"An orderly retreat: Dedifferentiation is a regulated process".

Mariko Katoh * ¹ , Chad Shaw *, Qikai Xu * ² , Nancy Van Driessche * ³ ,Takahiro Morio ¹  , Hidekazu Kuwayama ¹ , Shinji Obara ¹ , Hideko Urushihara ¹ , Yoshimasa Tanaka  ¹ ¶, and Gad Shaulsky * ^{2, 3} ||

*Department of Molecular and Human Genetics, ² Graduate Program in Structural and Computational Biology and Molecular Biophysics, and ³ Graduate Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030; and
¹ Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

¶To whom correspondence regarding the Japanese cDNA project should be addressed.
E-mail:   ytanaka@biol.tsukuba.ac.jp

||To whom correspondence should be addressed at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
E-mail:    gadi@bcm.tmc.edu

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state.

Introduction:

Dedifferentiation is the progression of cells from a more differentiated to a less differentiated state. It is observed in a variety of processes such as cancer, organ regeneration, and stem cell renewal, but it has been difficult to study because there are few experimentally tractable systems that dedifferentiate (1–4). Dedifferentiation in Dictyostelium is an experimentally tractable process. During development, starving cells aggregate and undergo synchronous morphological transitions until they form fruiting bodies after 24 hr (5). If the multicellular structures are disaggregated and incubated in nutrient medium, the cells dedifferentiate. Dedifferentiation is characterized by a loss of developmental markers and a subsequent gain of proliferative capacity (6, 7, 45), but these characteristics do not prove that dedifferentiation is a regulated process. The most convincing evidence in support of regulation has been the observation that a mutant strain (HI4) was defective in dedifferentiation (8, 9).

An argument against the idea that dedifferentiation is a regulated process comes from the observation that dedifferentiation occurs at different rates, depending on the developmental stage at which the cells were disaggregated (6, 10–13). This dependence may indicate that each developmental stage has a dedicated dedifferentiation program that is executed at a different rate, or that dedifferentiation is a stochastic event whereby cells lose developmental markers and regain growth markers.

The purpose of this work was to test whether dedifferentiation is a regulated process by comparing the molecular progression of cells from different developmental stages. We propose that if we found a common set of molecular changes, which is independent of the initial developmental stage, that finding would support the regulated process hypothesis. We used microarray transcriptional profiling to detect changes in the pattern of global gene expression during dedifferentiation. These transcriptional changes reveal physiological changes without prejudice as to what processes are involved (14–18), making them suitable for testing whether dedifferentiation from different developmental stages occurs in a regulated way. We followed the physiological changes that occur in cells during dedifferentiation from three developmental stages: aggregation, finger, and Mexican hat. These stages are quite different from each other in physiology and in morphology (5), yet we found that the dedifferentiating cells exhibited common transcriptional profiles. This finding indicates that
dedifferentiation is a regulated process. Examination of the coordinately regulated transcripts revealed genes that are also induced during development, raising the possibility that development and dedifferentiation are regulated by some common genes. We tested that possibility and found that one of the genes, dhkA, regulates both development and dedifferentiation.
...

Discussion:

Our data support the hypothesis that dedifferentiation is a regulated process because cells induced to dedifferentiate from three developmental stages underwent an identical set of transcriptional changes. In the first hour of dedifferentiation, developmental genes were still abundant in the cells and some dedifferentiation genes began to be expressed. During the second phase of dedifferentiation, the abundance of the developmental transcripts was greatly reduced, indicating a massive down-regulation of gene expression and a degradation of many transcripts. This process was also accompanied by the induction of 381 genes. Annotation of these genes revealed that some are probably related to mRNA metabolism and regulation of gene expression, likely relations in light of the massive changes in transcript abundance during phase II. Finally, in phase III, the cells began to express vegetative genes. The compelling finding is that the sequence of events and the specific regulation of several hundred dedifferentiation genes were independent of the initial developmental stage and the consequential variable rates of dedifferentiation. In addition, all of the measured parameters were coordinately regulated: the microarray profiles, DNA synthesis, cell division, and erasure. It is unlikely that such coordination could result from a stochastic unregulated process.

Annotation of the dedifferentiation genes revealed the possible involvement of several processes, including signal transduction, ubiquitin-mediated proteolysis, and regulation of transcription. The signal transduction genes are from the two-component system family (28, 29), including the histidine kinase gene dhkA, a hybrid histidine kinase essential for proper terminal differentiation (20, 27).

It is tempting to speculate about the function of a gene in dedifferentiation based on its annotation, but a functional test is better. We found that the signal transduction gene dhkA was essential for dedifferentiation, supporting the idea that dedifferentiation is a genetically regulated process because it is mutable. This finding also indicates that signal transduction is involved, and it adds confidence to the interpretation that the other gene groups may regulate dedifferentiation. The promise of manipulating the process genetically is supported by previous studies of a strain (HI4) defective in dedifferentiation (8, 9, 13). Unfortunately, the gene mutated in HI4 could not be cloned at the time and the strain has been lost. The dedifferentiation characteristics of dhkA^- cells also indicate that regulation of dedifferentiation is a stepwise process because dhkA is required only for cell division, not for DNA replication or for erasure.

Finally, our findings suggest that dedifferentiation and development have coevolved while using common genes, because dhkA is a regulator of both processes and several hundred genes are regulated during both processes. We propose that development consists of checkpoints that ensure a return path to the undifferentiated state in case development fails. The checkpoint could condition developmental progression on the accumulation of a protein, such as DhkA, that is essential for dedifferentiation. If development cannot proceed, the cells may attempt to reinitiate development as some strains undergo several cycles of development and dedifferentiation due to mutations in gene of the lagC pathway (30). However, dedifferentiation is not merely a reversal of development because it involves the coordinate regulation of 122 genes in patterns not seen in development.

The selective advantage of dedifferentiation is obvious, but the need for an intricately regulated process is intriguing. In vertebrate wound healing and regeneration, regulation may attenuate dedifferentiation to preserve information about tissue proportioning (31, 32). The lack of regulation may be a causative factor in cancer and other diseases (1–3, 33–41). Dictyostelium is a soil amoeba, and the sheath that surrounds the multicellular organism cannot protect it from all of the mechanical insults that the soil environment may inflict. Therefore, Dictyostelium dedifferentiation probably evolved under the pressure of occasional mechanical disaggregation during development. Regulating dedifferentiation allows the cells to reaggregate rapidly and continue to develop if starvation persists. It also provides an alternative: in the presence of food, cells can return to the more advantageous process of growth. Because Dictyostelium and higher eukaryotes share many
signal transduction mechanisms (42–44), further investigation of dedifferentiation in this model organism may help identify molecules that regulate dedifferentiation in vertebrates.
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1. Herstein PR, and Frenster JH, "Mated Models of Gene Regulation in Eukaryotes", "Embryonic and Fetal Antigens in Cancer", vol. 2, pp. 5-7, (Anderson NG, Coggin JH, eds.), National Technical Information Service, U.S. Dept. Commerce, Springfield, VA., 1972:

2. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming", RNA2004 (In Press).

3. Related Articles for Katoh M, et al (2004),  from PubMed.
 

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