Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A,B) Over-expression of kon in glia causes a very long VNC (A), and in neurons too, but to a lesser extent (B). RNAi knock-down of candidate genes could rescue these gain of function phenotypes, some examples are given. (C,D) Quantification of normalized VNC length shows rescue prominently by knock-down of most transmembrane phosphatases, the Notch-related Akap200 , and genes functionally related to the insulin signaling pathway ( Akt, lar and ia-2) , most prominently lar. Normalised measurements are given as a ratio of the VNC over total larval length. Kruskal-Wallis ANOVA p<0.0001, post-hoc Dunn’s test comparison to controls repo>kon or elav>kon . (C) N=2-28; (D) N=2-31. *Asterisks indicate multiple comparison post-hoc tests to controls: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For full genotypes and further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A,B) Exploratory quantitative real-time PCR (qRT-PCR), N=2 replicates each: (A) showing the change in mRNA levels for candidate genes upon kon RNAi targeted to either neurons (with elavGAL4 ) or glia (with repoGAL4 ). ia-2 mRNA levels increased at least 3 fold when kon was knocked-down in glia; (B) showing the effect of kon gain of function. kon over-expression in either neurons or glia decreased ia-2 mRNA levels. The first two columns have been left cut out as they are controls with the increase in kon mRNA with kon over-expression, which are very high compared to the rest. (C,D) Further replicates were carried out for a selected group of genes, and they validate that kon prominently regulates ia-2 expression. N=4 replicates each. (D) The first columns represent the very high increase in kon mRNA with kon over-expression, and they have been cut as they go well beyond this scale compared to the rest. For full genotypes and further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Expressing

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A) Quantitative real-time PCR (qRT-PCR) showing that gain of kon function reduced ia-2 mRNA levels by 25% (One Way ANOVA p=0.045), whereas loss of kon function in glia caused practically a 3-fold increase in ia-2 mRNA levels ( genotype: kon c452 /UASkonRNAi; repoGAL4/+ ; One Way ANOVA p<0.0001). Post-hoc Dunnett’s test multiple comparisons to control. N=4 replicates. (B) qRT-PCR showing that over-expression of ia-2 in glia downregulated kon mRNA levels. Left: Unpaired Student t-test with Welch correction p=0.457. Right: One Way ANOVA p<0.045, post-hoc Dunnett’s test multiple comparisons to control. N=4-6 replicates. (C) Ia-2 is functionally related to Notch: qRT-PCR showing that ia-2 mRNA levels increased in N ts mutant larvae at the restrictive temperature of 25°C. Unpaired Student t-test with Welch correction Left: p=0.4123; Right: p=0.2182. N=3 replicates. (D) ia-2 is functionally related to pros : qRT-PCR showing that over-expression of pros in glia increased ia-2 mRNA levels by 2-fold. Unpaired Student t-test with Welch correction. Left: p=0.1368; Right: p=0.0428. N=3 replicates. (E) qRT-PCR showing that UAS-ia-2 RNAi[TRIPHMS00536] knock-down in neurons (with elavGAL4 ) lowered ia-2 mRNA levels to 20%, whereas in glia it has no effect, meaning that ia-2 is expressed in neurons. A second UAS-ia-2RNAi[KK108555-VIE-260B] line lowered mRNA levels by 25%. One Way ANOVA p=0.0004, post-hoc multiple comparisons to control Dunnett’s test. N=3 replicates. (F,G,H) Fusion protein Ia-2YFP revealed expression exclusively in neurons, as all Ia-2YFP+ cells were also Elav+, but Repo — and Dpn — . Genotype: ia-2[CPTI100013]. N=4-16 larval VNCs. (I) Illustration showing that kon and ia-2 functions are restricted to glia and neurons, respectively, and they mutually exclude each other. (G) transverse views; (F,H) horizontal views; (H) higher magnification views). With more than two sample types, asterisks indicate multiple comparison post-hoc tests to controls: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For full genotypes and further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A,B) Loss of kon function in glia ( kon c452 /UASkonRNAi; repoGAL4/+) increased the number of Ia-2YFP+ cells along the midline. One Way ANOVA p<0.0001, post-hoc Tukey’s test. N=5-8 VNCs. (C) The ectopic Ia-2YFP+ cells in kon loss of function were Elav+ and not Repo+. N=5-7 VNCs. (D, E) Neither loss nor gain of ia-2 function affected the number of Eve+ neurons. One Way ANOVA p=0. 2374. N=7-12 VNCs. (F,G) Loss of ia-2 function (elavGAL4>UASia-2RNAi[TRIPHSM00536]) increased Pros+ cell number, and the excess cells were small. Kruskal-Wallis ANOVA p=0.0003, post-hoc Dunnett’s test. N=8-10 VNCs. (H) The small Pros+ cells in ia-2 knock-down did not have the glial marker Repo. (I,J) Dpn+ cells visualised at 120h AEL, after developmental neuroblasts have disappeared. Both loss and gain of ia-2 function increased Dpn+ cell number. All images are horizontal views, except for (I bottom row) which are transverse views. One Way ANOVA p=0.0002, post-hoc Dunnett. N=7-15. Asterisks indicate multiple comparison post-hoc tests to a fixed control: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Marker

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A) Neurons and their axonal fascicles are visualised with anti-FasII. N=7-11 VNCs. (B) Neurons and their dendrites are visualized with anti-BP102. N=9-10 VNCs. No abnormal phenotypes were observed. (A,B) Horizontal views; (A’,B’) transverse views. (C) Anti-Dpn visualised in larvae at 120h AEL, after disappearance of developmental neuroblasts. Loss of ia-2 function in neurons caused over-growths in thorax (arrowhead). N=7-12 VNCs.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques:

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A,B,E) Time course of crush-injury experiments, indicating the age of the larvae (After Egg Laying, AEL) when crush was applied (top arrows), followed by various recovery periods, and when they were fixed or processed (bottom arrows). (C) Drawing showing that crush injury induced ectopic Dpn+ cells. (D, F,G,H) Crush injury in the larval abdominal VNC at 74-76h after egg laying (AEL) caused: (D) an increase in the levels of ia-2 mRNA at 5-7h post-injury, which recovered homeostatically by 24h, detected by qRT-PCR. N=3 replicates. (F,G,H) Formation of ectopic Dpn+ neural stem cells (white arrowheads) by 5-7 hours post-injury, 74-80h AEL. Quantification in (H) shows number of Dpn+ in VNCs with ectopic Dpn+ cells (Penetrance 50% N=10 VNCs), as in some VNC cell loss caused by lesion was very severe. Dpn+ cells were Ia-2YFP — . (I,J,K) Crush injury in the larval abdominal VNC at 96h AEL caused ectopic Dpn+ cells by 6 hours post-injury (yellow arrowheads, penetrance 44% N=9 VNCs). Most Dpn+ cells were Ia-2YFP — , but some were Ia-2YFP + . At this stage, some developmental neuroblasts could still remain (white arrows), but dorsal ectopic Dpn+ were unequivocal (yellow arrowheads, J ). ( K) Student t-test p=0.0063. (L) Injury at 105h AEL visualised at 129h AEL, when no developmental neuroblasts remain, induced a significant increase in Dpn+ cells. Mann-Whitney U-test p=0.0375. (M,N,O) Crush injury in the larval abdominal VNC at 117h AEL caused ectopic Dpn+ cells by 12 hours post-injury (yellow arrowheads, 129h AEL, Penetrance 67% N=21, VNCs. Student t-test p=0.0302). Dpn+ cells were found in ectopic dorsal positions (yellow arrowheads, N ). This stage is devoid of developmental neural stem cells. N=9/32 VNCs. Student t-test. (P) Temporal profile of number of ectopic Dpn+ cells surrounding the lesions, in injured samples with ectopic Dpn+ cells, number in X axis indicate time-points of injury and fixation. (F,I,M) Horizontal views, (G,J,N) transverse views. (H,K,L,O) Graphs show quantifications in box-plots; (P) shows dot plots, with mean and error bars (±s.d.) indicated. *p<0.05, **p<0.01. For full genotypes and further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: All samples were analysed at 120h AEL, after disappearance of abdominal developmental neuroblasts. (A-C) Over-expression of ia-2 and dilp-6 , but not kon-full-length , increased Dpn+ cell number in the abdominal VNC. Both Ia-2 and Dilp-6 induced Dpn+ at the midline and in lateral positions: ia-2 most prominently, but not exclusively, along the midline (white arrowhead), and dilp-6 also prominently, but not exclusively, in lateral positions around the neuropile (yellow arrowheads). (C) Ectopic Dpn+ cells did not express Ia-2YFP (arrowheads). (D) Quantification of all abdominal VNC Dpn+ cells, and genetic epistasis analysis showing that: the increase in Dpn+ cell number caused by ia-2 over-expression was rescued by dilp-6 RNAi and kon-RNAi knock-down in glia, meaning that ia-2 requires Dilp-6 and glial Kon to induce Dpn; and preventing insulin signaling with InR DN in glia rescued the increase in Dpn+ cell number caused by dilp-6 over-expression, meaning that Dilp-6 induced Dpn via InR signalling in glia. One Way ANOVA p<0.0001, post-hoc Tukey’s test multiple comparisons all samples vs. all. N=7-13 VNCs. (E) Illustration showing that Ia-2 and Dilp-6 can induce Dpn via InR signalling in glial cells. (A) Horizontal views; (B) transverse views; (C) higher magnification. Graphs shows quantifications in box-plots. Asterisks refer to multiple comparison post-hoc tests: *p<0.05, ***p<0.0001, ****p<0.0001. For full genotypes and further statistical analysis details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: All samples were analysed at 120h AEL, after disappearance of abdominal developmental neuroblasts. (A-C’, E-G’) Cell proliferation was visualised with the S-phase marker PCNA-GFP, quantification in (D,H). dilp-6 expression was induced in all cells with heat-shock-GAL4 , raising the temperature to 37°C for 30 minutes at the end of the third instar larval stage at 110.5h AEL, then larvae were kept at 25°C for 9 hours, visualizing Dpn+ and PCNA-GFP at 120h AEL. (A-C’) Over-expression of dilp-6 resulted in Dpn+ PCNA-GFP+ cells laterally around the neuropile ( B,B’ white arrows) and along the midline ( C,C’ yellow arrowheads), showing that these ectopic Dpn+ cells were in S-phase. Quantification box-plots in (D) , Student t-test. There were also some Dpn+ cells that were not dividing (white arrows in C’). (E-G’) Over-expression of dilp-6 resulted in PCNA-GFP+ Wrp+ midline glia (yellow arrowheads) that therefore were dividing. In (G) there is a notable increase in the number of Wrp+ cells. In (E,E’) lateral PCNA-GFP+Wrp—Dpn+ cells around the neuropile (white arrows) most likely correspond to neuropile glia. (H) Quantification showing phenotypic penetrance: percentage of segmentally repeated Wrp+ cell clusters that contain PCNAGFP+ cells. Fisher’s Exact test p=0.0213. (I-L) Over-expression of ia-2 in glia with repoGAL4 mildly increased the number of pH3+ mitotic cells along the midline. The pH3+ cells lacked YFP (repoGAL4>his-YFP), consistently with corresponding to midline glia. (L) Quantification in box-plots, not significantly different from controls. One Way ANOVA p=0.0995. (A-C, E-G, I-K) horizontal views; (A’-C’, E’-G’, I’-K’) transverse views. For full genotypes, sample sizes and statistical details see Table Supplement 1.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Marker, Expressing, Over Expression

Journal: bioRxiv

Article Title: Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

doi: 10.1101/721498

Figure Lengend Snippet: (A) In the abdominal larval VNC, neurons have Ia-2, glia have Kon, and Ia-2 and Kon are mutually exclusive; non-midline glia have the transcription factor Repo and midline glia the membrane protein Wrapper. In the normal, uninjured abdominal VNC, InR is in glial cells and some neurons ; Ia-2 expression is constantly present in neurons; kon is switched off, and there are no neural stem cells (neuroblasts). (B) Drawing showing that Dilp-6 can be secreted from neurons, amplified and secreted by cortex glia, and received by all glial types. Dilp-6 production and secretion depend on Kon and Ia-2, which increase in injury. (C) Injury to the abdominal VNC provokes a dramatic surge in Ia-2 and Kon. This drives the initial secretion of Dilp-6 from neurons (1). Secreted Dilp-6 binds InR in glia, and InR signaling may facilitate cleavage and activation of Kon. Kon ICD activates glial proliferation, glial cell fate gene expression and expression of dilp-6 in cortex glia (2). In an autocrine Kon and InR dependent manner, Dilp6 sets off a positive feedback loop that amplifies Dilp-6 production from cortex glia (2). Once secreted, Dilp-6 and InR signaling cause the up-regulation of Dpn+ in neuropile glia - including Notch+ Pros+ lateral (astrocytes) and Wrp+ midline glia (3). Neuropile glia can stochastically switch on Dpn. Glial-derived Dpn+ neural stem cells can divide, and generate new neurons - although to a rather limited extent (4). After cell division, Kon may determine whether daughter cells become glia, to the exclusion of Ia-2. (D) Insulin signalling involving Ia-2, Dilp-6 and InR can increase cell number of various glial cell types - including cortex glia, neuropile astrocytes and midline glia - induce neural stem cells, and potentially generate new neurons. The neurogenic potential of glia may depend on the availability of Notch and Pros, and the downregulation of Kon. Together, these genes can potentially induce neurogenesis and gliogenesis, matching cell populations for regeneration.

Article Snippet: The construct was injected by BestGene Inc. to generate transgenic flies bearing UAS-ia-2 at the attP2 landing site.

Techniques: Expressing, Amplification, Activation Assay, Derivative Assay