A new look at the architecture and dynamics of the Hydra nerve net

Reviewer #1 (Public Review):

This work presents findings on the cellular and ultrastructural organization of the nervous system in the freshwater polyp Hydra. Although the work presents potentially important data, there are several points that need to be addressed:

1. The antibody has to be properly validated as a tool for detecting all neurons. As it stands, the antibody might not recognize a cadherin and it is not clear whether it is specific and labels all neurons.

2. The lack of communication between the two nerve nets is an interesting observation, but its implications are limited due to technical reasons. This should be investigated further.

3. The apparent lack of typical terminal synaptic contacts and the predominant presence of "en passant" contacts in the neurite bundles could be the central element of the paper but this would have to be supported by more thorough observations and experiments.

4. The authors should highlight the novelty of the findings as compared to previous work that had already addressed some of these points.

Reviewer #2 (Public Review):

In their manuscript, Keramidioti and co-authors investigate the cellular architecture of the nervous system in the freshwater polyp Hydra. Specifically, the authors attempt to improve the resolution, which is lacking in the previous studies, yet to generate a comprehensive overview of the entire nervous system's spatial organization and to infer communication between cells. To this end, Keramidioti et al. use state-of-the-art imaging approaches, such as confocal microscopy combined with the use of transgenic animals, transmission electron microscopy, and block face scanning electron microscopy. The authors present three major observations: i) A novel hyCADab antibody may be used to detect the entire nervous system of Hydra; ii) Nerve cells in the ectoderm and in the endoderm are organized in two separate nerve nets, which do not interact; iii) Both nerve nets are composed of bundles of overlapping nerve processes.

The manuscript addresses a long-standing and currently intensively studied question in developmental neurobiology biology - it attempts to reveal structural properties and principles that govern the function of the nervous systems in non-bilaterian animals. Hence, this study contributes to understanding the nervous system evolution trajectories. Therefore, the manuscript may represent interest to researchers interested in evolutionary and developmental neurobiology.

The manuscript reports a remarkably meticulous study and presents stunning imaging results. However, the manuscript would benefit from a more thorough presentation of immunochemical and electron microscopy data. The work would also greatly benefit from a more straightforward presentation of truly novel findings and a more concise summary of already-known aspects.

Major comments:

1. The novelty of findings.
   The authors present a lot of findings and illustrate them with numerous very impressive images. However, most observations have been actually reported before, and genuinely novel discoveries are obscured. For instance, the findings on the elongated morphology of the endodermal sensory cell (entire passage starting with "Figure 2B shows..."), qualitative ("Figure 3 shows..."), and quantitative estimation of neuronal densities in the different body compartments of Hydra - all these observations do not provide novel insights. Some co-authors of this manuscript or other authors have previously published all these features. A substantial advance would be performing in vivo experiments, addressing directly, for instance, the question of what is the function of sensory neurons reaching into the gastric cavity. What signals do they detect there? If the authors have access to such functional assays, any additional in vivo experiments will substantially improve the study.

2. The utility of the hyCADab as a pan-neuronal antibody.
   Most of the analysis in the manuscript relies on immunostaining of fixed polyps with a novel polyclonal antibody. The authors claim that this antibody recognizes a neuron-specific cadherin protein of Hydra and stains all neurons in the nerve net. However, a brief search in the publicly available resources (such as the Hydra Genome Portal: https://research.nhgri.nih.gov/HydraAEP/) indicates that the gene encoding a protein with a sequence similar to the epitope used by Keramidioti and co-authors is, in fact, not a neuron-specific. It is strongly expressed in nematocytes. Furthermore, the cytoplasmic staining hyCADab is puzzling. Given that the target Cadherin protein is a membrane-associated protein, one would anticipate the immunochemical signal to be localized on the cell's periphery, under the surface.

The authors compare the density of neurons related to epithelial cells detected in whole mounts by the antibody with counts on macerates. Perhaps, a more direct and accurate approach would be to stain macerates with the antibody. In this way, one would be able to identify neurons by their morphology and validate whether 100% of them are hyCADab-positive.

The nGreen strain used by the authors is a mosaic one (see Materials and Methods). Hence, not all neurons are, in fact, labeled by GFP. Therefore, the argument that 51/51 GFP-positive cells are also hyCADab-positive is not convincing and insufficient to claim that hyCADab is a pan-neuronal antibody.

Finally, it is truly surprising that transgenic GFP-positive neurons are, in most cases, hyCADab-negative. (It is particularly evident in Fig. 11B. If the hyCADab antibody is indeed a pan-neuronal one, the red signal in the transgenic neurons should be as high as in the surrounding cells, and the cells would appear yellow).

1. The apparent absence of contact between the ectodermal and endodermal nerve nets.
   A central claim of the manuscript is that there are no contacts between the nervous networks in the ectoderm and the endoderm. Therefore, the activities of these networks appear to be not coordinated. In support of these claims, the authors provide images of sections from the polyps' body column (Fig. 4). However, the mesoglea itself is not visible in these images.

Another limitation of the study by Keramidioti and co-authors is that they investigate sections only from the gastric region of a polyp. Earlier studies (for instance, Westfall, 1973) using TEM provided compelling evidence for communication between the ectodermal and endodermal nerve networks via neurites that cross the mesoglea. These neurites traversing mesoglea have been detected specifically in the hypostome of Hydra - the region not thoroughly investigated by Keramidioti et al. It is also surprising that transmesogleal bridges between ectodermal and endodermal epithelial cells, abundantly present not only in the hypostome but in the body column as well, can not be detected on any of the images provided by the authors. This suggests that their approach overall might be in general not suitable for addressing the question of connection and communication between the ectodermal and endodermal structures.

1. Formation of neurite bundles
   The most intriguing finding of the study by Keramidioti et al. is that neurites of nerve cells often run parallel to each other, forming conspicuous bundles in both ectodermal and endodermal nerve nets. The formation of such bundles per se is not surprising. It has already been documented by Takahashi-Iwanaga et al.,1994 (this study definitely did not escape the authors' attention) in Hydra's body column. Moreover, neurite bundles have been previously described in the hypostomes of other Hydra species (e.g., Davis et al., 1968; Grimmelikhuijzen, 1985; Yaross et al., 1986) and in other cnidarians (e.g., Mackie 1973, 1989; Garm et al., 2007). Hence, this appears to be a common, universal principle of the nervous system architecture in Cnidaria. I agree with the authors that such an organization of the nerve net is surprising and contrasts the neuronal architecture of most Bilateria. Could these observations, taken together, lead to a view of an alternative design of a nerve system? (a recently published description of the syncytial nerve net in Ctenophora is another revolutionary example of a nervous system architecture). The authors might compare the organization of the Hydra nerve plexus with the architecture of the vertebrate enteric nervous system - where bundles of neurites are also highly abundant, stimulating some thoughts on the evolutionary roots of the peripheral NS.

Another aspect worth discussing in this context is whether the nerve system of Hydra can be organized in any other way. Given the architecture of epithelia in Hydra, there's virtually no other way for the neurites to run other than to form bundles - they occupy the narrow spaces between the epithelial cells and between their muscular fibers. The growth of the neurites thus appears constrained.

Finally, the functional implications of such bundle formation appear extremely interesting. Do neurons really form contacts in these bundles? Unfortunately, the authors provide no evidence for synaptic contacts within the bundles. This is somehow surprising given that numerous studies have effectively localized chemical and electric synapses in Hydra cells (e.g., Westfall et al., 1971). Overlapping of neurites may suggest an alternative, non-synaptic mechanism of signal propagation - via ephaptic coupling. It would be beneficial if the authors provided more TEM data on the presence or absence of synapses between neurites in the body column of Hydra. Some experiments, such as the dye coupling approach, may also help probe the existence of synaptic connections between the neurons forming a bundle.

Reviewer #3 (Public Review):

In this paper by Keramidioti et al, the authors have characterized a polyclonal antibody from rabbit, which was raised against a peptide of the intracellular domain of the Hydra Cadherin. This antibody unexpectedly recognizes presumably all neurons in the Hydra polyp and indeed the specificity of the antibody is not fully convincing. Regardless, the antibody can be used to visualize and study the nerve net under a variety of conditions. The authors find that the endodermal and ectodermal nerve net do not make any contacts through the mesoglea, in contrast to earlier assumptions and data. They show that ectodermal neurons make close contacts to the myoepithelial muscles, in contrast to the endodermal muscles. Furthermore, they show that tentacle endoderm surprisingly does not have any neurons. Finally, a very nice tool to visualize the connections between the neurons is the staining of mosaic nGreen transgenic lines. This showed that the neurites align in parallel forming bundles of neurites over longer stretches, in particular in the ectoderm, which offers a mechanism how new neurons are added laterally to the existing nerve net. This has important implications about the way the neurons might communicate with each other.

Taken together, this paper adds to our knowledge of the Hydra nerve net and provides a new experimental tool. Although most of the study is rather descriptive the pictures are of spectacular quality, providing fascinating new insights into the arrangement and topology of the nerve net.