Dissection of Larval and Pupal Wings of Bicyclus anynana Butterflies

Banerjee, Tirtha Das; Monteiro, Antónia

doi:10.3390/mps3010005

Cited by 9 publications

(9 citation statements)

References 31 publications

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“…Thus, the complex interaction of these multiple genes results in the development of a single vein via a positional information mechanism. A recent study has proposed a similar mechanism to be in play in butterflies as well, where the Spalt expression domain is observed abutting both the dpp and the Optix expression domains (Banerjee and Monteiro, 2020b; Figure 5B ).…”

Section: Discussionmentioning

confidence: 61%

“…Pupation time was recorded using an Olympus tough tg-6 camera, and pupal wings were dissected at different timepoints after pupation under a Zeiss Stemi 305 microscope in 1x PBS at room temperature based on a protocol previously described (Banerjee and Monteiro, 2020b). Wings were fixed using 4% formaldehyde in fix buffer (see Table S3 for details), followed by four washes in 1x PBS, five mins each.…”

Section: Methodsmentioning

confidence: 99%

“…For the visualization of dpp expression in pupal wings of Bicyclus , pupation time was recorded, and the wings were dissected from 20-24 hrs old pupae in 1x PBS under a Zeiss stemi 305 microscope based on a previously described protocol (Banerjee and Monteiro, 2020b) and transferred to 1xPBST with 4% formaldehyde. After fixation for 30 min the wings were washed three times in 1x PBST for 5 mins each.…”

Section: Methodsmentioning

confidence: 99%

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Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

Banerjee

Seah

Monteiro

2021

Preprint

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optix, a gene essential and sufficient for eye development in Drosophila melanogaster, also plays important roles in the development of both the structure and pigmentation of butterfly wing scales. In particular, optix regulates wing scale lower lamina thickness and ommochrome pigment synthesis. Here we explore the role of optix in wing pattern development of Bicyclus anynana butterflies by examining its expression using immunostainings and testing its function via CRISPR-Cas9. We found Optix to be expressed in multiple domains, most prominently in the orange ring of the eyespots and in other scattered orange scales, and to regulate the pigmentation and the development of the upper lamina of the orange scales. We further explored the interaction of Optix with Spalt, a protein involved in the development of black scales in the eyespots, and expressed adjacent to the Optix domain. CRISPR knockouts of optix or spalt, followed by immunostainings, showed that Spalt represses optix expression in cells of the central black region of the eyespot. This regulatory interaction mimics that found in the anterior compartment of the wing disc where both genes respond to Decapentaplegic (Dpp) signaling and play a role in venation patterning. Using in situ hybridizations we show that dpp is expressed in the center of the eyespots and propose that this same circuit might have been recruited for eyespot development where Decapentaplegic acts as a central morphogen, activating optix and spalt at different concentration thresholds, and where spalt cross-regulates optix resulting in the formation of a sharp boundary between the two eyespot color rings.

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Section: Discussionmentioning

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

Banerjee

Seah

Monteiro

2021

Preprint

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“…B. anynana pupal wings at 24 h AP are fragile, translucent membranes with well defined trachae [ 26 ]. After cell dissocation from wing tissues using TrypLE, filtered cells stained with 0.4% trypan blue appear as a mixed population of different sized spherical cells ( Figure 3 ).…”

Section: Expected Resultsmentioning

confidence: 99%

“…A detailed video protocol for the dissection of pupal wings in Bicyclus anynana is available [ 26 ].…”

Section: Methodsmentioning

confidence: 99%

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Prakash

Monteiro

2020

MPs

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Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.

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Distal-lessandspaltare distal organisers of pierid wing patterns

Wee

Banerjee

Prakash

et al. 2021

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Simple spot patterns are proposed to be ancestral to more complex eyespot patterns found on the wings of butterflies. Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in two separate functions in establishing nymphalid butterfly eyespots: in the differentiation of their central signaling cells, or foci, necessary for eyespot ring formation, and in scale melanisation. However, little is known about the functions of these genes in the development of more primitive spot patterns. Here, we study the roles of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. We examined the expression pattern of both genes in developing larval and pupal wings and explored their functions using the CRISPR/Cas9 system. In P. canidia, both Dll and sal are expressed in the tips and along the margin of pupal wings, areas of future scale melanisation. Additionally, sal alone, is expressed in the future black spot patterns. CRISPR knockouts of Dll and sal showed that both genes are required for promoting melanic scales in the areas where they are expressed. Both Dll and sal also function as repressors of the pteridine pigment pathway. We conclude that both genes share a conserved role in promoting scale melanisation, across pierid and nymphalid butterflies, but are unlikely to be involved in differentiating spot centers. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct and independently evolved in each lineage.

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Dissection of Larval and Pupal Wings of Bicyclus anynana Butterflies

Cited by 9 publications

References 31 publications

Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Distal-lessandspaltare distal organisers of pierid wing patterns

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