EphrinB1 modulates glutamatergic inputs into POMC-expressing progenitors and controls glucose homeostasis

Proopiomelanocortin (POMC) neurons are major regulators of energy balance and glucose homeostasis. In addition to being regulated by hormones and nutrients, POMC neurons are controlled by glutamatergic input originating from multiple brain regions. However, the factors involved in the formation of glutamatergic inputs and how they contribute to bodily functions remain largely unknown. Here, we show that during the development of glutamatergic inputs, POMC neurons exhibit enriched expression of the Efnb1 (EphrinB1) and Efnb2 (EphrinB2) genes, which are known to control excitatory synapse formation. In vivo loss of Efnb1 in POMC-expressing progenitors decreases the amount of glutamatergic inputs, associated with a reduced number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits and excitability of these cells. We found that mice lacking Efnb1 in POMC-expressing progenitors display impaired glucose tolerance due to blunted vagus nerve activity and decreased insulin secretion. However, despite reduced excitatory inputs, mice lacking Efnb2 in POMC-expressing progenitors showed no deregulation of insulin secretion and only mild alterations in feeding behavior and gluconeogenesis. Collectively, our data demonstrate the role of ephrins in controlling excitatory input amount into POMC-expressing progenitors and show an isotype-specific role of ephrins on the regulation of glucose homeostasis and feeding.


2)
Given the absolutely minimal overlap of Pomc-GFP +ve and Pomc-Cre-tdTom +ve cells), how do the authors know that the Pomc-GFP transgenic mouse recapitulates any native Pomc expression at P14? It is not clear what the authors mean by "during postnatal day 14, the number of arcuate Pomc neurons is more important to that observed in adults".
We only tried to give an explanation (in addition of the change in cell fate) of the minimal overlap between Pomc-progenitors and Pomc-GFP in adults. At postnatal ages, the absolute number of POMC neurons is higher to that observed in adults. Consequently, at these ages, the overlap between Pomc progenitors and POMC neurons is more important when compared to adults. But we agree with the reviewer that Pomc-Cre mouse model is not a perfect model to exclusively target POMC neurons.
We chose the white color to illustrate the nuclear staining, as the blue color was too dark and was hiding the mRNA punctiform staining. Unfortunately, we did not have DAPI staining for the high magnifications. DAPI counterstaining was indeed performed on our sections as you can see in low magnifications but not acquired when using x63 objective because we quantified the vglut2+ inputs that were in contact with POMC somas. Regarding figure S1C, I deeply apologize for this missing information probably due to mistake during the acquisition or saving of this picture. The counterstaining would have been helpful to better distinguish the arcuate neurons. However, we cannot take this picture as the RNAscope staining do not persist over time. We are confident in the way we quantified the number of Efnb1 and Efnb2-positive spots as Pomc-GFP cytoplasmic labeling was distinguishable.
Nuclear staining in Fig. S4 is indicative of tissue deterioration.
We agree that the tissue seems to be degraded, specifically in areas surrounding the pituitary, probably due to retrieval step during the in situ hybridization, but the mRNA staining does not seem to be altered.
Reviewer #2: The authors have performed additional sutides which has addressed the Reviewers concerns in a satisfactory manner, which further strengthens the manuscript.
Reviewer #3: The authors have responded effectively to the majority of my comments and the comments of the reviewers. The manuscript is much improved. Unfortunately, the authors did not directly address my concerns about figure 3. I apologize if I was not clearbut my concerns remain. Here the authors show images of GFP labeled neurities and stain for vGlut2 a presynaptic marker. They show that there are decreases in this vGlut2 staining. These data would seem to suggest that in this figure, ephrin-B shRNAs are acting in the axon. However, for the rest of the manuscript, they claim that ephrin-Bs are acting postsynaptically. It seems that either the model system used in this figure is NOT appropriate for their studies (eg it looks at ephrin-B functions in AXONS, not dendrites) or ephrin-Bs are not functioning in axons in their studies. Based on the rest of their work, it seems likely that ephrin-Bs are postsynaptic in the POMC. This means that this assay is not a good model for their system. If the effects of ephrin-B are postsynaptic, the authors must look at postsynaptic markers to validate their shRNAs. In addition, no rescue controls are shown for these tools.
We agree with the reviewer comments and decided not to add these data in the manuscript. We therefore changed the text accordingly.