||School of Medicine, Institute for Biogenesis Research
||PhD (University of Chicago)
||(808) 956 7862 (office)
(808) 3893089 (mobile)
||(808) 956 6856
||1960 East-West Road Rm D104, Honolulu, HI 96822
Tom Humphreys's Curriculum Vitae
Current Research Projects:
Head Regeneration in the Marine Hemichordate, Ptychodera flava
Biological Roles of Stem Cells
Description of research:
Among animals with a dorsal hollow brain, only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordates and chordates, including humans. In spite of extensive sharing of fundamental structures and gene regulatory networks, humans regenerate little, while hemichordates exhibit robust regeneration. The limited regeneration found in higher animals may represent the evolutionary loss of only a few key signaling steps necessary to activate, guide and use the regenerative potential encoded in stem cell reprograming factor genes and in developmental gene regulatory networks of the animal genome. A premise of our work is that understanding these signals and how they work in hemichordate regeneration could provide insights important for developing effective reparative medicine in humans.
Figure 1: The Regenerative Process in Ptychodera flava. Head regeneration in P. flava with day from the time of transection marked on each frame; all views left side except day 11 more ventral. Uncut; intact animal, arrowheads mark site at posterior end of the gill basket where animal will be severed. Scale bar = 2mm all frames. Day 0; gapping anterior wound where body wall has been severed. Day 1; edges of the severed body wall are slightly swollen and smoothened and the wound has begun to close. Day 3; wound has closed and a tiny blastema can be detected slightly dorsal to the closure site. Day 5; blastema growing rapidly. Day 7; blastema shaped into nascent proboscis with collar. Day 10 head is well shaped and still growing rapidly. Day 11, new head, approaching final size to match original body, is attached to original anterior cut stump.
“The Role of Stem Cell Gene Reprogramming Factors in Hemichordate Head Regeneration”
Hemichordate acorn worms quickly regenerate a full-sized, functional head on the anterior wound surface wherever the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. We are examining the regeneration process by whole mount in situ hybridization (WISH) of sequences from stem cell genes reprogramming factors, as defined by genes inducing pluripotency in mammalian somatic cells, and organizer genes. We find a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begin to generate signal as the blastema is established and signal persists in the blastema as “organizer” and early development genes signal appears. These results suggest that activation of cells with regenerative capability involves the stepwise expression of stem cell gene in the cells that form a blastema, the expression of stem cell genes which maintain the blastema where expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. These results show that the stem cell genes currently emphasized in thinking about regenerative medicine play a central role in the dramatic regeneration of hemichordates.
Figure 2: Expression of the Pou domain reprogramming gene during head regeneration. Expression of Pf-Pou3f3/4(Oct4) A. Whole mount In situ hybridization of Pou3f3/4(Oct4). 0d and 1d. No signal is detected in the tissue at the severed site at the time of transection or one day later. 2d. Signal is first detected along the dorsal mid-line just posterior to the site of transection; note that at 2 days the wound edges are swollen (arrow heads) and the wound is still open to the gut (arrow). 3d. The wound has closed and signal along the dorsal midline has extended more anterior into the base of the nascent blastema. 5d, 7d, and 11d. Strong signal at the base of the blastema and forming head continues for the course of regeneration. During this time there is weak, but definite, signal throughout the blastema and forming head.
“The Stem Cell Population of the Hemichordate Adult Body”
In hydra and planarians, regeneration is based on activation of stem cells in the tissue to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells. The role of tissue dedifferentiation and stem cells in hemichordate regeneration is unknown and is an area of active research for the lab. Using DNA labeling and localization of dividing cells we are examining the cells first activated by the wound healing/regeneration signals, their division kinetics, their mobilization to the anterior wound and their pluripotent/multipotent states as they form the blastema and differentiate into the new head.
Figure 3: Dividing cells in the Anterior of a Regenerating Worm. BrdU labeling of nuclei during head regeneration. Green is BrdU labeled nuclei. Blue is general background fluorescence of animal tissues. A. Anterior sagital section of animal at 6 days of head regeneration. B. Enlargement of area from small square in A. A large population of nuclei synthesizing DNA are distributed in the blastema/regenerating head and in the cells of the ectodermal columnar epithelium around the blastema and extending considerably more posterior in the dorsal epithelium. Inset: Magnification of labeled nuclei with most label in the epithelium (arrow) but with a small set of mesenchymal labeled nuclei (arrowhead) below the basement membrane of the epithelium. C. Lateral dorsal quadrant of a body cross section posterior to a 5 day regenerating blastema. Nuclei incorporating BrdU are distributed in the dorsal lateral epithelium from the dorsal midline to the lateral edge. The ventral epithelium (not shown) contains few labeled nuclei. Label in the gut wall is the same as in non-regenerating animals. p, proboscis, dc, dorsal collar; vc, ventral collar, e, epithelium (ectodermal); bw, body wall (mesodermal); bc, body cavity; g, gut cavity.
- Novel pattern of Brachyury gene expression in hemichordate embryos. Kunifumi Tagawa, Tom Humphreys, Noriyuki Satoh. Mech. Develop. 75: 139-143 (1998)
- Characterization of a hemichordate fork head/HNF-3 gene expression. Taguchi S, Tagawa K, Humphreys T, Nishino A, Satoh N, Harada Y. Dev Genes Evol. 210(1):11-7 (2000)
- Molecular characterization of TgHBox4, a Drosophila Abd-B homolog found in the sea urchin Tripneustes gratilla. Vansant G, Humphreys T. DNA Cell Biol. 19(2):131-9 (2000)
- Characterization of gill-specific genes of the acorn worm Ptychodera flava. Okai N, Tagawa K, Humphreys T, Satoh N, Ogasawara M. Dev Dyn. 217(3):309-19 (2000)
- Developmental expression of the hemichordate otx ortholog. Harada Y, Okai N, Taguchi S, Tagawa K, Humphreys T, Satoh N. Mech Dev. 91(1-2):337-9 (2000)
- T-brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain. Tagawa K, Humphreys T, Satoh N. J Exp Zool. 288(1):23-31 (2000)
- Embryonic expression of a hemichordate distal-less gene. Harada, Y., Okai, N., Taguchi, S., Shoguchi, E., Tagawa, K., Humphreys, T., Satoh, N. Zoological Science 18 (1): 57-61 (2001)
- Molecular studies of hemichordate development: a key to understanding the evolution of bilateral animals and chordates. Tagawa K, Satoh N, Humphreys T. Evol. Dev. Nov-Dec;3(6):443-54 (2001)
- Group B sox genes that contribute to specification of the vertebrate brain are expressed in the apical organ and ciliary bands of hemichordate larvae. Taguchi, S., Tagawa, K., Humphreys, T., Satoh, N. Zoological Science 19(1): 57-66 (2002)
- A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny. Hiroki Oda, Hiroshi Wada, Kunifumi Tagawa, Yasuko Akiyama-Oda, Nori Satoh, Tom Humphreys, Shicui Zhang, and Shoichiro Tsukita. Evol Devlop. 4:426-34 (2002)
- Conserved expression pattern of BMP-2/4 in hemichordate acorn worm and echinoderm sea cucumber embryos. Harada Y, Shoguchi E, Taguchi S, Okai N, Humphreys T, Tagawa K, Satoh N. Zoolog Sci. 19:1113-21 (2002)
- Development and neural organization of the tornaria larva of the Hawaiian hemichordate, Ptychodera flava. Nakajima Y, Humphreys T, Kaneko H, Tagawa T. Zool. Sci. 21:69-78 (2004)
- Hemichordate embryos: procurement, culture, and basic methods. Lowe CJ, Tagawa K, Humphreys T, Kirschner M, Gerhart J. Methods Cell Biol. 74:171-94 (2004)
- An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model. Putnak RJ, Coller BA, Voss G, Vaughn DW, Clements D, Peters I, Bignami G, Houng HS, Chen RC, Barvir DA, Seriwatana J, Cayphas S, Garcon N, Gheysen D, Kanesa-Thasan N, McDonell M, Humphreys T, Eckels KH, Prieels JP, Innis BL. Vaccine. 23:4442-52 (2005)
- Cationic Polyamines Inhibit Anthrax Lethal Factor Protease. Mark Evan Goldman, Lynn Cregar, Glen Prosise, Dominique Nguyen, Jason Larson, Gary Thomas, Tom Humphreys. BMC Pharmacology. 6:8-11 (2006)
- Preparation and Immunogenic Properties of a Recombinant Subunit Vaccine for West Nile Virus. Michael M. Lieberman, David E. Clements, Steven Ogata, Gordon Wang, Gloria Corpuz, Teri Wong, Lynne Gilson, Watts DM, Tesh RB, Siirin M, Travassos da Rosa A, Humphreys T, Weeks-Levy C. Vaccine. 25:414-23 (2006)Development of a Dengue Virus Vaccine Based on a Recombinant Envelope Protein Subunit: Evaluation of Protection in Mice and Monkeys Immunized with a DEN-2 80%E Subunit. Beth-Ann G. Coller, David E. Clements, Gary S. Bignami, Iain D. Peters, J. Robert Putnak, Michael McDonell, and Tom Humphreys. Vaccine. 25:414-23 (2006)
- Development of a Recombinant Tetravalent Dengue Virus Vaccine: Immunogenicity and Efficacy Studies in Mice and Monkeys. David E. Clements, Beth-Ann G. Coller, ,Michael M. Lieberman, Steven Ogata, Gordon Wang, Kent E. Harada, J. Robert Putnak, John M. Ivy, Michael McDonell, Gary S. Bignami, Iain D. Peters, Julia Leung, Carolyn Weeks-Levy, Eileen T. Nakano and Tom Humphreys. Vaccine. 28:2705-15 (2010)
- Regeneration in the Hemichordate Ptychodera flava. Tom Humphreys, Akane Sasaki, Gene Uenishi, Kekoa Tamparra, Asuka Arimoto and Kuni Tagawa. Zool. Sci. 27:91-5 (2010)