Author Response
eLife assessment
This study reports the fundamental discovery of a novel structure in the developing gut that acts as a midline barrier between left and right asymmetries. The evidence supporting the dynamics, composition, and function of this novel basement membrane in the chick is in parts solid and in others convincing, but the investigation of its origin and impact on asymmetric organogenesis is not yet conclusive. This careful work is of broad relevance to anyone interested in patterning mechanisms, the importance of the extracellular matrix, and laterality disorders.
We extend our sincere gratitude to the editors at eLife for their meticulous evaluation of our manuscript, as well as the valuable insights shared in this Public Review. We also wish to convey our appreciation to the reviewers for their thought-provoking suggestions, which we are enthusiastic about integrating into our revised work. In this provisional response, our primary focus is to address the two main concerns raised: the necessity for functional data to elucidate the importance of the barrier, and the imperative to resolve uncertainties regarding its origin. We are dedicated to addressing these important points, and believe they will greatly enhance the quality and significance of our manuscript.
Joint Public Review:
When the left-right asymmetry of an animal body is established, a barrier that prevents the mixing of signals or cells across the midline is essential. Such a midline barrier preventing the spreading of asymmetric Nodal signaling during early left-right patterning has been identified. However, midline barriers during later asymmetric organogenesis have remained largely unknown, except in the brain. In this study, the authors discovered an unexpected structure in the midline of the developing midgut in the chick. Using immunofluorescence, they convincingly show the chemical composition of this midline structure as a double basement membrane and its transient existence during the left-right patterning of the dorsal mesentery, which authors showed previously to be essential for forming the gut loop and guiding local vasculogenesis. Labelling experiments suggest a physical barrier function, to cell mixing and signal diffusion in the dorsal mesentery. Cell labelling and graft experiments rule out a cellular composition of the midline from dorsal mesenchyme or endoderm origin and rule out an inducing role by the notochord. Based on laminin expression pattern and Ntn4 resistance, the authors propose a model, whereby the midline basement membrane is progressively deposited by the descending endoderm.
Laterality defects encompass severe malformations of visceral organs, with a heterogenous spectrum that remains poorly understood, by a lack of knowledge of the different players of left-right asymmetry. This fundamental work significantly advances our understanding of left-right asymmetric organogenesis, by identifying an organ-specific and stage-specific midline barrier. The complexities of basement membrane assembly, maintenance, and function are of importance in several other contexts, as for example in the kidney and brain. Thus, this original work is of broad interest.
Overall, reviewers refer to a strong and elegant paper discovering a novel midline structure, combining classic but challenging techniques, to show the dynamics, chemical, and physical properties of the midline. However, reviewers also indicate that further work will be necessary to conclude on the origin and impact of the midline for asymmetric organogenesis. Three issues have been raised to strengthen the claims:
- The function of the midline as a physical barrier requires clarification. Dextran injection here seems to label cells and not the extracellular space. By counting the proportion of dextran-labeled cells rather than dextran intensity itself, the authors do not measure diffusion per se, but rather cell mixing.
We agree that an additional means of showing the barrier function is important. We are currently addressing this using a fluorescently tagged derivative of the drug AMD3100 that we recently synthesized, per Poty et al. 2015. We previously showed that AMD3100 perturbs left sided CXCR4-dependent vasculogenesis when introduced on the left side of the dorsal mesentery (DM), but not when introduced on the right (Mahadevan et al. 2014). These data suggest that a midline barrier prevents diffusion of AMD3100 across the DM. We are currently characterizing the extracellular diffusion of this fluorescent derivative through the DM to complement our previous dextran data.
Additionally, we should emphasize that the dextran-injected embryos shown in Fig. 6 D-F were isolated two hours post-injection, a timeframe insufficient for cell migration to occur across the DM (Mahadevan et al., 2014). We also collected additional post-midline stage embryos ten minutes after dextran injections - too short a timeframe for significant cellular migration (Mahadevan et al., 2014). Importantly, the fluorescent signal in those embryos was comparable to that observed in the embryos in Fig. 6. Thus, we believe the movement of fluorescent signal across the DM when the barrier starts to fragment (HH20-HH23) is unlikely to represent cell migration. More than a decade of DNA electroporation experiments of the left vs. right DM by our laboratory and others have never indicated substantial cell migration across the midline (Davis et al., 2008; Kurpios et al., 2008; Welsh et al., 2013; Mahadevan et al., 2014; Arraf et al. 2016; Sivakumar et al., 2018; Arraf et al. 2020; and Sanketi et al., 2022). This is also shown in our current GFP/RFP double electroporation data in Fig. 2 G-H, and DiI/DiO labeling data in Fig. 2 E-G. Collectively, our experiments suggest that the dextran signal we observed at HH20 and HH23 is likely not driven by cell mixing.
- The descending endoderm zippering model for the formation of the midline lacks direct evidence. The claim of an endoderm origin is based on laminin expression, but the laminin observed in the midline with an antibody may not necessarily correspond to the same subtype assessed by in situ hybridization.
We have attempted to address this important issue by introducing several tagged laminin constructs, LAMB1-GFP, LAMB1-His, and LAMC1-His, to the endoderm via DNA electroporation to try to label the source of the basement membrane. However, despite endogenous laminin production and export within the endoderm, there appeared to be no export of any of the tagged proteins to the endodermal basement membrane. This experiment was further complicated by the necessarily large size of these constructs at 10-11kb due to the size of laminin subunit genes, resulting in low electroporation efficiency. Although we have not yet determined an alternative way to directly test the endodermal origin hypothesis, we are committed to exploring specific methods to help us test this in future experiments.
The midline may be Ntn4 resistant until it is injected in the relevant source cells.
Ntn4 has been shown to disrupt both nascently assembling and preformed mature basement membranes (Reuten et al., 2016). As such, we feel that this particular membrane’s resistance to degradation is likely not predicated by its stage of assembly.
Alternative origins could be considered, from the bilateral dorsal aortae or the paraxial mesoderm, which would explain the double layer as a meeting point of two lateral tissues.
We agree that alternate origins of the midline basement membrane cannot be ruled out from our existing data. We have indeed considered the bilateral dorsal aortae and the paraxial mesoderm as possibilities. However, at the earliest stages of midline basement membrane emergence, the dorsal aortae are already significantly distant from the nascent basement membrane, as are the somites, which have not yet undergone epithelial-to-mesenchymal transition. Fig. S2 G provides an example of a very early midline basement membrane without dorsal aortae or somite contact. Because this particular image is from a section that is fairly posterior in the HH12-13 embryo, it is thus less developed in pseudo-time and gives a window on midline formation in even earlier stage embryos. This is in contrast to the spatially close relationship of the midline basement membrane with the notochord and endoderm. In the context of potential dorsal aortae contributions, it is worth noting that the basement membrane of vascular endothelial cells has a distinct composition from a non-vascular basement membrane. For example, vascular endothelial cells produce only alpha 4 and alpha 5 laminin subunits but contain no alpha 1 subunit in any known species (reviewed in DiRusso et al., 2017). Thus, endothelial cell-derived basement membranes would not contain the alpha 1 laminin subunit that we used in our studies as a robust marker of the midline basement membrane. Note in Fig. 3 E-H and J-J’’’ the absence of dorsal aortae labeling using our laminin alpha 1 antibody. The dorsal aortae are also richer in fibronectin, as seen in Fig. S2, while the midline ECM exhibits far less fibronectin staining. While it may be possible that the converging aortae compress the midline ECM into a more compact structure, we feel direct contribution of basement membrane components is unlikely.
- The title implies a role of the midline in left-right asymmetric gut development. However, the importance of the midline is currently inferred from previously published data and stage correlations and will require more direct evidence.
We agree that we have not fully and directly demonstrated the extent of the role of the midline in enabling the asymmetry of DM compartments during gut development. We propose the following revised title: “An atypical basement membrane forms a midline barrier during left-right asymmetric gut development”. It is important to note that we have made diligent efforts to investigate the functionality of the midline basement membrane through various methods in which we are highly experienced. However, while targeting either the left or right side of the DM is relatively straightforward, accessing the midline presents substantial challenges. We attempted physical perturbation using in vivo laser ablation, but we observed no significant effect or stable disruption of the midline. Additionally, our attempts at ablation using diphtheria toxin proved to be too harsh on the endoderm, preventing reliable and consistent data interpretation. We have tried electroporating MMP9 and MMP2 into the DM, but these did not produce any appreciable effect on the midline. We are also concerned that directly injecting MMPs or other enzymes may lead to injection-related tissue damage to the embryo that may be difficult to separate from direct MMP digestion of the matrix. However, we firmly believe that our inference regarding the involvement of the midline ECM in the asymmetry of DM compartments is robust, based on the functionally distinct yet closely positioned cell populations of the DM, and the timing of the midline in relation to the establishment of these asymmetric compartments. Notably, recent research conducted in our laboratory has highlighted the vital necessity of maintaining the separation of diffusible signaling molecules, such as Bmp4, from these neighboring cell populations, which would otherwise be in direct contact if not for the presence of the midline basement membrane (Sanketi et al., 2022). We will continue developing specific methods to perturb the midline in preparation of a revised manuscript.
