Morphogenesis of the Drosophila antennal auditory structure in the pupa, with a focus on the connection between the sensory organ and the exoskeleton

Technical Summary: Morphogenesis of the Drosophila Antennal Auditory Structure

Problem Statement
In Drosophila melanogaster, hearing relies on the Johnston's Organ (JO), a mechanosensory array located in the second antennal segment (a2). The JO detects sound-induced vibrations of the third antennal segment (a3) via the rotation of the a2-a3 joint. While the functional importance of the a2-a3 joint and its connection to the JO is well-established, and genetic mutations (e.g., atonal, spalt, cut) known to disrupt these structures cause deafness, the morphogenetic processes occurring during the intervening pupal stage remain largely unknown. Specifically, the mechanisms governing the formation of the elaborate joint structure, the invagination of the epidermis, and the establishment of the physical connection between the sensory organ and the exoskeleton prior to cuticle deposition have not been characterized.

Methodology
The study utilized Drosophila pupae to track antenna development over a period of approximately 5 days. Key methodological approaches included:

• Genetic Drivers and Markers: The authors employed specific GAL4 drivers (e.g., Dll-GAL4 for general epidermis, pros-GAL4 for scolopale cells, bib-GAL4, fng-GAL4, and neur-GAL4 for Notch signaling targets) to express membrane-targeted fluorescent proteins (GFP, RFP, mCherry).
• Structural Visualization: The dendritic cap (extracellular matrix connecting neurons to the cuticle) was visualized using a nompA-GFP transgene. Actin-rich structures (scolopale rods and cap rods) and the epidermal apical cortex were stained with phalloidin. Chitin deposition was visualized using Calcofluor White.
• Imaging and Analysis: Confocal microscopy (Olympus FV-3000) was used to capture 3D images of dissected pupal heads. Dendritic cap lengths were quantified using the Simple Neurite Tracer (SNT) plugin in ImageJ.
• Functional Assays: To assess gene function, bib was knocked down via RNAi driven by bib-GAL4.

Key Results

1. Epidermal Morphogenesis of the a2-a3 Joint:

   • The antennal primordium undergoes deformation starting around 12 hours after puparium formation (APF).
   • The a2-a3 joint initially appears as a simple epithelial constriction. Between 23 and 38 hours APF, the joint invaginates dorsally into the a2 segment. This invagination is asymmetric, with the lateral region invaginating deeper than the medial region.
   • By 54 hours APF, the joint forms a ring-like structure with an inner diameter of approximately 10 µm.
   • Cuticle formation (chitin deposition) does not begin until approximately 54 hours APF. Prior to this, at 47 hours APF, chitin is only detectable on external bristles, not within the joint itself.

2. Development of the Johnston's Organ (JO) and Attachment:

   • Scolopale cells (visualized via pros-GAL4) develop actin-rich protrusions containing scolopale rods as early as 10 hours APF.
   • The dendritic cap, marked by GFP-NompA, is initially intracellular but begins to extend extracellularly by 14 hours APF.
   • By 21 hours APF, the tips of the dendritic caps reach the apical actin network of the epidermis, establishing contact with the a2-a3 joint epidermis well before cuticle formation.
   • A period of rapid elongation of the dendritic cap occurs between 38 and 44 hours APF.
   • The study observed that in GFP-NompA flies, a subset of scolopidia detach from the epidermis starting at 27 hours APF, a phenomenon likely caused by the interference of GFP with NompA-mediated adhesion.

3. Gene Expression in the Joint Epidermis:

   • The study screened for Notch signaling target genes expressed in the a2-a3 joint epidermis.
   • big brain (bib), a target of Notch signaling, was found to be expressed in a circular band of epidermal cells distal to the JO at 12–17 hours APF. Later (17–19 hours APF), expression expanded to both the distal and proximal sides of the epidermal invagination, remaining restricted to the epidermis and excluding the JO.
   • fringe (fng) expression was observed in the a2 epidermis but excluded from the joint regions.
   • neuralized (neur) was expressed in JO neurons but not in the epidermis, contrasting with its expression pattern in leg joints.
   • Knockdown of bib via RNAi did not result in apparent morphological abnormalities in the a2-a3 joint or JO under the conditions tested.

Significance and Claims
The paper provides the first detailed temporal and spatial description of the morphogenetic processes that establish the Drosophila auditory structure during the pupal stage. The authors claim that their findings clarify:

• The sequence of epidermal invagination that forms the a2-a3 joint.
• The timing of the physical connection between the sensory organ (JO) and the epidermis, which occurs prior to cuticle deposition.
• The rapid elongation dynamics of the dendritic cap.

The authors suggest that the JO may actively influence the epidermal invagination, potentially through mechanical pulling or biochemical signaling, as the dendritic cap tips remain precisely positioned at the deepest point of the invagination. Conversely, they propose that the a2-a3 joint epidermis is crucial for the maintenance of the JO, as scolopidia require anchorage to the epidermis before the cuticle forms. The identification of bib expression in the joint epidermis offers a starting point for exploring the genetic pathways regulating the JO-exoskeleton connection, though the specific functional role of bib in this context requires further investigation. The study establishes a morphogenetic baseline against which the defects caused by known hearing-related mutations (such as ato, ct, and salm) can be better understood.