Genomics Genetic Engineering To mass-produce proteins To give species new characteristics

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.


A.Overview:


- So, cell specialization is really a very special type of gene regulation; the regulation of genes that influence the developmental fate of a cell.


A.Overview:


- 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


A.Overview:


- 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?


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).


A.Overview:



- 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.


A.Overview:





The zygote’s genome produces proteins that react to these gradient in two ways:


A.Overview:






- 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.


A.Overview:






- 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.

- hox genes (shortened from homeobox genes) determine the developmental fate of cells in a particular segment.


A.Overview:



- 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.


- 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)





On chromosome 3, in order…

Antennapedia cluster (Ant-C)

Bithorax cluster (Bx-C)

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.





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….





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 only on in thoracic segments. (antennaepedia)





Activaton of bithorax in third thoracic segment …

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



Synpolydactyly in humans, caused by a mutation in HOXD13



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 Pax6 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 VPGQPTqdgcqqqegggeN-----------------------TNSISS--------NGED 210 Sbjct 192 TTAHLTLPPAASVVTSPANLSGQADRDDVQKRELQFSVEVSHTNSHDSTSDGNSEHNSSG

251 Query 211 SDEAQMRLQLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQV 270 Sbjct 252 DEDSQMRLRLKRKLQRNRTSFSNEQIDSLEKEFERTHYPDVFARERLADKIGLPEARIQV 311

Query 271 WFSNRRAKWRREEKLRNQRRQA 292Sbjct 312 WFSNRRAKWRREEKMRTQRRSA 333

Query = toy (twin of eyeless gene)Subject = human Pax6

Red = Paired-box domain – DNA binding siteGreen = 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 Pax6 gene is inserted.

Likewise, a regulatory element upstream from ey can restore Pax6 activity in vertebrates.

So they gene (Pax6/ey) and the regulation of the gene, have been conserved through metazoan evolution.

Pax6 / Ey


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

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)

NICD

Mycp21


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)

NICD

Mycp21


How such signals coordinate gene expression has been determined for the vinegar worm, Caenorhabditis elegans.


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?



Initially, they both produce delta signal protein and notch receptor protein…



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.


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 1o vulva genes.


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 1o vulva genes.In the closest cell, a protein is produced that activates the lin-12 gene in the neighboring cells, which arrests 1o vulvar cell development – they become 2o vulvar cells.

Organogenesis in flowers

Arabidopsis thaliana

Thale kress Mustard family

Organogenesis in flowersFour whorls


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


Wild = s,p,st,c APETALA2 = c,st,st,c PISTILLATA = s,s,c,c AGAMOUS = s,p,s,p

S = sepal, p = petal, st = stamens, c = carpels

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.

One class of Homeobox genes are HOX genes. They were the first class of homeobox genes found. They are only in animals.

Parahox

NK

MADS-box

58 aa sequence


A.Overview:


cells/tissues

B.Genetic control of Body Plan:C.Co-ordination of cell differentiation with other cellsD.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 (Dnmt3) using RNAi

(Dnmt3 is an important enzyme in methylating CpG islands.)

http://www.sciencemag.org.libproxy.furman.edu/search?author1=R.+Kucharski&sortspec=date&submit=Submit

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

http://www.sciencemag.org.libproxy.furman.edu/content/319/5871/1827/F2.large.jpg

Figure 4. Expression profile of an alternatively spliced and differentially methylated gene GB18602 in queen and worker brains.(A) The CpG methylation pattern indicating the level of methylation for individual CpGs (blue squares, workers; red squares, queens). (B) Gene model of GB18602 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 up-regulated 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 S4.

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)

More methylation in workers, except in some genes in brain with respect to one gene.

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1000506

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1000506

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

5-Aza is a known methylation inhibitor. When The green fluorescent protein gene is heavily methylated, 5-Aza reduces methylation BUT 10HDA 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.

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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.


A.Overview:


cells/tissues

B.Genetic control of Body Plan:C.Co-ordination of cell differentiation with other cellsD.Epigenetic Effects on Development

- Heritable (maternal effects):snail coiling: dextral vs. sinistral

- Environmental : - Heritable Epigenetic Effects: Dutch Hunger Winter

From Nov. 1944 – May 1945: - Hard winter in northern Europe - Germany blockaded Netherlands - People ate ~ 30% of necessary caloric intake – 22,000 died.

From Carey, N. 2013. The Epigenetic Revolution.

Babies conceived in October, starved during late development – born small.

Babies conceived in February, starved early – caught up late in development when food was available to their mothers – born average size.

Stayed small as adults; lower incidence of obesity

Higher incidence of obesity than population at large

And their children has higher levels of obesity, too. A heritable change in epigenetic modification?

IGF2 DMR methylation

Mean methylation fraction (SD)Relative change

exposed

Difference in SDs P

Exposed (n = 60) Controls (n = 60)

Average 0.488 (0.047) 0.515 (0.055) −5.2% −0.48 5.9 × 10−5

CpG 1 0.436 (0.037) 0.470 (0.041) −6.9% −0.78 1.5 × 10−4

CpG 2 and 3 0.451 (0.033) 0.473 (0.055) −4.7% −0.41 8.1 × 10−3

CpG 4 0.577 (0.114) 0.591 (0.112) −2.3% −0.12 .41

CpG 5 0.491 (0.061) 0.529 (0.068) −7.2% −0.56 1.4 × 10−3

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.

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.

Genomics Genetic Engineering To mass-produce proteins To give species new characteristics

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