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Combinatorial interactions of Hox genes establish appendage diversity of the amphipod crustacean Parhyale hawaiensis

Erin Jarvis Alberstat, Kevin Chung, Dennis A Sun, Shagnik Ray, Nipam H. Patel

Preprint posted on 27 March 2022 https://www.biorxiv.org/content/10.1101/2022.03.25.485717v1

and

Distinct regulation of Hox genes by Polycomb Group genes in a crustacean

Dennis A Sun, Yuri Takahashi, Rebecca J Chang, Nipam H Patel

Preprint posted on 28 March 2022 https://www.biorxiv.org/content/10.1101/2022.03.27.485719v1

A double feature on crustacean Hox regulation

Selected by Olivia Tidswell

Background

Hox genes act as master regulators of anterior-to-posterior (AP) axial identity in nearly all animals. Precise spatial and temporal regulation of these genes during embryogenesis is crucial for establishing a normal body plan. The regulation of Hox genes is tightly linked to their genomic organisation; they tend to be clustered in the genome, and the order of Hox genes in a cluster mirrors their order of expression along the AP axis (a phenomenon known as “spatial collinearity”). .

Hox gene regulation has perhaps been best characterised in the animal in which Hox genes were first discovered, Drosophila.  In Drosophila, Hox gene expression boundaries are initially established by genes of the segmentation cascade. They are then reinforced and maintained by Polycomb group (PcG) and Trithorax group (TrxG) genes, which introduce and maintain chromatin modifications, and by cross-regulation between Hox genes. Whether these phases of regulation are representative of the arthropods in general, or are specific to some insects (including Drosophila), remains unclear.

The two preprints I have chosen to highlight here reveal aspects of Hox gene regulation in a non-insect arthropod, the amphipod crustacean Parhyale hawaiensis. Like other crustaceans, Parhyale sports one pair of appendages per segment – this makes it quick and easy to spot homeotic phenotypes using appendage morphology as a readout. The first preprint, by Alberstat et al., focuses on cross-regulation between Hox genes, while the second preprint by Sun et al. focuses on the regulation of Hox genes by PcG and TrxG chromatin modifying enzymes. Both preprints hail from Nipam Patel’s lab, and use a combination of CRISPR/Cas9 mutagenesis and in situ hybridisation to infer regulatory interactions.

Key Findings

Preprint 1 – Posterior prevalence and the Hox code

The first preprint, by Alberstat et al., focuses primarily on cross-regulation of the posterior Hox genes, Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B). These genes are encoded and expressed at the posterior of the Hox cluster and embryo, respectively. In Drosophila, these genes display a property known as posterior prevalence; posterior genes are phenotypically dominant to more anterior genes. This dominance can emerge through transcriptional or post-transcriptional suppression. By knocking down each of the posterior Hox genes individually and in combination with each other, Alberstat et al. revealed that the situation in Paryhale is a little more complex.

Some Parhyale Hox genes do interact in a manner consistent with the posterior prevalence model. The authors found that Ubx is transcriptionally repressed by the most posterior gene of the cluster, Abd-B, and that Ubx is functionally repressed by its posterior neighbour, abd-A, although this is repression is not at the transcriptional or translational level. They also showed that Ubx, abd-A and Abd-B are all capable of repressing the more anterior Hox gene Sex combs reduced (Scr). However, abd-A does not appear to be repressed transcriptionally or otherwise by any of the other posterior Hox genes. In fact, it is co-expressed with its posterior neighbour, Abd-B, in the swimming appendages, where the two genes act co-operatively to specify appendage identity. Furthermore, abd-A seems to have a dose-dependent effect on appendage identity, with different levels of expression generating different appendage types.

In summary, cross-regulation between Hox genes appears to be essential for normal axial patterning in Parhyale, but does not entirely align with a simple posterior prevalence model.  The authors suggest that Parhyale utilises a “Hox code” to pattern its appendages, wherein both the dosage and combination of Hox genes present determine appendage fate. If conserved more broadly amongst the crustaceans, this model might help to explain the huge diversity of appendage types in this lineage.

 

Figure 1. The combinations and dosages of Hox genes proposed to code for each appendage type in Parhyale. Adapted from Figure 9B in the preprint by Alberstat et al.

Preprint 2 – Polycomb in Parhyale

In the second preprint, by Sun et al., the authors focus on the role of Polycomb Group (PcG) and Trithorax Group (TrxG) genes in regulating Parhyale Hox genes. They found that the genome of Parhyale contains homologs of all core PcG and TrxG genes known in Drosophila. As observed in other animals, knocking out PcG genes generated homeotic transformations and misexpression of Hox genes. They also observed a number of non-homeotic defects in limb patterning, cuticle deposition, and plate formation, suggesting that PcG genes play pleiotropic roles in Parhyale development. Surprisingly, TrxG knockouts did not display any homeotic transformations. It may be that TrxG genes play a more subtle role in regulating Hox gene expression in Parhyale than in Drosophila.

Sun et al. also demonstrate that the interactions between individual PcG and Hox genes are highly gene- and tissue-dependent in Parhyale. Knocking out different PcG genes resulted in different degrees of de-repression for different Hox genes, in a tissue-specific manner. For example, the PcG gene Sce seems to repress Ubx strongly in the nervous system and appendages; abd-A weakly in the nervous system, but not appendages; and Abd-B strongly in the nervous system and very weakly in the appendages. The timing of de-repression also varies between Hox genes – for example, Ubx is initially expressed normally in PcG knockouts, and only subsequently becomes ectopically expressed, while ectopic expression of Abd-B is observed at the same time as the wild-type domain is established.

In summary, Sun et al. show that PcG, but not necessarily TrxG, genes are required for Hox regulation in Parhyale. However, the interactions between PcG and Hox genes are not one-size fits all, and depend on the individual PcG gene and Hox gene in question, and the tissue where they are expressed.

Why I think these two preprints are important

Our understanding of Hox gene regulation is dominated by research from a very small number of model organisms – in particular, Drosophila and mice. By developing detailed models of Hox gene regulation in other organisms, we can make inferences about how it has evolved over time and how this influences axial patterning.

Based on these preprints, it seems likely that the three broad phases of Hox gene regulation identified in Drosophila – PcG-independent anterior boundary establishment, PcG-dependent boundary maintenance, and Hox cross regulation – are conserved in Parhyale, and were a common feature of the ancestor of insects and crustaceans. However, both preprints highlight complexities in regulation that might help Parhyale to fine tune its Hox gene expression and generate its diverse complement of appendages.

Tags: crustacean, hox, hox regulation, parhyale, polycomb, trithorax

Posted on: 14 April 2022

doi: https://doi.org/10.1242/prelights.31822

(1 votes)

Author's response

Nipam Patel shared about Combinatorial interactions of Hox genes establish appendage diversity of the amphipod crustacean Parhyale hawaiensis

Your triple Hox knockouts are really fantastic! Are there efforts underway to do similar analyses for the anterior Hox genes? If so, which interactions are you most intrigued to investigate?

We would like to extend these studies, with double and triple knockouts combined with Hox gene expression analysis, to the more anterior Hox genes, particularly to further refine the functions and interactions of Scr, Antp, and Ubx.  One challenge is that these more anterior appendages are more difficult to image and analyze due to their small size and position tucked into the head.

You note that abd-A has been also found to act co-operatively with Ubx and Abd-B to specify regional identity in other arthropod species. Do you think that abd-A shows an unusual propensity for co-operating with other Hox genes to specify identity, and why might this be?

abd-A does seem to hold the key to several interesting transformations during evolution.  It appears that both changes in abd-A’s expression domain, and “escape” from posterior prevalence, have been key to several transitions in arthropod body plans, and may also be key to the origin of early insects without abdominal limbs.  We are quite interested in pursuing many of the unique properties of abd-A in Parhyale and other arthropods to understand its special role.

and

Dennis Sun shared about Distinct regulation of Hox genes by Polycomb Group genes in a crustacean

In your preprint, you reveal that different Parhyale PcG proteins have varying levels of repressive ability depending on the tissue they are expressed in and the Hox gene they are targeting. Do you think that this level of specificity is likely to be unique to crustaceans, or more generally representative of arthropods? In particular, to what extent to Drosophila PcG genes seem to act in a tissue- or Hox gene-specific manner?

The original work on Drosophila Polycomb genes showed that there were differences in the effects of individual Polycomb genes on the expression of each Hox gene, although the precise differences in tissue or Hox specificity seem to have only been lightly investigated. We now have more advanced techniques for visualizing gene expression at higher resolution, such as reporter lines or fluorescent in situ hybridization. It would be great to revisit the Hox misexpression phenotypes of PcG mutations in Drosophila and to explore their function in other genetically tractable insects such as Tribolium. I think such studies would likely reveal that insect PcG genes also have tissue- and Hox-specific mechanisms. It’s likely that this specificity is ancestral to pancrustaceans and understanding the ancestral mechanisms of this specificity could provide insight into the evolution of the insect Hox cluster.

Significantly, your findings suggest that PcG genes are not required for the initial establishment of Hox gene boundaries, as is the case in insects. In insects, this phase of regulation is instead driven by segmentation genes, most prominently the gap genes – none of which appear to be expressed at the right time for this role in Parhyale. Do you have any theories as to which genes might be responsible for establishing early Hox boundaries in Parhyale?

We have some preliminary data that a few Parhyale orthologs of some gap genes play a role in segmentation and Hox regulation. We expect that those genes, as well as other genes found to have gap gene-like activity in non-Drosophila insects (such as nubbin in Tribolium), would be good candidates for the factors involved in the PcG-independent boundary formation in Parhyale. As Parhyale is quite phylogenetically distant from insects, it would also be important to identify candidates in a more unbiased fashion, such as by using single-cell or spatial sequencing of early developmental stages to identify regionally-expressed factors that might contribute to this pre-PcG phase of Hox regulation. Identifying such factors would also provide useful insights into how the Hox regulatory mechanisms of insects and crustaceans may have diverged.

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