Cellcycleandregeneration

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Cell Cycle Regulation and Regeneration
Article in Current Topics in Microbiology and Immunology · December 2012

DOI: 10.1007/82_2012_294 · Source: PubMed
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Book Title
Series Title 82
Chapter Title Cell Cycle Regulation and Regeneration
Copyright Year 2012
Copyright HolderName Springer-Verlag Berlin Heidelberg
Corresponding Author Family Name Heber-Katz
Particle
Given Name Ellen
Suffix
Division
Organization Wistar Institute
Address 3601 Spruce St., 19104, Philadelphia, PA, USA
Email heberkatz@wistar.org
Author Family Name Zhang

Particle
Given Name Yong
Suffix
Division
Email
Author Family Name Bedelbaeva
Particle
Given Name Khamila
Suffix
Division
Email
Author Family Name Song
Particle
Given Name Fengyu
Suffix
Division Department of Oral Biology, Indiana University School of Dentistry
Organization Indiana University-Purdue University
Address 46202, Indianapolis, IN, USA
Email
Author Family Name Chen
Particle
Given Name Xiaoping
Suffix
Division Department of Biology, School of Science
Address 723 W. Michigan St., 46202, Indianapolis, IN, USA
Email
Author Family Name Stocum
Particle
Given Name David L.
Suffix
Division Department of Biology, School of Science
Address 723 W. Michigan St., 46202, Indianapolis, IN, USA
Email
Abstract Regeneration of ear punch holes in the MRL mouse and amputated limbs of the axolotl show a number of
similarities. A large proportion of the fibroblasts of the uninjured MRL mouse ear are arrested in G2 of the
cell cycle, and enter nerve-dependent mitosis after injury to form a ring-shaped blastema that regenerates
the ear tissue. Multiple cell types contribute to the establishment of the regeneration blastema of the
urodele limb by dedifferentiation, and there is substantial reason to believe that the cells of this early
blastema are also arrested in G2, and enter mitosis under the influence of nerve-dependent factors supplied
by the apical epidermal cap. Molecular analysis reveals other parallels, such as; (1) the upregulation of
Evi5, a centrosomal protein that prevents mitosis by stabilizing Emi1, a protein that inhibits the
degradation of cyclins by the anaphase promoting complex and (2) the expression of sodium channels by
the epidermis. A central feature in the entry into the cell cycle by MRL ear fibroblasts is a natural
downregulation of p21, and knockout of p21 in wild-type mice confers regenerative capacity on non-
regenerating ear tissue. Whether the same is true for entry into the cell cycle in regenerating urodele limbs
is presently unknown.
Layout: T1 Standard SC Series ID: 82 MS No.: HeberKatzHeberKatz
Chapter No.: 294 Date: 28-11-2012 Page: 1/24
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1 Cell Cycle Regulation and Regeneration
2 Ellen Heber-Katz, Yong Zhang, Khamila Bedelbaeva, Fengyu Song,

3 Xiaoping Chen and David L. Stocum
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4 Abstract Regeneration of ear punch holes in the MRL mouse and amputated
5 limbs of the axolotl show a number of similarities. A large proportion of the
6 fibroblasts of the uninjured MRL mouse ear are arrested in G2 of the cell cycle, and
7 enter nerve-dependent mitosis after injury to form a ring-shaped blastema that
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8 regenerates the ear tissue. Multiple cell types contribute to the establishment of the
9 regeneration blastema of the urodele limb by dedifferentiation, and there is sub-
10 stantial reason to believe that the cells of this early blastema are also arrested in
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11 G2, and enter mitosis under the influence of nerve-dependent factors supplied by
12 the apical epidermal cap. Molecular analysis reveals other parallels, such as; (1)
13 the upregulation of Evi5, a centrosomal protein that prevents mitosis by stabilizing
14 Emi1, a protein that inhibits the degradation of cyclins by the anaphase promoting
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15 complex and (2) the expression of sodium channels by the epidermis. A central
16 feature in the entry into the cell cycle by MRL ear fibroblasts is a natural
17 downregulation of p21, and knockout of p21 in wild-type mice confers regener-
18 ative capacity on non-regenerating ear tissue. Whether the same is true for entry
19 into the cell cycle in regenerating urodele limbs is presently unknown.
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E. Heber-Katz (&) Y. Zhang K. Bedelbaeva

Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA
e-mail: heberkatz@wistar.org
F. Song
Department of Oral Biology, Indiana University School of Dentistry,
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Indiana University-Purdue University, Indianapolis, IN 46202, USA

X. Chen D. L. Stocum
Department of Biology, School of Science, Indiana University-Purdue University,
723 W. Michigan St., Indianapolis, IN 46202, USA
Current Topics in Microbiology and Immunology

DOI: 10.1007/82_2012_294
Ó Springer-Verlag Berlin Heidelberg 2012
E. Heber-Katz et al.
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20 Abbreviations:
21 AEC Apical epidermal cap
22 AGP Anterior gradient protein
23 APC Anaphase promoting complex
24 CDK Cyclin-dependent kinase
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25 EGF Epidermal growth factor
26 FGF Fibroblast growth factor
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27 G Gap
28 GGF Glial growth factor
29 HGF Hepatocyte growth factor
30 IGF Insulin-like growth factor
31 M Mitosis
32 PDGF Platelet-derived growth factor
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33 RP Restriction point
34 S DNA synthesis
35 TGF Transforming growth factor
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Contents
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37
38 1 Introduction: The Cell Cycle ..................................................................................................

39 2 Regeneration of Mouse Ear Tissue ........................................................................................
40 2.1 Events of Regeneration ..................................................................................................
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41 2.2 G2/M Arrest and DNA Damage.....................................................................................

42 2.3 G2/M Arrest and Cytokinesis .........................................................................................
43 3 Regeneration of the Amphibian Limb....................................................................................
44 3.1 Blastema Formation and Growth ...................................................................................
45 3.2 Regulation of Go/G1 and Progression Through G1 .......................................................
46 3.3 Regulation of G2/M ........................................................................................................
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47 3.4 Role of Nerves and Wound Epidermis in Blastema Cell Cycling ...............................
48 4 Similarities, Differences, and Unanswered Questions Between the Urodele Limb
49 and MRL/MpJ Mouse Ear Regeneration................................................................................
50 5 Summary ..................................................................................................................................
51 References......................................................................................................................................
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52
53 1 Introduction: The Cell Cycle

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54 Cell proliferation is an absolute requirement for tissue regeneration, and regulation

55 of cell cycle entry and progression are obviously important for this process. Most
56 studies on cell proliferation in regeneration involve either compensatory hyper-
57 plasia (as in liver regeneration) or stem cell/progenitor proliferation of epithelia.

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Fig. 1 The phases of the cell cycle and their regulation by cyclin:CDK complexes. G1 gap 1,
S DNA synthesis, G2 gap 2, M mitosis, CDK cyclin-dependent kinase, Emi1 early mitotic
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inhibitor, Evi5 ecotropic viral integration site 5, PLK1 Polo-like kinase 1, APC anaphase
promoting complex. The blue arrows indicate degraded cyclins. The green arrow indicates
stabilization of Emi1 by Evi5. Dashed red arrows/bars indicate inhibitory interactions
58 More recently, research has been initiated on cell cycle regulation as part of the
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59 regenerative mechanism of complex multi-tissue structures (Arthur et al. 2010).

60 The MRL mouse ear and the urodele limb are two complex structures that share
61 some common features of regeneration after tissue loss. This chapter presents data
62 and ideas that can further our understanding of how cell cycle regulation might
63 explain some aspects of the regenerative ability displayed in these two systems.
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64 The cell cycle has four phases (Fig. 1) that take place sequentially when the cell
65 is activated from its quiescent state, termed Go. These phases are G1 during which
66 the cell is rendered competent to synthesize DNA; S-phase, where the DNA is
67 replicated; G2 during which the cell is certified ready to divide; and M, during
68 which the chromosomes condense and the successive steps of mitosis take place
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69 (prophase, metaphase, anaphase, and telophase) to produce two daughter cells

70 (Lodish et al. 2008).
71 Checkpoints controlled by regulatory proteins inhibit entry into S and M until
72 molecular sensing systems determine that all requirements for DNA synthesis and
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73 mitosis are satisfied. Two major mechanisms are used to regulate the cell cycle:
74 protein phosphorylation and ubiquitin-mediated degradation (Lodish et al. 2008).
75 The regulatory proteins belong to three classes of molecules: the cyclins, the
76 cyclin-dependent kinases (CDKs), and the CDK inhibitors. Cyclins are regulatory
77 units and CDKs are catalytic units that must interact with one another for each to
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78 function. The cyclin:CDK complexes that drive each phase of the cycle are cyclin
79 D: CDKs 4 and 6 in mid G1, cyclin E:CDK2 in late G1, cyclin A:CDK2 in S-phase,
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80 Cyclin A:CDK1 in G2, and cyclin B:CDK1 in M-phase. To exit S or M, cyclins
81 must be degraded and this occurs through the action of the anaphase promoting
82 complex (APC).
83 CDK inhibitors act primarily in G1 and belong to two families, the cip/kip
84 (CDK interacting protein/Kinase inhibitory protein) family, and the INK4a/ARF
85 (Inhibitor of Kinase4/Alternative Reading Frame) family. The cip/kip family
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86 includes the genes for p21, p27, and p57. The INK4a/ARF family includes
87 p16INK4a, which binds to CDK4, and p19ARF, which prevents p53 degradation
88 and thus maintains p21.
89 The competence of quiescent G0 cells to enter G1 requires signaling by growth
90 factors (Morgan and Pledger 1992). These growth factors activate preexisting
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91 transcription factors that induce the transcription of early response genes such as
92 c-Fos and c-Jun, which in turn induce the transcription of delayed response genes
93 encoding the mid and late G1 and S-phase cyclin-CDKs, as well as the E2F
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94 transcription factor that activates the genes involved in DNA synthesis (Lodish
95 et al. 2008). The CDKs are inhibited for most of G1 by the CDK inhibitors p21,
96 p27, and p57, and E2F acts as a transcriptional repressor because it is bound to the
97 hypo-phosphorylated retinoblastoma (Rb) protein. At a point in late G1 called the
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98 restriction point (RP), the concentration of cyclin:CDKs reaches a level where the
99 CDK inhibitors are saturated. As the concentration of cyclin:CDK rises further, it
100 inactivates Rb by hyper-phosphorylation. This frees E2F to now transcriptionally
101 activate the expression of over 800 genes involved in DNA synthesis. E2F also
102 induces the transcription of cyclin E, which interacts with CDK2 to bring about the
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103 G1-S transition, and induces the transcription of S-phase cyclin. Before the cells
104 get to S-phase, DNA damage, oxidative stress, and other DNA damaging events
105 can activate the p53 protein. The target gene of p53 is p21Cip1/Waf1 (el-Deiry et al.
106 1993; Xiong et al. 1993) and the p21Cip1/Waf1 protein will bind to the cyclin/CDK2
107 complex to block phosphorylation of Rb and thus prevent S-phase transit until
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108 DNA repair occurs.

109 During DNA synthesis, S-phase cyclin A:CDK complexes phosphorylate and
110 activate proteins of pre-replication complexes assembled during G1, and various
111 enzymes collaborate to replicate DNA strands. Upon completion of DNA synthesis,
112 the cell is temporarily arrested in G2, which serves as a second checkpoint to detect
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113 any DNA damage that needs to be repaired before transiting to M, or if repair
114 cannot be accomplished, to activate apoptotic pathways. The Emi1 (early mitotic
115 inhibitor 1) and Evi5 (ecotropic viral integration factor 5) proteins prevent a pre-
116 mature entry into M. Expression of these proteins is stimulated by E2F at the G1/S
117 transition, after which they accumulate and localize to the centrosome. Emi1 is an

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cytoplasm Evi5
Rab11
Yap/
TAZ
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transporting
p53BP2/ASPP
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nucleus
Yap/ P
TAZ Differentiation
signal
time Dedifferentiation
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signal
P21 Expression time
Cell cycle regulation
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Fig. 2 Potential mechanism of regulation by Evi5 of the Hippo pathway, which plays a role in
the cell cycle, cell differentiation and cell dedifferentiation. p53BP2 tumor suppressor p53-
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binding protein 2, also known as Apoptosis-stimulator of p53 protein 2 (ASPP2), TAZ
transcriptional coactivator with PDZ-binding motif, and Yap Yes-associated protein. Red dashed
lines indicate negative regulation; green solid lines indicate positive regulation; blue dashed lines
indicate effects of p21 and Evi5 on cell cycle regulation
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118 F-box protein that interacts with Cdc20 to inhibit the E3 ligase APC and prevent the
119 ubiquitination and degradation of the APC substrates, cyclins A and B, thus
120 maintaining the cell in G2 (Eldridge et al. 2006). Cells lacking Emi1 suffer DNA
121 damage induced by replication stress and undergo apoptosis (Verschuren et al.
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122 2007). Interphase Evi5 is a 110 kDa protein that stabilizes Emi1 and cyclin A. A
123 second protein, Pin1, has also been shown to stabilize Emi1 (Bernis et al. 2007). AQ2
124 Evi5 also serves as a GAP (GTPase Activating Protein) to inactivate the small
125 GTPase vesicle trafficking protein Rab11 (Dabbeekeh et al. 2007; Westlake et al.
126 2007). Vesicle trafficking regulates the Hippo pathway by controlling translocation
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127 of the Yes-Associated Protein (YAP) from the cytosol to the nucleus (Fig. 2),
128 where YAP can act as a co-activator of Smad-7 to activate genes involved in the
129 cell cycle such as E2F and survivin (Zhao et al. 2011). In addition, dephospho-
130 rylated YAP has been shown to locate in the cytosol and to be upregulated in
131 pluripotent stem cells, whereas inactivated (phosphorylated) YAP is detected in
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132 the nucleus and is associated with cell differentiation (Lian et al. 2010). Thus Evi5
133 might help restrain cells from entering mitosis by inhibiting Rab 11, which would
134 prevent phosphorylated YAP from localizing to the nucleus and acting as a mitotic
135 and differentiation signal, thereby promoting and maintaining a dedifferentiated
136 state.
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137 Evi5 survives during mitosis and is redistributed from the centrosome as a
138 90 kDa protein and a 20 kDa protein that are formed by cleavage of the 20 kDa
139 form off the far end of the C-terminal (Faitar et al. 2006). The 90 kDa protein is
140 distributed throughout the cell during prophase, but co-localizes at metaphase with
141 a-tubulin of the mitotic spindle. During anaphase 90 kDa Evi5 localizes at the mid
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142 zone, where it associates with the chromosomal passenger complex (CPC) con-
143 sisting of aurora B kinase, INCENP, survivin, and borealin (Ruchaud et al. 2007).
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144 During late telophase and cytokinesis, Evi5 dissociates from the CPC and forms
145 two parallel disk-like structures in the region where new membrane formation will
146 separate the daughter cells. Evi5 is required for this separation, as shown by the
147 fact that its knockdown prevents cytokinesis (Faitar et al. 2006). There is evidence
148 that Evi5 regulates the formation of new cell membrane formation at cytokinesis
149 by a mechanism other than GTPase activity (Westlake et al. 2007).
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150 Once sensing systems have established that all the requirements for mitosis
151 have been met, including repair of any DNA damage, Emi1 and Evi5 are phos-
152 phorylated by Polo-like kinase 1 (PLK1) and Emi1 is degraded, allowing the
153 degradation of cyclins A and B by the APC and progression through prophase,
154 metaphase, and anaphase (Eldridge et al. 2006). Interestingly, a recent study
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155 indicated that p21 downregulates Emi1, activating the APC in cells arrested in G2
156 by DNA damage. However, this resulted in stable G2 arrest, rather than progres-
157 sion into mitosis, indicating that Emi1 plays a role in a novel p21-dependent
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158 mechanism to maintain G2 arrest after DNA damage (Lee et al. 2009).
2 Regeneration of Mouse Ear Tissue

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159
160 2.1 Events of Regeneration
In 1998, the MRL/lpr and MRL/Mpj mice were shown to have the unique char-
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161
162 acteristic of closing ear hole punches (Clark et al. 1998) (Fig. 3). Further analysis
163 showed that this occurred without the formation of a scar, with normal tissue
164 architecture, and with the replacement of elastic cartilage. Many of the features
165 described for limb regeneration in amphibians also appeared in this system. Since
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166 then many laboratories have confirmed and extended the finding that healing in the
167 MRL mouse is unique and regenerative (e.g. Balu et al. 2009; Buhimschi et al.
168 2010; Rai et al. 2012; Thuret et al. 2009).
169 One of the early events in amphibian regeneration is the epidermal response. In
170 the amphibian, rapid coverage of the wound occurs within the first 12–24 h. The
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171 epidermis migrates rapidly to re-cover the edges of punch wounds In the MRL
172 mouse and thickens within 3–5 days to form a cap of epidermis resembling the
173 apical epidermal cap (AEC) that forms on the amputated amphibian limb (see
174 ahead). This cap forms a ring around the edge of the punch hole. By contrast,
175 5–10 days are required to re-cover the wound in the non-regenerating B6 mouse

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Fig. 3 Punch injuries in the ear pinna lead to different outcomes in MRL versus C57BL/6 ears.
a Macroscopic visualization of punch holes in C57BL/6 (left panels) and MRL (right panels) at
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1 day post-injury (upper panels) and 1 month post-injury (lower panels). The hole has been
completely closed by regeneration in the MRL by 30 days. b Histology of regeneration in MRL
ear tissue. At day 1, inflammatory cells can be seen lining up at the edge of the ear punch (arrow).
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By day 2, re-epithelialization is complete. By day 5 hair follicles are forming in the new
epidermis (upper arrow) and cells are accumulating beneath the epidermis in a blastema-like
structure (lower arrow). By day 10, the blastema (arrow) has expanded significantly
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176 and a structure like the AEC does not form. A mesenchymal ring blastema forms
177 under the thickened MRL wound epidermis and then undergoes a rapid growth
178 phase via cell proliferation. In the amphibian, no basement membrane re-forms to
179 prevent communication between epithelial and mesenchymal cell populations. In
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180 the MRL mouse a basement membrane is seen by day 3 and is eliminated by day 5,
181 but is maintained in non-regenerative mice. Cells then accumulate under the
182 ‘‘AEC’’ and proliferate centripetally until the hole is closed (Gourevitch et al.
183 2003). In vitro, cells derived from the ears of MRL mice were found to be highly
184 proliferative compared to cells from the ears of B6 mice. These cells are mes-
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185 enchymal-like fibroblasts that potentially contribute to the blastema population.

186 To determine the genes involved in the MRL regenerative response, extensive
187 mapping studies of genes expressed early after wounding have been performed.
188 The MRL was bred to different non-regenerating mice. MRL is the result of
189 crossbreeding 4 inbred strains, LG/J (75 %), C3H (12 %), AKR-(12 %), and
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190 C57BL/6 (0.3 %). The LG/J mouse was shown to contribute the healing phenotype
191 and genetic studies were carried out using this strain as well to compare it to the
192 non-regenerator strain SM/J. In these studies, recombinant RI strains and advanced
193 intercross lines were used for fine mapping and intervals were significantly
194 (20–40-fold) narrowed (Cheverud et al. 2012).
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195 2.2 G2/M Arrest and DNA Damage
196 Cell cycle analysis was carried out on multiple sources of regenerator cell
197 populations compared to multiple sources of non-regenerator cell populations.
198 Thus, we compared regenerator MRL, LG/J, RI (LxS) line 6, and a B6 healer
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199 congenic to non-regenerative B6, SM/J, and RI (LxS) line 33. In all cases using
200 regenerative cells, G2/M arrest was seen in a mean of 45 % of the population as
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201 compared to 12 % in the non-regenerative lines. These data suggested a DNA
202 damage response in the regenerators.
203 An early marker of DNA damage is the recruitment and the formation of H2AX
204 foci in the nuclei, which in the regenerator cells was significantly increased with
205 more nuclear foci and more foci/cell. Also p53, the major DNA damage sensor,
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206 showed clear upregulation in regenerator cells. These results were found not just in
207 cells in culture but in regenerating tissue in vivo. To demonstrate actual DNA
208 damage, comet assays were carried out which demonstrated shortened DNA
209 fragments in an electric field. Such analysis showed that 80 ? % of MRL
210 cells were comet-positive compared with only 6 % of non-regenerator cells
211 (Bedelbaeva et al. 2010).
212
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A major target of p53 activation is p21Cip1/waf1 (Xiong et al. 1993; el-Deiry
213 et al. 1993), which is responsible for G1 arrest, and a first step to senescence. P21 is
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214 activated upon DNA damage, but examination of the regenerator cell lines showed
215 that p21 was, contrary to expectation, reduced or not expressed as determined by
216 Western analysis. This finding supported the idea that lack of p21 led not to G1
217 arrest but rather G2 arrest. Further support for this idea came from the finding that
218 the p21-/- B6 mouse is a complete regenerator similar to the MRL mouse. Thus
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219 elimination of p21 conferred the ability for ear hole regeneration on a normally
220 non-regenerating mouse (Bedelbaeva et al. 2010). The p21 protein binds to the
221 cyclin E:CDK2 complex as well as other cyclin:CDK complexes, preventing the
222 phosphorylation of Rb, thus blocking E2F family members from activating genes
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223 involved in DNA synthesis. Without p21, Rb is phosphorylated, E2F is released

224 and activates these genes.
225 Why p53 is not inducing the expression of p21 in the MRL mouse remains to be
226 determined, but brings into question the role of p53 in regeneration. Previous
227 studies suggested that p53 was required for hair follicle regenerative growth
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228 (Ruzankina et al. 2009). In addition, limb regeneration is blocked in the axolotl by
229 administration of a p53 inhibitor, which inhibited MDM2 (a negative regulator of
230 p53) and GADD45 (a stress response gene upregulated by p53) (Villiard et al.
231 2007; see ahead). To determine the effect of a p53 deletion in mice, p53 knockout
232 mice were bred to either B6 or B6/129 non-regenerator mice and to MRL mice. In
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233 the first case, p53 deletion in a non-regenerator strain in a heterozygous or

234 homozygous state had no effect; i.e., the tissue did not regenerate after ear punch
235 hole wounding. When backcrossed to an MRL, the p53 deletion had no effect on
236 regeneration in MRL males, but did have a positive effect on regeneration in MRL

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237 females. Thus, p53 does not appear to be necessary for MRL regeneration and its
238 deletion may have a positive effect, at least in females (Arthur et al. 2010).
239 To determine the expression of other genes in the p21Cip1/waf1 pathway, we
240 examined other knockout mice and found that a TGFb deletion, on a RAG-/-
241 background to reduce the level of inflammation and promote neonatal survival,
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242 showed partial ear hole closure. TGFb is a potent stimulator of p21 and its
243 elimination should reduce p21 activity and lead to regenerative healing. A recent
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244 study identifying an ENU mutant that could close its ear holes proved to be a
245 mutation in TGFbR1 (Liu et al. 2011). Whether this mouse can activate p21
246 through SMAD activation is unclear. A SMAD3 deletion mutant that could not
247 close its ear holes (Ashcroft et al. 1999) but had inflammatory problems similar to
248 those of the TGFb mutant, could potentially mask a regenerative response. A study
249 using a SMAD7-overexpressing mouse that inhibited SMAD3, enhanced liver
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250 regeneration, suggesting that elimination of TGFb signaling and thus p21
251 expression enhances regeneration (Zhong et al. 2010).
252 Preliminary evidence (Bedelbaeva, MS in preparation) indicates that Evi-5 is
253 upregulated in the MRL as assessed by immunohistochemistry in both normal ear
254 tissue (found in hair follicle stem cells, endothelial cells, and muscle cells) and in
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255 injured ear tissue (in blastema cells directly under the AEC and potentially in
256 epidermis as well) (Fig. 4).
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257 2.3 G2/M Arrest and Cytokinesis

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258 Two genes are candidates for implication in G2/M arrest. Exonuclease 1 (exo1) on
259 chr 1 was found to be mutated in the MRL mouse and this mutation was found to
260 be carried in the congenic regenerator. This molecule interacts with MSH2,
261 removes DNA mismatches during homologous recombination and thus is involved
262 with mismatch repair. In the MRL mouse, mismatch repair leads to metaphase
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263 arrest and thus may be involved in the regenerative process (Namiki et al. 2003).
264 A second candidate gene identified on chr 9, Kif23 or MKLP1, a microtubule
265 motor protein, is recruited to the spindle mid-body by INCENP, is phosphorylated
266 by Aurora B kinase and is necessary for cytokinesis (Zhu et al. 2005; Guse et al.
267 2005). This protein may play an important role in the regenerative response. KIF23
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268 expression is upregulated in the MRL and LG mouse and has several coding
269 sequence changes that may lead to a gain or loss of function and to enhanced or
270 reduced cytokinesis (Cheverud et al. 2012). Such a result could lead to either a
271 rapid 4–2 N shift and increase in cell number during regeneration, or it could be
272 responsible for the G2/M arrest that is seen. Hepatocytes of regenerating adult liver
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273 show a high percentage (approximately 70 %) of either mononuclear or binuclear

274 polyploid cells (Michalopoulos and DeFrances 1997; Guidotti et al. 2003).
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MRL 20x MRL 40x
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C57BL/6 20x C57BL/6 40x

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Fig. 4 Evi5 expression in the mouse ear post-injury. Antibody to Evi5 (red) and DAPI (DNA,
blue) was used to stain ear tissue from MRL and C57BL/6 ear tissue 5 days after injury. Evi5
(circumscribed by yellow ovals) was found directly under the epidermal cap in the MRL and may
also include cells in the epidermis. There were a few positive cells in the C57BL/6 ear tissue
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distal to the injury
275 3 Regeneration of the Amphibian Limb

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276 3.1 Blastema Formation and Growth
277 The urodele (salamanders and newts) limb regenerates after amputation at any
278 level. Amputation induces the formation of a mound-shaped blastema (Fig. 5)
279 consisting of undifferentiated progenitor cells derived by the histolytic liberation
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280 and dedifferentiation of dermal and other fibroblasts, Schwann cells, cartilage, and
281 mononucleate myofiber fragments and satellite cells. Within 3–4 days after
282 amputation, the wound epidermis thickens apically to form an AEC several layers
283 thick that has both protective and signaling functions (Christensen and Tassava
284 2000). Nerve axons and blood vessels regenerate into the blastema and the AEC as

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Fig. 5 Blastema formation in the axolotl limb after amputation through the distal radius (R) and
ulna (U). Left panel accumulation blastema stage at 4 days post-amputation showing the
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aggregation of intensely stained dedifferentiated cells (blue arrow) under the wound epidermis.
Right panel three days later, the blastema is a cone-shaped structure as the result of continued
dedifferentiation and a significant increase in mitosis. The AEC is indicated by the red arrow
285 they form. The blastema then enters a growth phase and undergoes patterned
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286 differentiation that replicates the amputated limb parts. Genetic marking experi-
287 ments have shown that the blastema cells redifferentiate primarily into their
288 parental phenotype of derivation (i.e., muscle into muscle, cartilage into cartilage,
289 epidermis into epidermis), although blastema cells derived from dermal fibroblasts
290 exhibit transdifferentiation into cartilage and tendons, but never into muscle (Kragl
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291 et al. 2009).

292 During blastema formation, a substantial percentage of blastema cells synthe-
293 size DNA. Pulse labeling with 3H-thymidine indicated that *30 % of blastema
294 cells are labeled by the accumulation blastema stage (Tassava and McCullough
295 1978), but continuous labeling experiments indicated that as many as 80 % of
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296 blastema cells have entered the cell cycle by that stage (Barger and Tassava 1985;
297 Tassava et al. 1987). The cells do not all cycle synchronously, because there is a
298 transiently quiescent population that appears to feed cells into the actively cycling
299 population. Despite the high labeling index, the cells exhibit a very low mitotic
300 index of *0.4 %, as assessed by counting mitotic figures in sections. The cell
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301 cycle is *40–45 h in length, with mitosis taking only *1 h, and varies little
302 throughout regeneration (Tassava and McCullough 1978; Tassava and McCul-
303 lough 1978). The low mitotic index cannot be ascribed just to the long period
304 spanning G1–G2 (39–44 h, most of which is taken up by S), or asynchronous
305 cycling, since entry into S begins within 2 days after amputation and it takes 3–5
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306 more days (depending on limb size) to form the blastema, by which time the
307 number of cells entering mitosis would statistically be expected to be much higher
308 than indicated by mitotic counts. The fact that blastema cells have a high level of
309 DNA replication and very low level of mitosis during blastema formation suggests
310 that most of them involved in forming the accumulation blastema arrest in G2 after
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311 completing DNA replication where they stay until the growth phase begins
312 (Mescher and Tassava, 1976; Tassava and Mescher 1975).
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313 Formation and growth of the blastema requires signals from the AEC that are
314 nerve-dependent, as shown by the fact that either denervation of the limb or
315 preventing the wound epidermis from contacting underlying tissues inhibits both
316 blastema formation and the mitosis required for growth (Mescher and Tassava
317 1976; Tassava and Garling 1979). Interestingly, the dedifferentiating cells of
318 amputated and denervated or AEC-deprived limbs synthesize DNA at control rates
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319 and exhibit zero mitosis during blastema formation (Tassava and McCullough
320 1978), and there is no difference in the array of proliferation genes upregulated
321 during formation of the accumulation blastema in control and denervated axolotl
322 limbs (Monaghan et al. 2009). The blastema cells of denervated limbs appear to
323 exit the cell cycle and to either disappear (apoptosis?) or form scar tissue.
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324 Denervation performed after the blastemal growth phase has begun results in
325 failure of AEC function and both DNA synthesis and mitotic index fall to zero. In
326 this case, however, scar tissue does not form and the blastema cells constitute a
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327 mass sufficient to organize into a miniature regenerate (Singer and Craven 1948;
328 Powell 1969; Tassava et al. 1987).
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329 3.2 Regulation of Go/G1 and Progression Through G1
330 The fact that limb cells enter the cell cycle and synthesize DNA during blastema
331 formation, along with the low mitotic index, suggests that growth factor signaling
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332 stimulates entry into and progression through G1. The growth factors would pre-
333 sumably be the same ones required to exit G0 by mammalian cells, such as PDGF,
334 FGF-2, EGF, and IGF-1 (Morgan and Pledger 1992). Intraperitoneal injection of
335 IGF-1 has been shown to shorten the time required for blastema formation in newts
336 (Fahmy and Sicard 1998), and insulin was shown to be crucial to 3H-thymidine
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337 incorporation by cultured newt blastemas (Vethamany-Globus et al. 1978; Kesik

338 et al. 1986). These signaling molecules may act in combination to stimulate the
339 G0/G1 transition. They are probably derived from a combination of sources:
340 platelets, the degradation of ECM, which sequesters growth factors or the inactive
341 forms of growth factors during tissue formation, the wound epidermis and nerves,
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342 and the systemic circulation.

343 Newt and mammalian myotube nuclei differ in their ability to re-enter the cell
344 cycle in response to serum growth factors. In both mammalian and newt myo-
345 blasts, pRb is hyperphosphorylated and inactive, allowing them to proliferate in
346 response to serum. As myoblasts fuse and differentiate into myotubes, pRb

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347 becomes hypophosphorylated and actively suppresses myonuclear cell cycle

348 activity. Myonuclei of normal mammalian myotubes will not re-enter the cell
349 cycle in response to serum or growth factor stimulation, suggesting that pRb
350 inactivation is not sufficient to reverse cell cycle suppression. In fact, the ARF
351 gene of the mammalian p16Ink4a locus, which is not expressed in urodeles, must
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352 also be inactivated or deleted in mammalian muscle cells to obtain cell cycle re-
353 entry in response to serum (Pajcini et al. 2010).
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354 By contrast, cell cycle re-entry in cultured newt myoblasts and myotubes is
355 promoted by thrombin, which appears to activate a factor present in serum that
356 leads to hyperphosphorylation and inactivation of the Rb protein. Inactivation of
357 Rb is sufficient to allow passage through the RP and to initiate DNA synthesis
358 (Tanaka et al. 1997; Straube and Tanaka 2006). Consistent with the low mitotic
359 index of the forming blastema, the thrombin-activated factor is not sufficient to
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360 drive the myonuclei into mitosis, and they arrest in G2. Cell cycle re-entry is
361 independent of myotube cellularization, since cell cycle-inhibited myotubes
362 implanted into newt limb blastemas cellularize (Velloso et al. 2000). The mech-
363 anism of myotube or myofiber fragmentation into single cells is not known, nor is
364 it known whether the thrombin-activated factor is also necessary to drive other cell
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365 types as well, or whether this is a feature unique to myofibers. Thus it would
366 appear that muscle cells must be in a mononucleate state to respond to growth
367 factor stimulation, which is consistent with cellularization that takes place during
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368 the histolysis of limb regeneration.
369 The serum factor that promotes phosphorylation of Rb in urodele myonuclei
370 has not been identified, but if limb cells use a mechanism similar to that of
371 mammalian cells to inactivate Rb, one would expect that cyclin: CDKs would
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372 increase during G1 to negate a CDK inhibitor such as p21, resulting in Rb phos-
373 phorylation. The Cip/Kip family in most vertebrates consists of p21Cip1/Waf1,
374 p27Kip1, and p57. In Xenopus, three CDK inhibitors of the Cip/Kip family have
375 been identified, p28Xic1, p16Xic2, and p17Xic3 (Daniels et al. 2004). The latter two
376 are homologs of mammalian p21Cip1 and p27Kip1, respectively. The p27Kip1 pro-
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377 tein has been identified in the axolotl (Habermann et al. 2004), so it is likely that
378 one or more Cip/Kip proteins is involved in regulating progress through G1. One of
379 these may be p16, because injection of plasmid carrying the human p16Ink4a gene
380 microinjected into nuclei of newt myotubes inhibited uptake of BrdU after serum
381 stimulation (Tanaka et al. 1997). We have detected one CDK inhibitory protein in
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382 our proteomic analysis of blastema formation in axolotl limbs (Rao et al. 2009),
383 but it remains unidentified because of poor statistical rank. No protein comparable
384 to p21 has been identified in the axolotl. We have tried to clone axolotl p21
385 without success using primers from human p21; however, there is a 1713 bp Cip/
386 Kip contig (ID C0207013) on the Sal-Site database (Ambystoma Genetic Stock
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387 Center, University of Kentucky; www.ambystoma.org) that is only 44 % identical

388 to human p21, but shares a cyclin:CDK inhibitory site that may offer an oppor-
389 tunity for cloning (Table 1).
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Table 1 p21 Homologies, Accession numbers of the protein sequences are as follows: Axolotl
(Ambystoma mexicanum) ID: C0207013); Xenopus laevis (NP_001087933); Xenopus tropicalis
(XP_002935823); mus (NP_001104569); humanp21 (NP_000380)
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390 3.3 Regulation of G2/M D
391 In mammals, the p53 gene is mutated in approximately 50 % of all human cancers.
At the G2 checkpoint, p53 protects against DNA damage by stimulating the
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392
393 expression of p21, and if the damage is irreparable, the p53 binding protein 2
394 (p53BP2) mediates apoptosis through p53 (Levine et al. 1991). Villard et al.
395 (2007) recently cloned the axolotl p53 gene. They found that endogenous axolotl
396 p53 was activated following DNA damage by UV irradiation or treatment with an
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397 alkylating agent. Inhibition of axolotl p53 with pifithrin-a inhibited limb regen-
398 eration, as measured by the inhibition of expression of p53 target genes such as
399 Mdm2 (p53 binding protein 2) and Gadd45. However, pifithrin-a may have other
400 targets as well (Komarova et al. 2003). Interestingly, axolotl p53 shows (and
401 tolerates) amino acid changes that mimic those found in p53 variants of human
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402 cancer cells. We have detected the p53 binding protein 2 during hind limb blas-
403 tema formation in Xenopus stage 60 by both immunofluorescence and proteomic
404 analysis (F. Song, X. Chen, unpublished results), but have not examined the
405 expression of the p53 protein.
406 In our proteomic study of blastema formation during limb regeneration in the
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407 axolotl, we found that Evi5 was the most highly upregulated protein at 1, 4, and
408 7 days post-amputation (Rao et al. 2009). Since the number of cells undergoing
409 mitosis is so low during blastema formation, the Evi5 detected is most likely not
410 the 90 kDa fragment associated with the cytokinetic bridge, but is rather the full-
411 size 110 kDa mammalian protein that accumulates during G1 and S to stabilize
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412 Emi1 and prevent premature entry into M. The high levels of Evi5 during blastema
413 formation again suggest that prior to the growth phase, blastema cells are arrested
414 in G2. Using a mammalian Evi5 antibody, we found that Evi5 is expressed in both
415 mesenchymal blastema cells and in cells of the wound epidermis/AEC of the

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416 regenerating axolotl limb (F. Song, X. Chen, unpublished results). Our current
417 working hypothesis is that Emi1, stabilized by Evi5 (Eldridge et al. 2006) and
418 perhaps Pin1 (Bernis et al. 2007), may restrain cells from entering mitosis until
419 they have dedifferentiated and accumulated sufficiently to constitute a blastema,
420 possibly through interactions with regulatory molecules such as Rab11 (Rao et al.
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421 2009) through the Hippo pathway. Another hypothesis is that Evi5 plays a role in
422 the fragmentation of myofibers into mononucleate cells, comparable to its role in
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423 cytokinesis during cell division. In this case, we would expect the 90 kDa form of
424 Evi5 to be expressed during dedifferentiation and blastema cell accumulation.
425 There is no convincing proof as yet that dedifferentiating cells of the regen-
426 erating urodele limb arrest in G2. In mammals, cell cytometry has been used to
427 distinguish cells in different phases of the cell cycle on the basis of their DNA
428 content (Van Dilla et al. 1969). Cell cytometry has been applied to quantify the
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429 DNA content of regenerating control and denervated urodele limbs, but no dif-
430 ference in 2C and 4C populations was found between the two (Mescher and
431 Tassava 1976). This problem could be profitably re-investigated with propidium
432 iodide DNA labeling and flow cytometry analysis to compare cell cycle profiles, as
433 described for the MRL/Mpj mouse ear (Bedelbaeva et al. 2010), and also by the
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434 use of G2 markers such as cH3, cyclins A and B1, Evi5 and Emi1, to determine the
435 proportion of cells in G2 at the accumulation blastema stage.
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436 3.4 Role of Nerves and Wound Epidermis in Blastema

437 Cell Cycling
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438 As outlined above, dedifferentiating cells of amputated urodele limbs can enter the
439 cell cycle in the absence of either nerves or wound epidermis, but axon-dependent
440 factors expressed by the wound epidermis are necessary for cell cycling during
441 blastema growth. Recently, a protein expressed by epidermal gland cells was
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442 identified whose expression by the AEC is axon-dependent, and which can pro-
443 mote complete regeneration in denervated and amputated adult newt limbs after
444 electroporation of its gene into dedifferentiating limb tissue (Kumar et al. 2007).
445 The protein is the anterior gradient protein (AGP), a ligand for the blastema cell
surface receptor Prod1. Prod1 is a member of the Ly6 family of three-finger
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446
447 proteins anchored to the cell surface by a glycosylphosphatidylinositol linkage
448 (Morais da Silva et al. 2002; Brockes and Kumar 2008; Garza-Garcia et al. 2009).
449 Neuregulin (GGF-2), along with other growth factors produced by platelets and
450 macrophages (FGFs, PDGF, TGF-b, IL -1, 2, 6) was shown to be mitogenic for
451 Schwann cells in transected mammalian peripheral nerves (Davies 2000). A newt
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452 neuregulin gene cloned from spinal cord is expressed in newt dorsal root ganglia,
453 and recombinant human GGF-2 infused into denervated axolotl limb blastemas
454 maintained the DNA labeling index at control levels and was reported to support
455 regeneration to digit stages (Wang et al. 2000). However, little data were given to
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456 support the report that GGF-2 supported complete regeneration in these
457 experiments.
458 AGP is strongly expressed in the distal-most Schwann cells of regenerating
459 newt limbs at 5 and 8 days post-amputation, when histolysis and dedifferentiation
460 are underway (Kumar et al. 2007). By 10 days post-amputation in the newt limb,
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461 when dedifferentiated cells are accumulating to form the blastema, AGP expres-
462 sion shifts from Schwann cells to sub-epidermal secretory gland cells of the AEC
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463 (Kumar et al. 2007). The wound epithelium of the axolotl does not have sub-
464 epidermal gland cells and here AGP expression is observed in the Leydig cells.
465 Both sets of gland cells appear to discharge secretions by a holocrine mechanism
466 (Kumar et al. 2010). The expression of AGP by Schwann and gland cells is axon-
467 dependent, as shown by the fact that it is abolished in denervated limbs.
468 Like HGF in liver regeneration (Michalopoulos and De Frances 1997), AGP is
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469 a complete mitogen for limb regeneration. Clearly, it binds to Prod1 on the
470 blastema cell surface and is all that is required for DNA synthesis in vitro and in
471 vivo (Kumar et al. 2007). However, FGFs 1, 2 and 8 have also been shown to
472 individually promote proliferation in vitro and in vivo (Chew and Cameron 1983;
473 Boilly et al. 1991; Zenjari et al. 1997; Han et al. 2001; Christensen et al. 2001,
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474 2002; Yokoyama et al. 2000, 2001; Dungan et al. 2002; Giampaoli et al. 2003).
475 What the functional relationship of AGP is to other mitogens of the AEC in vivo is
476 unclear. AGP might regulate the expression of FGFs by the AEC, or AGP and
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477 FGFs might represent redundant mechanisms for proliferation, like the HGF,
478 TGFa/EGF, and TNF-a pathways in liver regeneration, although none of the FGFs
479 have been shown to promote regeneration to digit stages in vivo. Presumably, AGP
480 would function by some intracellular pathway to induce dedifferentiating cells to
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481 traverse G1 to the RP. Answers to these questions will require a great deal more
482 experimentation. The temporal analysis of AGP expression is incomplete, having
483 been carried out for only two time points (stages) of regeneration. Whether or not
484 AGP regulates the expression of FGFs by the AEC might be answered by
485 denervation/AGP therapy experiments. Denervation strongly downregulates FGF-
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486 8 expression (Christensen et al. 2001, 2002) and presumably other FGFs of the
487 AEC in regenerating axolotl limbs, but whether AGP expression in denervated
488 limbs restores the expression of these growth factors is unknown.
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489 4 Similarities, Differences, and Unanswered Questions

490 Between the Urodele Limb and MRL/MpJ Mouse
491 Ear Regeneration
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492 The MRL mouse ear tissue and the urodele limb exhibit both similarities and
493 differences with regard to regeneration (Fig. 6). Punch hole injury in the MRL
494 mouse removes a disk of skin, loose connective tissue, and cartilage, whereas
495 amputation of the urodele limb removes whole segments of skin and

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Axolotl MRL mouse

Injury
DNA Damage
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Interphase (G1 arrest)
p21
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Interphase (G2M arrest)
EVI-5 EVI-5
Metaphase Telophase (Cytokinesis)
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MKLP-1
EVI-5 EVI-5
Fig. 6 Cell cycle similarities between MRL mouse and axolotl. The MRL mouse shows a DNA
damage response leading to p53 upregulation. However, the G1 cell cycle checkpoint regulatory
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gene p21 CIP1/WAF1 is not expressed and the cells do not arrest in G1. Instead, like axolotl cells,
MRL cells arrest in G2 and both species show upregulation of Evi5, a molecule that enforces G2
arrest by stabilizing the APC inhibitor Emi1. Cytokinesis, when it does occur, requires both Evi5,
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upregulated in axolotl and MRL, and MKLP1 which is upregulated in the MRL
496 musculoskeletal tissue. Fibroblasts appear to be a major source of the blastema in

497 the ear, though infiltrating bone marrow-derived cells probably contribute as well
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498 (unpublished data), and the urodele limb blastema, though the latter gets contri-
499 butions from muscle (including satellite cells), cartilage, and Schwann cells as
500 well (Stocum 2012, for review). Both form a mesenchymal blastema under a
501 wound epidermis, ring-shaped in the MRL mouse ear, and conical in the ampu-
502 tated urodele limb. While a basement membrane initially forms in the MRL, it
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503 degrades after a few days, and is continually degraded in the regenerating limb,
504 allowing communication between the wound epidermis and subjacent mesen-
505 chyme. What the molecular nature of this communication in the MRL is, we do not
506 know, but in the limb the wound epidermis secretes AGP and FGFs that act as
mitogens for blastema cells. The function of the wound epidermis in the limb is
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507
508 nerve-dependent. Recent experiments indicate that denervation of the MRL/MpJ
509 mouse ear prevents regeneration after a punch hole wound, so there may be a
510 similar nerve-wound epidermis circuit operating in the MRL mouse (Buckley et al.
511 2012).
512 Over 40 % of uninjured MRL ear fibroblasts are arrested in G2, and are thus
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513 poised to repair DNA damage and enter M, or initiate apoptosis, immediately after
514 injury. This is not an unusual feature among regeneration-competent tissues. Stem
515 cells in general exhibit a preference for G2 arrest (Hong et al. 2007; Chuykin et al.
516 2008; Galvin et al. 2008). Epithelial cells and interstitial gland cells from
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517 regenerative hydra are arrested in G2 (Schmidt and David 1986; Dubel and
518 Schaller 1990; Holstein et al. 1991), and G2 arrest is a dominant feature in adult
519 mammalian hepatocytes (Michalopoulos and De Frances 1997; Guidotti et al.
520 2003). Cells of the unamputated limb are not preferentially in G2 arrest as far as we
521 know, but collect there as cells liberated by histolysis enter the cell cycle during
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522 blastema formation.
523 The downregulation of p21CIp1/Waf1 appears to be sufficient for uninjured MRL
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524 ear fibroblasts to phosphorylate Rb and pass the RP checkpoint into S and progress
525 to G2. The p21 knockout confers regeneration competence on the ear tissue of the
526 non-regenerating wild-type mouse and this could very well be due to the fact that
527 the lack of p21 leads to G2 arrest instead of arrest in G1. We would like to know
528 whether p21 or its equivalent is downregulated in regenerating urodele limbs as
529 well, and also whether in MRL ear tissue thrombin activates a factor that leads to
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530 downregulation of p21 and phosphorylation of Rb. Deletion of the major regulator
531 of p21, p53, does not confer regenerative ability in B6 mice, but does have a
532 positive effect when deleted in female MRL mice. Inhibition of p53 in the urodele
533 limb, however, may prevent regeneration, suggesting that it is essential for
534 regenerative competence. D
535 The G1-S checkpoint relies on p53 as a sensor for DNA damage, leading in the
536 worst-case scenario to p21 mediated initiation of apoptosis. In this vein, an
537 interesting finding is that in the tails of Xenopus tadpoles (Tseng et al. 2007) and
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538 the South American knifefish (Sirbulescu and Zupanc 2009) there is massive
539 apoptosis during the first 24 h after amputation. This apoptosis is obligatory for
540 Xenopus tail regeneration (it is not known if the same is true for the fish tail)
541 because regeneration is prevented if apoptosis is inhibited. Thus upregulation of
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542 p53 and p21 might be expected in the first 24 h after tail amputation in these
543 species. Apoptosis has not been detected in limb regenerate tissue examined at
544 more than 1-day post- amputation (Mescher et al. 2000; Atkinson et al. 2006), but
545 has not been examined within the first 24 h after amputation.
546 Interestingly, another requisite of tail and limb regeneration is influx of sodium
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547 ions and efflux of hydrogen ions (Borgens et al. 1977, 1979; Adams et al. 2007).
548 Inhibition of either sodium or proton channels results in the prevention of
549 regeneration. Xenopus tails prevented from regenerating by shRNA directed at the
550 NaV1.2 sodium channel mRNA can be rescued by mis-expression of the human
551 NaV1.5 sodium channel gene to transiently create a sodium current (Tseng et al.
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552 2011). The connection of these ionic currents with apoptosis or other early events
553 of regeneration is unknown. Preliminary studies show that the NaV1.2 channel is
554 upregulated in the epidermis in the MRL mouse ear (Fig. 7) and parallels the
555 amphibian tail or limb by the occurrence of apoptosis or ionic currents in the first
556 24 h after punch injury is unknown.
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Fig. 7 Expression of sodium ion channel in the MRL and C57BL/6 ear 3 days post-injury. The
ear tissue was stained with antibody to the Na ion channel NavV1.2. Significant immunohis-
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tochemical staining was seen in the epidermis and in hair follicles in the MRL (right panel,
arrows) but not in the C57BL/6 (left panel)
557 5 Summary D
558 There are multiple parallels presented in this paper showing similarities in the
MRL regenerating ear hole and the axolotl regenerating limb. The two major cell
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559
560 cycle pathways involve G2 arrest and cytokinesis (Fig. 6). First, the axolotl shows
561 clear Evi5 upregulation leading to G2 arrest stabilization; the MRL downregulates
562 p21, also show Evi5 upregulation, and G2 arrest. G2 could very well provide a state
563 in which the accumulating blastema cells are epigenetically modifying their
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564 chromatin to attain the state required to respond to growth stimuli.

565 The next step is mitosis leading to cytokinesis with the upregulation of two
566 molecules during MRL mouse regeneration, specifically Evi5 and MKLP1, and
567 Evi5 in axolotl limb regeneration. Cytokinesis requires three critical complexes
568 associated with the midzone/midbody: the first is associated with microtubules and
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569 involves PRC1 and KIF4, the second in central spindling and involves CYK-4 and
570 MLKP1; and the third is the CPC and involves INCENP, Aurora B, Borealin, and
571 Survivin where Evi5 is functioning (Glotzer 2005). There is further evidence from
572 another study showing that knocking out the cytokinesis gene mps-1 leads to
defects in zebra fish fin regeneration (Poss et al. 2002). However, it is not known
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573
574 whether any modulation of this response such as a delay in cytokinesis contributes
575 to regeneration such as might occur in the liver (Margall-Ducos et al. 2007).
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576 References
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Cell Cycle Regulation and Regeneration

Article in Current Topics in Microbiology and Immunology · December 2012

Ellen heber-katz Kamila Bedelbaeva

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Fengyu Song Xiaoping Chen

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The user has requested enhancement of the downloaded file.

Author Family Name Zhang

1 Cell Cycle Regulation and Regeneration

2 Ellen Heber-Katz, Yong Zhang, Khamila Bedelbaeva, Fengyu Song,

E. Heber-Katz (&) Y. Zhang K. Bedelbaeva

Indiana University-Purdue University, Indianapolis, IN 46202, USA

Current Topics in Microbiology and Immunology

38 1 Introduction: The Cell Cycle ..................................................................................................

41 2.2 G2/M Arrest and DNA Damage.....................................................................................

53 1 Introduction: The Cell Cycle

54 Cell proliferation is an absolute requirement for tissue regeneration, and regulation

Cell Cycle Regulation and Regeneration

59 regenerative mechanism of complex multi-tissue structures (Arthur et al. 2010).

69 (prophase, metaphase, anaphase, and telophase) to produce two daughter cells

108 DNA repair occurs.

Cell Cycle Regulation and Regeneration

Cell cycle regulation

2 Regeneration of Mouse Ear Tissue

160 2.1 Events of Regeneration

Cell Cycle Regulation and Regeneration

185 enchymal-like fibroblasts that potentially contribute to the blastema population.

195 2.2 G2/M Arrest and DNA Damage

223 involved in DNA synthesis. Without p21, Rb is phosphorylated, E2F is released

233 the first case, p53 deletion in a non-regenerator strain in a heterozygous or

Cell Cycle Regulation and Regeneration

257 2.3 G2/M Arrest and Cytokinesis

273 show a high percentage (approximately 70 %) of either mononuclear or binuclear

C57BL/6 20x C57BL/6 40x

distal to the injury

275 3 Regeneration of the Amphibian Limb

276 3.1 Blastema Formation and Growth

Cell Cycle Regulation and Regeneration

291 et al. 2009).

329 3.2 Regulation of Go/G1 and Progression Through G1

337 incorporation by cultured newt blastemas (Vethamany-Globus et al. 1978; Kesik

342 and the systemic circulation.

Cell Cycle Regulation and Regeneration

347 becomes hypophosphorylated and actively suppresses myonuclear cell cycle

387 Center, University of Kentucky; www.ambystoma.org) that is only 44 % identical

Cell Cycle Regulation and Regeneration

436 3.4 Role of Nerves and Wound Epidermis in Blastema

489 4 Similarities, Differences, and Unanswered Questions

Cell Cycle Regulation and Regeneration

Axolotl MRL mouse

Metaphase Telophase (Cytokinesis)

496 musculoskeletal tissue. Fibroblasts appear to be a major source of the blastema in

Cell Cycle Regulation and Regeneration

564 chromatin to attain the state required to respond to growth stimuli.

628 GTPase-activating protein motif for RAB11. Oncogene 26:2804–2808

Cell Cycle Regulation and Regeneration

669 libraries. Genome Biol 5:R67

Cell Cycle Regulation and Regeneration

775 by a transient sodium current. J Neurosci 30:13192–13200

Author Query Form

Schaller (1990), respectively so that these citations matches the

Mescher and Tassava (1975), Tassava and Mescher (1976),