Peer review of: Glial type specific regulation of CNS angiogenesis by HIFα-activated different signaling (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Recent studies have suggested a major role for oligodendrocyte in the regulation of CNS angiogenesis. 
Specifically, it has been proposed that oligodendrocyte lineage cells promote the proliferation of CNS 
endothelial cells through a hypoxia inducible factor alpha (HIFa) pathway that is mediated by Wnt but 
not VEGF signaling. In the current manuscript the authors have reexamined this hypothesis using a 
variety of genetic approaches. Consistent with earlier studies the authors demonstrate that deletion of 
HIFa using Cnp-Cre to target oligodendrocyte lineage cells results in a reduction in CNS blood vessel 
density and endothelial proliferation. Conversely, blocking HIFa degradation results in increased CNS 
vasculature. The stabilization of HIFa in Cnp+ cells did not however lead to the predicted elevation in 
Wnt expression, a result that was confirmed using Sox-10 targeting. Furthermore, blocking Wnt 
signaling in either OPCs or oligodendrocytes did not alter CNS angiogenesis suggesting that Wnt 
signaling is not a critical component of oligodendrocyte mediated CNS angiogenesis. By contrast to 
earlier work the authors provide data suggesting the primary downstream signaling pathways from 
HIFa in OPCs/oligos is mediated by VEGF and that Wnt signaling is largely derived from GFAP+ 
astrocytes. 

Understanding the interplay between glial populations and developing vasculature in the CNS is of 
significant interest. The data in the current manuscript is extensive and consistent with the 
interpretation of the authors. There are however a number of issues with the manuscript. 
First, while the authors have used some established targeting Cre’s to deliver cells specific changes in 
gene expression there is no data clearly demonstrating the specific cell targeting in the Cre-lox 
system. It is known that cellular targeting with Cnp and other Cre’s may be less specific than originally 
described. This is even more important where the authors are using inducible constructs and yet the 
level of induction is poorly defined. Since the data hinges so strongly on cell type localization this 
would seem to be a critical issue. 

Second, the data in Fig 1-3 appear to be largely confirmatory and add little new insights. 

Third, the conceptual advance in the manuscript is greatly over exaggerated. What the authors 
suggest is that different signaling pathways may mediate glial type specific angiogenesis. While this is 
an advance in our understanding it does not constitute a paradigm shift and the potential relevance in 
a clinical/pathological situation is not immediately obvious. 

Reviewer #2 (Remarks to the Author): 

This is an interesting manuscript that addresses the role of oligodendrocyte and astrocyte HIFa-
activated signaling in angiogenesis. The data, in general, are clearly presented and convincing. A 
strength is that most assessments are done multiple ways, adding to the strength of the findings. 
Some concerns are noted as follows: 

1. In multiple experiments the effects of HIFa are determined by genetically disrupting VHL, rather 
than directly disrupting HIFa. Success of the stabilization of HIFa is assessed by evaluation of its 
downstream genes and proteins. It would be helpful and more direct to know if deletion of HIFa would 
similarly not have effects on Wnt/B-catenin activity. 

2. Figure 2 D should be improved to better substantiate the assertion that HIF1a is stabilized. 

3. In Figure 4 the lack of effect of deletion of VHL is indicated on B-catenin in the Cnp mice, but not in 
experiments looking at Sox10 or Pdgfra mice. Instead readouts of Wnt/B-catenin signaling are 

indicated. It would be more direct to look at B-catenin in these mice as well. 

4. Please indicate whether tamoxifen was administered to both the controls as well as the inducible 
floxed mice. 

5. In Figure 2D it does not look as though HIF1a is in the nucleus, as indicated in the legend. Figure 
7E should be improved to represent the observation. Figure 9A should be improved to illustrate that 
EYFP and S100B are co-localized. 

6. In the text Supplementary Figure 3 was not discussed. 

Reviewer #3 (Remarks to the Author): 

The manuscript by Zhang et al, addresses the important question of glio-vascular communication. 
The current state of knowledge is based on a manuscript published five years ago, which suggested a 
role of HIF-induced WNT expression in oligodendrocyte progenitors, as the signal regulating in an 
autocrine manner the inhibition of OL differentiation and in a paracrine manner promoting 
angiogenesis. 
Using a variety of genetic tools, this manuscript challenges the current dogma, and propose that 
oligodendroglia (progenitors and mature OL) and astroglia cooperate in regulating angiogenesis via 
the HIF-mediated secretion of VEGFA. There are a number of strengths in this manuscript, including 
the use of several lines of transgenic mice and a detailed analysis of the angiogenesis phenotype in 
vivo, in spinal cord and cortex. It is also important that, similar to Yuen et al., the authors 
demonstrate that stabilization of HIF (due to ablation of VHL either using Cnp-cre or Sox10-cre 
drivers) results in increased angiogenesis, as indicated by increased PECAM and proliferation of ERG+ 
endothelial cells (Figs. 2 and 3), while the ablation of HIF function results in decreased proliferation 
and angiogenesis (Fig.1). 

However there are a number of MAJOR CONCERNS that need to be addressed in order to strengthen 
the overall message: 

1. The current study challenges HIF-regulation of WNT as paracrine mechanism of regulation of 
angiogenesis, based on at least three lines of evidence in CnpCreVhlfl/fl mice : similar levels of Wnt7a 
and its target genes in spinal cord and forebrain tissues at distinct time points and lack of activity 
using a Wnt reporter transgenic mouse. Importantly, as it was previously described that Wnt7a 
expression declines as OPC differentiate , the authors also report similar gene expression also in tissue 
from two additional lines Sox10CreVhlfl/fl and PdgfraCreERT2Vhlfl/fl . While the data are convincing, 
one would argue that a more direct approach is needed to challenge the mechanism of autocrine 
regulation. Assessing the levels of Wnt7a and its target genes as well as Hk2 (as control for HIF 
activity) in progenitor cells either directly sorted from the brain and spinal cord or cultured from the 
neonatal brain is truly essential. 

2. Part of the rationale in support of previously suggested paracrine regulation of angiogenesis by 
WNT-secretion from OPC, was based on the results on pharmacological inhibitors of the palmitoyl 
acyltransferase Porcupine. This study adopts a genetic approach targeting a distinct secretory pathway 
involving Wntless (Wls). Since these two secretory pathways may differentially impact WNT secretion, 
it is very important for the authors to provide clear evidence of impaired WNT secretion in their triple 
mutant. One could achieve this, for instance, by measuring WNT levels in the culture medium of OPC 
isolated from Sox10CreERT2Vhlfl/fl Wlsfl/fl.. Without this critical piece of information , is difficult to 
assess whether Wnt secretion was truly impaired in these mice, and therefore any interpretation of 
the mouse phenotype should be carefully considered. 

3. While the demonstration that VEGFA secretion by oligodendroglia is very convincing, one would 
want to understand why the authors would test the hypothesis of astroglial secreted VEFGA as 
regulator of angiogenesis, by analyzing p60 tissue. Evaluation of the effect on blood vessels during the 
early postnatal period is absolutely necessary. 

Minor points: 

1. it would be important to show that CnpCre behaves like no-Cre control, at least in one 
supplemental figure 
2. Fig. 3 B, D, E have typos in the Y axis