Dynamin 2–dependent endocytosis differentially regulates ligand and receptor contributions to Notch signaling during neural precursor cell fate determination
Curation statements for this article:-
Curated by eLife
eLife Assessment
This useful study examines the role of Dynamin-2 in regulating neural progenitor behavior and fate by regulating Notch ligand and receptor function. Whilst the study is of interest because of the importance of these questions, the analyses are incomplete, and there are significant areas requiring additional experimentation in order to provide full confidence in the findings.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
A critical event in the development of the highly structured cerebral cortex is the appropriate generation of differentiated daughter cell populations from asymmetric precursor cell divisions. Internalization of extracellular receptors and ligands in the process of endocytosis is key to the establishment of asymmetry. However, the detailed mechanisms mediating this exchange are incompletely understood. The dynamin family of membrane remodeling GTPases is considered critical for many forms of clathrin-mediated endocytosis (CME) but its role in brain development is unexplored. Here we found that dynamin 2 (Dnm2), a protein involved in CME through vesicle release from the plasma membrane, is essential for the maintenance of neural stem cells. Conditional deletion of Dnm2 leads to early exhaustion of neural precursor cells and premature neurogenesis, resulting in gross structural abnormalities, periventricular hemorrhaging and increased perinatal mortality. Notably, Notch ligands accumulate at the cell surface and cleaved NICD is reduced in neural stem cells, consistent with impaired ligand-mediated activation. In contrast, inhibition of CME in receptor-expressing cells increases cell autonomous Notch signaling, likely by promoting receptor accumulation at the plasma membrane. Further, dynamin 1, 2, and 3 can rescue loss of Dnm2 to different extents. Taken together, these findings reveal that Dnm2 is the essential isoform for physiological non-cell autonomous Notch-mediated neural stem cell maintenance, and that CME exerts fundamentally distinct roles in Notch signaling, promoting ligand activity while restricting receptor signaling through control of surface receptor abundance.
Article activity feed
-
-
-
eLife Assessment
This useful study examines the role of Dynamin-2 in regulating neural progenitor behavior and fate by regulating Notch ligand and receptor function. Whilst the study is of interest because of the importance of these questions, the analyses are incomplete, and there are significant areas requiring additional experimentation in order to provide full confidence in the findings.
-
Reviewer #1 (Public review):
Summary:
This study explores the role of Dynamin 2 (Dnm2)-mediated clathrin-mediated endocytosis (CME) in the fate determination of neural progenitor cells in the embryonic cerebral cortex, with particular attention to its asymmetric regulation of ligand and receptor functions in the Notch signaling pathway. Using conditional knockout mice, in vitro co-culture systems, in utero electroporation, and luciferase reporter assays, the authors propose that Dnm2 is required for neural stem cell maintenance and that CME plays a dual role in Notch signaling.
Strengths:
The authors systematically demonstrate the critical role of Dnm2 in embryonic cerebral cortical development. They show that Dnm2 is the predominant dynamin isoform expressed in the embryonic central nervous system, and that its conditional knockout …
Reviewer #1 (Public review):
Summary:
This study explores the role of Dynamin 2 (Dnm2)-mediated clathrin-mediated endocytosis (CME) in the fate determination of neural progenitor cells in the embryonic cerebral cortex, with particular attention to its asymmetric regulation of ligand and receptor functions in the Notch signaling pathway. Using conditional knockout mice, in vitro co-culture systems, in utero electroporation, and luciferase reporter assays, the authors propose that Dnm2 is required for neural stem cell maintenance and that CME plays a dual role in Notch signaling.
Strengths:
The authors systematically demonstrate the critical role of Dnm2 in embryonic cerebral cortical development. They show that Dnm2 is the predominant dynamin isoform expressed in the embryonic central nervous system, and that its conditional knockout disrupts ventricular zone (VZ) architecture, causes premature depletion of radial glia (RG), and leads to precocious neurogenesis, accompanied by periventricular hemorrhage and perinatal death.
Weaknesses:
I believe that some conclusions of this study should still be treated with caution. Specific comments follow:
(1) scRNA-Seq analysis of the Dnm2-cKO cortex at E11.5 and E13.5 is needed, or alternatively, additional LGE-MGE scRNA-Seq should be performed, to examine whether the key gene expression changes are conserved across the MGE-CGE-LGE and cortex.
(2) Regarding Figure 5 (Loss of Dynamin 2 leads to abnormal accumulation of Dll1 and altered NICD distribution in the E11.5 cortex), I do not understand why Dll1 accumulation occurs primarily in the medial cortex.
(3) A detailed schematic model is needed to illustrate the function and mechanism of action of Dnm2.
(4) Dnm2-cKO exhibits extensive cell death and vascular abnormalities. The authors should explain the possible specific causes.
(5) Clinical relevance: Are there specific forebrain phenotypes associated with Dnm2 mutations in humans? What are the possible reasons? This study appears to have limited relevance to Dnm2-related human disease.
-
Reviewer #2 (Public review):
Summary:
Breuig et al. studied the effect of Dnm2 conditional knockout in the developing neocortex. The authors generated CNS-specific Dnm2 conditional knockout (cKO) mice using Nestin-Cre, Emx1-Cre, and Nex1-Cre drivers. Confirming existing findings of previous research, they reported that Dnm2 was the predominant dynamin isoform in the embryonic forebrain by immunoblot, as well as by analysis of published single-cell and spatial transcriptomic atlases. The authors went on to demonstrate that deletion of Dnm2 in progenitors causes disorganization of the ventricular zone, depletion of Sox2+ progenitors, an excess of Tbr2+ intermediate progenitor cells and Tuj1+ immature neurons, as well as periventricular hemorrhage and perinatal lethality. On the contrary, deletion of Dnm2 in postmitotic neurons produces no …
Reviewer #2 (Public review):
Summary:
Breuig et al. studied the effect of Dnm2 conditional knockout in the developing neocortex. The authors generated CNS-specific Dnm2 conditional knockout (cKO) mice using Nestin-Cre, Emx1-Cre, and Nex1-Cre drivers. Confirming existing findings of previous research, they reported that Dnm2 was the predominant dynamin isoform in the embryonic forebrain by immunoblot, as well as by analysis of published single-cell and spatial transcriptomic atlases. The authors went on to demonstrate that deletion of Dnm2 in progenitors causes disorganization of the ventricular zone, depletion of Sox2+ progenitors, an excess of Tbr2+ intermediate progenitor cells and Tuj1+ immature neurons, as well as periventricular hemorrhage and perinatal lethality. On the contrary, deletion of Dnm2 in postmitotic neurons produces no detectable phenotype. In the mutant cortex, Dnm2 conditional knockout led to accumulation of Dll1, reduced cleavage of NICD, and reduced Hes1/Hes5 expression. To separate ligand- from receptor-side effects, they utilized a neural stem cell co-culture assay. They tested dynamin function in the in vitro assay using a variety of molecules and probes, including a dominant-negative Dnm2 (K44A), an shRNA, and the tamoxifen-inducible deletion in reprogrammed Dnm2-floxed neural stem cells. They further performed postnatal electroporation with Hes5-EGFP and Dll1-mOrange2 reporters and an in vivo dual-luciferase assay to test the effect of receptor side effects. In summary, they showed that inhibiting or downregulating Dnm2 increases Notch signaling levels, while the involvement of Dll1 remains unclear depending on the experimental condition.
Strengths:
The topic of the manuscript, which is the molecular mechanisms and the signal transduction of neural stem cell maintenance, is of fundamental importance to the field of neurodevelopment. The conditional knockout strategy is compelling.
Weaknesses:
The central claims of the current manuscript are not sufficiently supported by experimental results. The rigor of the experiments conducted also needs to be significantly improved.
(1) Quantification and statistical analyses of the core in vivo phenotypes are essential to establish the central claim of progenitor depletion and premature neurogenesis. Only representative images of Sox2, Ki67, Tbr2, Ngn2, Tuj1, and cleaved Caspase-3 are shown (Figures 2, 3, 4, S2, S3), as is the EM observation of increased clathrin-coated vesicles (Figure 3G-H).
(2) The authors report that "the brain of mutant animals appears indistinguishable from control littermates". However, based on the representative images of Figure 2, at 11.5 the disorganization of the neocortex and the disruption of the radial columns have already manifested. The authors should carefully describe the aspects of the brain that they deemed indistinguishable between control and mutant animals, and by what criteria.
(3) The transition from Dnm2 conditional knockout (cKO) to Notch signaling appears somewhat abrupt. The authors should provide additional experimental evidence or literature precedents to logically bridge this gap.
(4) The claim that dynamin inhibition in receptor-expressing cells increases Notch signaling is not supported by the comparisons shown. In Figure 7D, the significant receptor-side comparison is K44A-receiver + Dll1 versus K44A-receiver without ligand, which tests whether ligand-driven activation survives dynamin inhibition, not whether it is enhanced. The in vivo results are marked p = 0.07 (Fig. 8F), p = 0.07 (Fig. 8L), and n.s. (Fig. 8Q), yet the text states the increase was significant, and the Figure 8 title asserts it. This is the paper's most novel claim and the basis of the ligand/receptor dichotomy in the title and Abstract. As stated, it is not supported at conventional significance.
(5) Since the prominent phenotype of Dnm2 cKO was a reduction in radial glial cells, the subsequent reduction of signaling molecules (Hes1, Hes5, etc.) may be due to the reduced progenitor pool, rather than cell-autonomous reduction of Notch signaling. Quantitative results, such as the ones in Figure 6, should take changes in cellular composition into consideration and normalize to the reduced proportion of apical progenitors.
(6) The authors stated that Notch ligands "accumulate at the cell surface," but all immunostaining appears to be on permeabilized tissue, and no internalization assay is reported. The proposed mechanism is a block in ligand endocytosis. Accumulated Dll1 in permeabilized sections is equally consistent with intracellular retention in stalled endocytic intermediates, which the EM in Figure 3G-H might actually favor.
(7) Inconsistency in the cortical regions examined (Anterior vs posterior, medial vs lateral, etc.). Are the observations and experiments conducted in the same region of the cortex? If so, which region and why?
(8) Statistical tests should be described in sufficient detail in the methods section. The number of samples or replications needs to be reported for all experiments. The "Imaging and Quantification" section also did not provide enough information (the authors only stated that "Cell counts were performed on matched cortical regions across genotypes and developmental stages"). Figure legends report significance (Figs. 6B, 6C, 7D, 7F, 8F, 8L, 8Q, S6) without specifying what was compared or how in the corresponding main text or methods.
(9) The main text attributes the cNICD (Val1744) result to Figure 5A-B, but the Figure 5 legend describes the red channel as "NICD (red; total Notch intracellular domain)," which is the antibody that the main text explicitly distinguishes. Figs. 4 and 5 appear to show overlapping Dll1/NICD data.
-
