Genetics contributes to size. Bundschu et al., 2005, JBC Shingleton et al., 2005, PLOS

Size: px

Start display at page:

Download "Genetics contributes to size. Bundschu et al., 2005, JBC Shingleton et al., 2005, PLOS"

Maria Bishop
5 years ago
Views:

1 Matters of size

2 Genetics contributes to size Bundschu et al., 2005, JBC Shingleton et al., 2005, PLOS

5 Advantages to examining growth mechanisms in the zebrafish fin The fin and body are easy to measure The fin grows throughout the lifetime of the fish When amputated, the fin grows back (regeneration) The fin is comprised of relatively few tissues Zebrafish are easy to manipulate genetically

6 Fin length mutants provide opportunities to evaluate abnormal growth

7 Identification of the mutation causing the sof phenotype reveals a gene required for normal bone growth in fins WT sof Mutations in the gap junction gene connexin43 cause the short fin phenotype The short fin mutant exhibits less connexin43 activity, which leads to less growth and shorter bony segments

8 100 Trillion = cells/human body; >200 different cell types

9 Cells are Organized in Tissues and Organs Outer layers of cells in close contact basement membrane

10 Cells in tissues communicate by the exchange of small molecules via gap junction channels Gap Junctions Development Differentiation Cell and Tissue Function - Heart beat - Onset of labor - Conduct neuronal signals through electrical synapses gap junctions

11 Appearance of Gap Junctions n n n n Immunofluorescence 400nm 100 Thin Section 100 Negative Stain

12 Gap junctions facilitate the exchange of small molecules between cells Ca2+ Electrical Currents Second Messengers (IP3) Proteins Nucleic Acids 7nm 1.5nm 17 nm

in bone shape and size sof Paznekas et al.

13 Mutations in mammalian CX43 also cause skeletal malformations delayed bone development Lecanda et al., 2000 Human ODDD Mouse ODDD WT abnormalities in bone shape and size sof Paznekas et al., 2003 Flenniken et al., 2005 How do mutations in the connexin43 gene lead to bone defects?

Strategy for determining the role of connexin genes in causing disease phenotypes Identify defects at cellular level: cell differentiation proliferation gene expression

14 Strategy for determining the role of connexin genes in causing disease phenotypes Identify defects at cellular level: cell differentiation proliferation gene expression Comprehensive understanding of how direct cell-cell communication regulates tissue growth Identify defects in connexin function: assembly dye coupling ionic coupling develop in vivo assay

15 The fin ray is comprised of multiple tissues transverse section longitudinal section 1 e m bone forming cells collagen-like fibers BrdU dividing/undifferentiated cells calcein e bone forming cells collagen-like fibers dividing/undifferentiated cells In which cells does connexin43 function?

16 Different cells use different sets of genes alcohol dehydrogenase collagen myosin muscle liver bone Expressed genes generate a message or mrna, that gets translated into protein

17 Detecting gene expression in situ hybridization Permits localization of gene-specific mrnas in a complex tissue

18 Fins regenerate and grow rapidly following amputation Akimenko et al., Dev Dyn. e Wound healing Little cell division Blastema Outgrowth ~48hpa Fin outgrowth This facilitates monitoring gene expression and other events during fin growth

19 cx43 is expressed in dividing cells and in bone-forming cells at segment boundaries bm 8 dpa e e e e lepidotrichia/ bone matrix Does Connexin43-based communication influence cell division?

Ways to monitor cell proliferation BrdU H3P

20 Ways to monitor cell proliferation BrdU H3P Bromo-deoxy-uridine is a thymidine analog incorporates into DNA during S-phase. Acts as a persistent marker for recently divided cells. Use of antibody for detection. Histone-3-phosphate is an antibody that detects Serine10 only when phosphorylated. Ser10 is only phosphorylated during MITOSIS.

Fins of short fin mutants grow more slowly and

35 140 Rate growth ( g g(cm/day) 0.3 0.25 0.

05 wild-type short fin # H3P positive cells

2 3 4 5 6 7 8 9 10 days post amputation 0 0 1

21 Fins of short fin mutants grow more slowly and exhibit reduced levels of cell proliferation Rate growth ( g g(cm/day) wild-type short fin # H3P positive cells wild-type short fin days post amputation days post amputation wild-type short fin wild-type short fin 3dpa 3dpa

Fewer progenitor cells proliferate in short fin fins wild-type short fin 50 H3P + 0.9 cm 25 H3P + 0.

22 Fewer progenitor cells proliferate in short fin fins wild-type short fin 50 H3P cm 25 H3P cm wild-type short fin Suggests Cx43 plays a role in initiating cell proliferation or instructing cells to divide

23 Model for Cx43-mediated cell proliferation Which genes mediate Cx43 function?

24 Microarray analysis detects differences in gene expression from whole tissues Method: Different genes are spotted on chips glass slides the size of a postage stamp

25 The another long fin mutant (alf) exhibits phenotypes opposite of the short fin mutant overlong bony segments increased cx43 mrna wild-type alf

26 To find genes mediated by cx43, identify genes regulated in opposing manners in short fin and alf i.e. genes down-regulated in short fin AND up-regulated in alf Pathways downregulated in short fin =underexpression of cx43 ****** Pathways upregulated in alf =overexpression of cx43 Total genes on zebrafish microarray: Identified by overlap 43,000 genes 180 genes

physiological processes transport cell biogenesis

27 Categorizing microarray data limits the number of genes for further analysis metabolism physiological processes transport cell biogenesis cell proliferation cell cycle mobility cell death cell growth

Genes of further interest will be expressed in the same cells as connexin43 Examples from list of genes involved in Cell Proliferation: - Insulin-like growth factor binding protein 2 (igfb2) -

28 Genes of further interest will be expressed in the same cells as connexin43 Examples from list of genes involved in Cell Proliferation: - Insulin-like growth factor binding protein 2 (igfb2) - Insulin-like growth factor 1 (igf1) - Cyclin-selective ubiquitin carrier protein E2-C - Vascular endothelial growth factor c (vegfc) - Platelet-derived growth factor alpha polypeptide (pdgfa) -dkc1 protein

29 Cx43 function is required to establish the correct number of progenitor cells during fin growth overlying epithelium Cx43 positive, dividing cells growing segment osteoblasts completed segment Expression of connexin43 in dividing cells coordinates expression of other genes required for cell proliferation. In the absence of sufficient cell-cell communication in this population, fewer cells enter the cell cycle. This leads to reduced bone growth. Evaluating the expression and function of genes acting in response to Cx43 function will reveal more precisely how bone length is regulated by cell-cell

30 Acknowledgements Iovine lab - Lehigh University Jake Fugazzotto* Rebecca Jefferis Sarah Nielsen* Isha Jain* Ken Sims* Sarah Gerhardt Andrew Brown Erica Fratz Tom Neill Doug Trembly Technical support: Jutta Marzillier Funding: NIDCR and NICHD

Diseases of the skeleton

Diseases of the skeleton Flexibility Protection of vital organs Strength Skeletal defects impact human health Developmental diseases Degenerative diseases Fins regenerate and grow rapidly following amputation