Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings

Reviewer #1 (Public Review):

In their article, "Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings," Tendolkar and colleagues explore Ubx regulation in butterflies. The authors investigated how Ubx expression is restricted to the hindwing in butterflies through a series of genomic analyses and genetic perturbations. The authors provide evidence that a Topoologiacally Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin around Ubx, largely through an apparent boundary element. CRISPR mutations of this boundary element led to ectopic Ubx expression in forewings, resulting in homeotic transformation in the wings. The authors also explore the results of the mutation in two non-coding RNA regions as well as a possible enhancer module. Each of these induces homeotic phenotypes. Finally, the authors describe a number of homeotic phenotypes in butterflies, which they relate to their work.

Together, this was an interesting paper with compelling initial data. That said, I have several items that I feel would warrant further discussion, presentation, or data.

First, I would not state, "Little is known about how Hox genes are regulated outside of flies." They should add "in insects" since so much in known in vertebrates

For Figure 1, it would aid the readers if the authors could show the number of RNAseq reads across the locus. This would allow the readership to evaluate the frequency of the lncRNAs, splice variants, etc.

How common are boundary elements within introns? Typically, boundary elements are outside gene bodies, so this could be explored further. This seems like an interesting bit of biology which, following from the above point, it would be interesting to, at a minimum, discuss, but also relate to how transcription occurs through a possible boundary element (are there splice variants, for example?).

The CRISPR experiments led to compelling phenotypes. However, as a Drosophila biologist, I found it hard to interpret the data from mosaic experiments. For example, in control experiments, how often do butterflies die? Are there offsite effects? It's striking that single-guide RNAs led to such strong effects. Is this common outside of this system? Is it possible to explore the function effects at the boundary element - are these generating large deletions (for example, like Mazo-Vargas et al., 2022)?

For the mosaic experiments, how frequent are these effects in nature or captive stocks? Would it be possible to resequence these types of effects? At the moment, this data, while compelling, was hard to put into the context of the experiments above without understanding how common the effects are. Ideally, there would be resequencing of these tissues, which could be targeted, but it was not clear to me the general rates of these variants.

In sum, I enjoyed the extensive mosaic perturbations. However, I feel that more molecular descriptions would elevate the work and make a larger impact on the field.

Reviewer #2 (Public Review):

Summary:
The existence of hox gene complexes conserved in animals with bilateral symmetry and in which the genes are arranged along the chromosome in the same order as the structures they specify along the anteroposterior axis of organisms is one of the most spectacular discoveries of recent developmental biology. In brief, homeotic mutations lead to the transformation of a given body segment of the fly into a copy of the next adjacent segment. For the sake of understanding the main observation of this work, it is important to know that in loss-of-function (LOF) alleles, a given segment develops like a copy of the segment immediately anterior to it, and in gain-of-function mutations (GOF), the affected segment develops like a copy of the immediately posterior segment. Over the last 30 years the molecular lesions associated with GOF alleles led to a model where the sequential activation of the hox genes along the chromosome result from the sequential opening of chromosomal domains. Most of these GOF alleles turned out to be deletions of boundary elements (BE) that define the extent of the segment-specific regulatory domains. The fruit fly Drosophila is a highly specialized insect with a very rapid mode of segmentation. Furthermore, the hox clusters in this lineage have split. Given these specificities it is legitimate to question whether the regulatory landscape of the BX-C we know of in D.melanogaster is the result of very high specialization in this lineage, or whether it reflects a more ancestral organization. In this article, the authors address this question by analyzing the continuous hox cluster in butterflies. They focus on the intergenic region between the Antennapedia and the Ubx gene, where the split occurred in D.melanogaster. Hi-C and ATAC-seq data suggest the existence of a boundary element between 2 Topologically-Associated-Domain (TAD) which is also characterized by the presence of CTCF binding sites. Butterflies have 2 pairs of wings originating from T2 (forewing) specified by Antp and T3 specified by Ubx (hindwing). Remarkably, CRISPR mutational perturbation of this boundary leads to the hatching of butterflies with homeotic clones of cells with hindwings identities in the forewing (a posteriorly oriented homeotic transformation). In agreement with this phenotype, the authors observe ectopic expression of Ubx in these clones of cells. In other words, CRISPR mutagenesis of this BE region identified by molecular tool give rise to homeotic transformations directed towards more posterior segment as the boundary mutations that had been 1st identified on the basis of their posterior oriented homeotic transformation in Drosophila. None of the mutant clones they observed affect the hindwing, indicating that their scheme did not affect the nearby Ubx transcription unit. This is reassuring and important first evidence that some of the regulatory paradigms that have been proposed in fruit flies are also at work in the common ancestor to Drosophilae and Lepideptora.

Given the large size of the Ubx transcription unit and its associated regulatory regions it is not surprising that the authors have identified ncRNA that are conserved in 4 species of Nymphalinae butterflies, some of which also present in D.melanogaster. Attempts to target the promoters by CRISPR give rise to clones of cells in both forewings and hindwings, suggesting the generation of regulatory mutations associated with both LOF and GOF transformations. The presence of clones with dual homeosis suggests the targeting of Ubx activator and repression CRMs. Unfortunately, these experiments do not allow us to make further conclusions on the role of these ncRNA or in the identification of specific regulatory elements. To the opinion of this reviewer, some recent papers addressing the role that these ncRNA may play in boundary function should be taken with caution, and evidence that ncRNA(s) regulate boundaries in the BX-C in a WT context is still lacking.

Strengths:
The convincing GOF phenotype resulting from the targeting of the Antp-Ubx_BE.

Weaknesses:
The lack of comparisons with the equivalent phenotypes obtained in D.melanogaster with for example the Fub mutation.