pair-rule, segment polarity genes) create segments in the embryo by defining body regions (gap), then dividing the embryo into units two segments wide (pair-rule), and then an active segment polarity gene splits each segment into front and backs.

B. Genetic Control of Body Plan: - 1. There are two genomes involved: - 2. There is strong homology among animals in these genes: Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates the development of neurons and sex determination. In mammals, it is needed for bone deposition in segments. The most dramatic examples of developmental effects are in the hox genes: On chromosome 3, in order… Antennapedia cluster (Ant-C) Bithorax cluster (Bx-C)

B. Genetic Control of Body Plan: - 1. There are two genomes involved: - 2. There is strong homology among animals in these genes: Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates the development of neurons and sex determination. In mammals, it is needed for bone deposition in segments. The most dramatic examples of developmental effects are in the hox genes: These genes are in all cells, but each is only active in it’s respective segment - switching on a host of specific genes that lead to cell, tissue, and organ differentiation. On chromosome 3, in order… Antennapedia cluster (Ant-C) Bithorax cluster (Bx-C)

B. Genetic Control of Body Plan: - 1. There are two genomes involved: - 2. There is strong homology among animals in these genes: Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates the development of neurons and sex determination. In mammals, it is needed for bone deposition in segments. The most dramatic examples of developmental effects are in the hox genes: Each gene codes for a transcription factor. Each gene has a 180 bp homeobox region that encodes a 60 AA homeodomain in the protein that takes a Helix-Loop Helix configuration….

B. Genetic Control of Body Plan: - 1. There are two genomes involved: - 2. There is strong homology among animals in these genes: Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates the development of neurons and sex determination. In mammals, it is needed for bone deposition in segments. The most dramatic examples of developmental effects are in the hox genes: And if you turn them on in the wrong segment, you get an incorrect pattern of body segment development! a. Normal fly head, with normal antennae. b. Fly with legs for antennae, cause by activating a hox gene for leg development that is normally on in thoracic segments. (antennaepedia)

And the most dramatic examples of homology are in the hox genes, as well.

And the most dramatic examples of homology are in the hox genes, as well. And, human diseases have been identified as hox mutants by identifying homology with fruit fly hox genes, and they have been found in the genome with ss-DNA (probe) from the fly homeobox region of that gene. Cleinocranial dysplasia (CCD), caused by a dominant mutation in the runt homeotic gene

And the most dramatic examples of homology are in the hox genes, as well. And, human diseases have been identified as hox mutants by identifying homology with fruit fly hox genes, and they have been found in the genome with ss-DNA (probe) from the fly homeobox region of that gene. Synpolydactyly in humans, caused by a mutation in HOXD 13

And the most dramatic examples of homology are in the hox genes, as well. And, human diseases have been identified as hox mutants by identifying homology with fruit fly hox genes, and they have been found in the genome with ss-DNA (probe) from the fly homeobox region of that gene. And, because of evolution and common ancestry, we can use model organisms like flies to learn about how heredity and development work in all animals…including humans.

Ey (eyeless): a selector gene in Drosophila. Turn it ON and get eyes in novel places… fully functional.

Homologous to the Pax 6 gene in vertebrates, that affects eye and brain development. Query 5 HSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQTHADAKVQVLDNQNVSN 64 Sbjct 30 HSG INQLGGVYVNGRPLPDSTRQKIVELAHSGARPCDISRILQ-------- VSN 75 Query 65 GCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECPSIFAWEIRDRLLSEG 124 Sbjct 76 GCVSKILGRYYETGSIKPRAIGGSKPRVATTPVVQKIADYKRECPSIFAWEIRDRLLSEQ 135 Query 125 VCTNDNIPSVSSINRVLRNLASEKQQMGA---DGMYDKLRMLNGQTGSWGTRPGWYPGTS 181 Sbjct 136 VCNSDNIPSVSSINRVLRNLASQKEQQAQQQNESVYEKLRMFNGQTGGW----AWYPSNT 191 Query 182 VPGQPTqdgcqqqeggge. N------------TNSISS----NGED 210 Sbjct 192 TTAHLTLPPAASVVTSPANLSGQADRDDVQKRELQFSVEVSHTNSHDSTSDGNSEHNSSG 251 Query 211 SDEAQMRLQLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQV 270 Sbjct 252 DEDSQMRLRLKRKLQRNRTSFSNEQIDSLEKEFERTHYPDVFARERLADKIGLPEARIQV 311 Query 271 WFSNRRAKWRREEKLRNQRRQA 292 Sbjct 312 WFSNRRAKWRREEKMRTQRRSA 333 Query = toy (twin of eyeless gene) Subject = human Pax 6 Red = Paired-box domain – DNA binding site Green = homeodomain DNA binding site Probability of similarity occurring by chance: 3. 2 x 10 -77

In fact, the homology is so good that knock-out flies lacking the eyeless gene can make eyes when the mammalian Pax 6 gene is inserted. Likewise, a regulatory element upstream from ey can restore Pax 6 activity in vertebrates. So they gene (Pax 6/ey) and the regulation of the gene, have been conserved through metazoan evolution. Pax 6 / Ey

B. Co-ordination of cell differentiation with other cells Notch genes code for a transmembrane signal receptor protein that binds another transmembrane protein called Delta…. So this binding occurs between cells: Extracellular domain (NECD) Transmembrane domain (NTMD) Intracellular domain (NICD) Myc p 21 NICD

B. Co-ordination of cell differentiation with other cells When bound, NICD is cleaved and binds to the Su(H) protein – which passes into the nucleus and binds and stimualtes transcription factors. Extracellular domain (NECD) Transmembrane domain (NTMD) Intracellular domain (NICD) Myc p 21 NICD

B. Co-ordination of cell differentiation with other cells How such signals coordinate gene expression has been determined for the vinegar worm, Caenorhabditis elegans.

B. Co-ordination of cell differentiation with other cells How such signals coordinate gene expression has been determined for the vinegar worm, Caenorhabditis elegans. This is a great developmental model, because the animal has a set number of somatic cells (959) and the fate of each has been mapped:

Organogenesis of the vulva in C. elegans There are two cells; one will become the gonadal anchor cell, and the other will become the first uterine cell. What determines their fate?

Organogenesis of the vulva in C. elegans There are two cells; one will become the gonadal anchor cell, and the other will become the first uterine cell. What determines their fate? Initially, they both produce delta signal protein (Lag-2) and notch receptor protein (lin-12). By chance, these cells won’t produce exactly the same amount of signal… the one bombarded by MORE signal produces more receptor. . . which causes the genetic cascade that results in the cell becoming the uterine cell.

Organogenesis of the vulva in C. elegans The anchor cell now interacts with 6 epidermal cells. The anchor cell produces another protein, lin-3. In the closest cells where exposure to this protein are greatest, receptors trigger a signal transduction pathway that turns on 1 o vulva genes. In the closest cell, a protein is produced that activates the lin-12 gene in the neighboring cells, which arrests 1 o vulvar cell development – they become 2 o vulvar cells.

Organogenesis in flowers Arabidopsis thaliana Thale kress Mustard family

Organogenesis in flowers Four whorls

Organogenesis in flowers ‘A’ genes are usually on in first and second whorls. When active alone they specify sepal development. When active with B genes, petals develop.

Organogenesis in flowers

Organogenesis in flowers S = sepal, p = petal, st = stamens, c = carpels Wild = s, p, st, c APETALA 2 = c, st, c PISTILLATA = s, s, c, c AGAMOUS = s, p, s, p

Homeobox Genes: - have a 180 bp sequence, which codes for 60 aa’s in the protein called the homeodomain. This sequence creates the DNA binding site of the protein. As such, it has been highly conserved over evolutionary history, and all multicellular organisms (fungi, animals, and plants) have them… for regulating gene activity in cells that specialize for different functions. Parahox One class of Homeobox genes are HOX genes. They were the first class of homeobox genes found. They are only in animals. NK MADS-box 58 aa sequence

XIII. Developmental Genetics A. Overview: - This requires two more levels of genetic regulation: - regulation of cell specialization/differentiation - co-ordination of this specialization with other cells/tissues B. Genetic control of Body Plan: C. Co-ordination of cell differentiation with other cells D. Epigenetic Effects on Development - Heritable (maternal effects): snail coiling: dextral vs. sinistral - Environmental : Female worker or female Queen?

KAMAKURA, M. 2011. Royalactin induces queen differentiation in honeybees. Nature. 473: 478 -483.

Nutritional Control of Reproductive Status in Honeybees via DNA Methylation, R. Kucharski*, et al. Science 2008. 1. Silencing of DNA methyltransferase (Dnmt 3) using RNAi (Dnmt 3 is an important enzyme in methylating Cp. G islands. )

2. Silence methylation in embryos, get a queen phenotype with larger ovaries. (suggests that royal jelly may disrupt methylation make queen)

Figure 4. Expression profile of an alternatively spliced and differentially methylated gene GB 18602 in queen and worker brains. (A) The Cp. G methylation pattern indicating the level of methylation for individual Cp. Gs (blue squares, workers; red squares, queens). (B) Gene model of GB 18602 showing the two spliced variants S (short protein) and L (long protein) and the positions of PCR primers used for variant-specific amplifications. The green and orange arrows indicate the positions of two alternative Stop codons. (C) Relative expression of the two spliced variants in brains of queens and workers examined by real-time PCR. The level of transcript S (green) encoding the truncated protein is significantly upregulated in the queen brain, whereas the L variant (orange) is expressed at the same level in both castes. The queen expression represents a combined set of data from three independent queen samples: 4 mo old (1 brain), 12 mo old (2 brains), and swarm queens of unknown age (2 brains). Workers were 8 d old (6 brains in 3 replicates). The reference gene was calmodulin [2]. Whisker-box plot of expression ratio values: dotted line, median value; box, inter-quartile range of values; whiskers, outer 50% of observations. For more details, see Table S 4. More methylation in workers, except in some genes in brain with respect to one gene. Ratio = 1, like orange, means same amount in queen/worker brains. More green in queen. The Honey Bee Epigenomes: Differential Methylation of Brain DNA in Queens and Workers, Lyko, et al. 2010)

Spannhof, et al. 2011. EMBO 12: 238 -243. Histone deacetylase inhibitor activity in royal jelly might facilitate caste-switching in bees. Fas is methylated and off in K-ras cells. RJ releases inhibition So does HDA

But it helps 5 -Aza turn on genes No demethylation by itself 5 -Aza is a known methylation inhibitor. When The green fluorescent protein gene is heavily methylated, 5 -Aza reduces methylation BUT 10 HDA does NOT. (see bar charts, and see the number of fluorescing cells in the images). So, 10 HAD is not really a methylation inhibitor – it must be stimulating transcription another way.

10 -HDA is structurally similar to a known Histone deacetylase inhibitor (HDACi)…. (don’t let the common HDA letters fool you – they stand for different things!) AND, as HDA concentration increases (from right to left), there is MORE HDAC inhibition.

Spannhof, et al. 2011. EMBO 12: 238 -243. Histone deacetylase inhibitor activity in royal jelly might facilitate caste-switching in bees. What does RJ do? - 5% of RJ is 10 -hydroxy-2 -decenoic acid (HDA) - acts as a histone deacetylase inhibitor (HDACi) - histone deacetylases (HDAC’s) remove acetyl groups from lysine amino acids in the histone proteins – this causes the histones to bind DNA more tightly, inhibiting polymerases. - by Inhibiting HDAC’s, histones are relaxed – genes can turn on Queen. - So, although 10 -HDA is not a methylation inhibitor ITSELF, it relaxes histones and allows access to genes (and access to demethylating agents, too, like in the 5 -Aza experiments). - Royalactin stimulates growth and reproduction.

From Nov. 1944 – May 1945: - Hard winter in northern Europe - Germany blockaded Netherlands - People ate ~ 30% of necessary caloric intake – 22, 000 died. Babies conceived in October, starved during late development – born small. Stayed small as adults; lower incidence of obesity Babies conceived in February, starved early – caught up late in development when food was available to their mothers – born average size. Higher incidence of obesity than population at large And their children has higher levels of obesity, too. A heritable change in epigenetic modification? From Carey, N. 2013. The Epigenetic Revolution.

60 years later, children starved early in development had lower methylation of IGF -2 than their older or younger siblings. Those starved late in development – no difference. IGF 2 DMR methylatio n Mean methylation fraction (SD) Exposed (n = 60) Controls (n = 60) Relative change exposed Difference in SDs P Average 0. 488 (0. 047) 0. 515 (0. 055) − 5. 2% − 0. 48 5. 9 × 10− 5 Cp. G 1 0. 436 (0. 037) 0. 470 (0. 041) − 6. 9% − 0. 78 1. 5 × 10− 4 Cp. G 2 and 3 0. 451 (0. 033) 0. 473 (0. 055) − 4. 7% − 0. 41 8. 1 × 10− 3 Cp. G 4 0. 577 (0. 114) 0. 591 (0. 112) − 2. 3% − 0. 12 . 41 Cp. G 5 0. 491 (0. 061) 0. 529 (0. 068) − 7. 2% − 0. 56 1. 4 × 10− 3 Heijmans et al. 2008. Persistent epigenetic differences associated with prenatal exposure to famine in humans.

So, perhaps changes incurred during a lifetime can cause a heritable change to the genome. How long these epigenetic changes are imprinted remains to be seen. Also, they create variation on which selection can act. Still, it may be somewhat Lamarckian.