3.1 Micro-Arrays
3.1.4 Array Data Sorted into Functional Categories
Judging the two genes which are regulated at all time points is difficult, only knowing that they are transcribed loci. Further analysis using the “blast” software tool on the PubMed webpage did also not lead to satisfying results. Judging their regulation pattern, upregulated at all three time points, and knowing of the very specific actions needed for regeneration, could classify them as a byproduct of tissue damage, reflected e.g. in upregulated metabolism.
The very limited information about the 10 overlapping genes between the 12 hour and 11days time points only allows suggestions. Genes which are regulated not at 6 hours post-lesion but later might not have “immediate early” characteristics or an immediate stress response but would more likely fall into e.g. axon elongation or pathfinding.
Figure 6: Venn diagram showing the minimal overlapping of regulated genes at all three time points after lesion (6 hours, 12 hours and 11 days).
Figure 7: Graphical display of regulated genes sorted in catagories after an optic nerve crush at three different time points: 6 hours, 12 hours and 11days.
Unidentified 56%
Signaling 3%
Stress/Rep air/
M etabolism 30%
Cy toskeleton 2%
Clock / Photorecep tor
3%
Transcrip tion Factors
6%
Axon guidance 3%
Clock / Photorecep tor
4%
Cy toskeleton 3%
Differentiation 6%
Unidentified 63%
Stress/Rep air/
M etabolism 8%
Signaling 7%
Transcrip tion Factors
6%
Signaling 6%
Differentiation 2%
Regulation of translation
2%
Axon Guidance
8%
Unidentified 47%
Stress/Rep air/
M etabolism 12%
Cy toskeleton 15%
Clock/
Photorecep tor 1%
Transcrip tion factors 7%
Unidentified 56%
Signaling 3%
Stress/Rep air/
M etabolism 30%
Cy toskeleton 2%
Clock / Photorecep tor
3%
Transcrip tion Factors
6%
Axon guidance 3%
Clock / Photorecep tor
4%
Cy toskeleton 3%
Differentiation 6%
Unidentified 63%
Stress/Rep air/
M etabolism 8%
Signaling 7%
Transcrip tion Factors
6%
Signaling 6%
Differentiation 2%
Regulation of translation
2%
Axon Guidance
8%
Unidentified 47%
Stress/Rep air/
M etabolism 12%
Cy toskeleton 15%
Clock/
Photorecep tor 1%
Transcrip tion factors 7%
Explanations for categories:
Transcription factor:
Genes that code for proteins that bind to regulatory regions and control gene expression Signaling:
Intra- and extracellular signaling molecules Axon guidance:
Cell surface molecules potentially involved in axon guidance
Clock / Photoreceptor:
Molecules in photoreceptors, some of which are involved in the day/night cycle control Cytoskeleton:
Includes cytoskeletal genes and those that interact with the cytoskeleton
Differentiation:
Genes involved in cell fate and development Regulation of Translation:
Influence translation
Stress/Repair/Metabolism:
These genes represent acute responses to injury
b) 12 hours post lesion
c) 11 days post lesion a) 6 hours post lesion
Every time point had specific categories which are mainly regulated, e.g. at 6 hpl 30% of the regulated genes belong to the stress/repair/metabolism group, whereas at 12 hpl this group only represented 8% of the regulated genes.
At 12 hpl the group of axon guidance genes, which was not detectable at 6 hours at all, increased to 3%.
Looking at 11dpl the amount of axon guidance genes increased further to 8% and the group of cytoskeleton related genes reached 15%, compared to 2% at 6 hours and 3% and 12 hours.
The 4 most strongly regulated functional groups, judged by the highest change in percentage (see Figure 7) comparing the 6 hour time point and the 11 days time point, were arranged graphically in Figure 8 below to show their development over the three time points.
Figure 8: Changes in the percentage of genes belonging to the 4 most strongly, comparing 6 hpl and 11 dpl, regulated ontological groups (axon guidance, cytoskeleton, signaling and stress/repair/metabolism) at the three time points after the optic nerve lesions
Some of the regulated genes found to be up-regulated at the 11day post lesion time point had already been described in the literature, e.g.: Thy-1 (Deininger, Rajendran et al. 2003), Gap-43 (Bormann, Zumsteg et al. 1998) and Gefiltin (Asch, Leake et al. 1998).
5%
10%
15%
20%
25%
30%
6h 12h 11d
0%
AG: Axon Guidance
CY: Cytoskeleton
S: Signaling
SRM: Stress/Repair/Metabolism
[ T ]
Percentileofgenesinfunctionalcategory
SRM SRM
SRM SRM
SRM SRM CY CY
CY CY CY
CY
AG AG
AG AG AG
AG S S
S
S SS
Description Gene Name Fold Change Previously reported in nerve injury models
Thymosin, beta Dr.19380.1.S1_at 25,39 RGCs, zebrafish (Roth et al., 1999) Thy-1 Dr.20019.1.S1_at 17,99 RGCs, zebrafish (Stuermer et al., 2004) GAP 43 Dr.92.1.A1_at 14,72 RGCs, zebrafish (Bormann et al., 1998);
RGCs,rat (Doster et al., 1991) Tubulin, beta 5 Dr.4416.3.A1_at 14,43
Sox 11b Dr.5112.1.S3_at 8,115 RGCs, zebrafish (Goldman et al., 2007);
DRGs, mouse (Tanabe et al., 2003) Plasticin Dr.263.2.S1_x_at 7,956 RGCs, zebrafish (Asch et al., 1998);
SMNs, rat (Troy et al., 1990) ATF 3 Dr.14282.1.S1_at 7,805 RGCs, rat (Takeda et al., 2000);
DRGs, rat (Tsujino et al., 2000) Uchl 1 Dr.8724.1.S1_at 6,112
Sulfatase 2 Dr.12717.1.S1_at 5,196 Tubulin, alpha 8 like 3 Dr.20214.1.A1_at 5,135
Gefiltin Dr.264.1.S1_at 4,873 RGCs, zebrafish (Asch et al., 1998) Tubulin beta, III Dr.7928.1.A1_at 4,349
Adcyap 1b Dr.10739.2.S1_a_at 4,126
MARCKS Dr.3153.1.A1_at 3,923 MNs, rat (McNamara et al., 2000) junction plakoglobin Dr.25119.1.S1_s_at 3,274
Galectin 1-like 2 Dr.13015.1.S1_at 3,113 CRMP 5a (dpysl 5a) Dr.21550.1.S1_at 2,929
ALCAM/Neurolin Dr.20912.1.S2_at 2,886 RGCs, goldfish (Stuermer at al., 1992) ICAT Dr.1102.1.S1_at 2,763
FABP 3 Dr.6814.1.S1_at 2,738
jun Dr.7608.1.A1_at 2,554 RGCs, zebrafish (Goldman et al., 2007) Mibp 2 Dr.781.1.S1_at 2,51
Ppia Dr.9654.1.A1_at 2,507
CRMP 4 (dpysl 3) Dr.16753.2.A1_at 2,435 DRGs, chick (Yazan et al., 2007) Caspase 3 Dr.4796.1.A1_at 2,359
HuD Dr.424.1.S1_at 2,321 Tubulin, alpha 2 Dr.26381.1.A1_at 2,277 Reticulon 1 Dr.4188.2.S1_at 2,199 Eef 2l Dr.908.1.S1_at 2,147 Acsl 4 Dr.16391.1.A1_at 2,125 Selenoprotein W1 Dr.10201.1.S1_at 2,084 Aanat 2 Dr.8142.1.S1_at 2,083 CD99 l2 Dr.25120.3.S1_at 2,066 Fibronectin 1 Dr.19965.1.S1_at 2,038
Sox 11a Dr.4763.3.S1_at 2,03 RGCs, zebrafish (Goldman et al., 2007)
Table 4: Table of the most strongly regulated annotated genes at 11days post lesion. DRG: Dorsal Root Ganglion; MN: Motor Neuron; RGC: Retina Ganglion Cell; SMN: Spinal Motor Neuron
The complete list of regulated genes is depicted in the appendix (section 5.6). Since no data for 6 hours and 12 hours after a CNS lesion in zebrafish has been published to date, no regulated genes for comparison with our gathered data was available.
The analysis revealed a regeneration-specific time course of gene regulation after injury in the zebrafish retina. Most interesting for this analysis were the RGCs, capable of axon regrowth in vivo, which are part of the retina. Hence, some potentially interesting genes were chosen from the list (see appendix 5.6) to be cloned and tested by in situ hybridizations to confirm their regulation and determine the cell type in which these genes were up-regulated.
Our main focus was axon growth, differentiation and transcription factors while still including other (internal group decisions based on literature and database research) genes with different functions. The final decisions were made, taking function, biological relevance and regulational strength into account. The list of these chosen genes is set out in Table 4.
Name Fold
Change
Gene Name
Baseline Raw Data
Regulated
Raw data Function Cloned in Vector
In-situ probe
Thra 3,3 Dr.454.1.S1_at 64 205 TF Yes Yes
Her 4.2 2,4 Dr.5372.7.A1_at 14 34 TF Yes Yes
Wnt-7b 4,4 Dr.22588.1.A1_at 23 101 S Yes Yes
Ndrg1l 0,4 Dr.8090.1.A1_at 251 108 S Yes Yes
Tf12 5,3 Dr.10346.1.S1_at 14 89 TF Yes Yes
Nf1 5,3 Dr.15268.1.A1_at 178 958 TF Yes Yes
Lim-only 2,9 Dr.18163.2.S1_at 26 76 TF Yes Yes
Sp5 2,7 Dr.23472.1.S1_at 13 22 TF Yes Yes
Vsx1 2,7 Dr.558.1.S1_at 185 489 TF Yes Yes
close to plexin B3 2,1 Dr.22796.1.A1_at 95 196 AG Yes Yes
Aanat 2 13,6 Dr.8142.1.S1_at 37 459 CP Yes Yes
Bmpr1a 8,1 Dr.8154.1.S1_at 18 150 D Yes Yes
Thra 4,1 Dr.454.1.S1_at 64 282 D Yes Yes
Ndrg1l 3,9 Dr.8090.1.A1_at 251 992 D Yes Yes
Rx3 2,6 Dr.540.1.S1_at 48 122 D Yes Yes
CRMP-5a 2,9 Dr.21550.1.S1_at AG Yes Yes
CRMP-4 2,4 Dr.16753.2.A1_at AG Yes Yes
CRMP-5b Not reg Dr.11133.1.A1_at AG Yes Yes
Jun 2,6 Dr.7608.1.A1_at 56 92 TF Yes Yes
T-Box1 2,2 Dr.10723.1.S1_at 13 143 TF Yes Yes
Sox11b 8,1 Dr.5112.1.S3_at 204 1654 TF Yes Yes
Sox11a 2,0 Dr.4763.3.S1_at 422 870 TF Yes Yes
Alcam 2,9 Dr.20912.1.S2_at 40 118 AG Yes Yes
Aanat 2,1 Dr.8142.1.S1_at 37 94 CP Yes Yes
12hours postlesion6hours postlesion11days postlesion
Table 5: The list depicts the chosen candidates for in-situ probe making sorted by function; AG: Axon Guidance, CP: Clock/Photoreceptor, D: Differentiation, S: Signaling, TF: Transcription Factor