ABS 103: The Role of Ptx-1 in Midgut and Nervous System Morphogenesis in Drosophila melanogaster

Cailyn J. Lee ¹ , Jeffrey Matthew ¹ , SeYeon Chung ¹

¹ Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

Van Wickle (2025) Volume 1, ABS 103

Introduction: Pituitary homeobox 1 (Ptx-1) is a conserved transcription factor that regulates gene expression and cell functions. It is involved in pituitary development, muscle protein quality control, and lifespan. We use Drosophila as a model to understand its role during tissue morphogenesis. During Drosophila embryogenesis, ptx-1 is highly expressed in several tissues, including the central nervous system (CNS) and the gut. Particularly, ptx-1 shows dynamic expressions in the endoderm and the developing gut. At stage 12, the posterior midgut shows high expression. During stages 13-16, strong expression is maintained in the midgut, with a strong correlation between high ptx-1 expression patterns and constriction sites that form the different gut chambers. These patterns suggest a potential role for ptx-1 in regulating midgut chamber formation.

We first analyzed the dynamic localization of the Ptx-1 protein using a transgenic line expressing the functional fusion protein of Ptx-1 with a green fluorescent protein (GFP) tag inserted into the ptx-1 locus. Analysis of the Ptx-1-GFP line revealed high expression in the CNS and midgut chamber constriction. To test the role of ptx-1 in midgut formation during embryogenesis, we also generated ptx-1 null mutant lines using CRISPR/Cas9, deleting the region containing the open reading frame (ORF). To analyze the ptx-1 null mutant phenotypes, we used immunohistochemistry with markers for the apical domain (Crb), the CNS (BP102), and the peripheral nervous system (22C10). Initial analysis revealed that ptx-1 mutant embryos had an overall normal morphology but showed subtle defects in the gut morphology at stages 16 and 17. Detailed analysis of the mutants is underway. Investigating how ptx-1 regulates midgut morphogenesis will help clarify its role in gut chamber formation and tissue patterning and may provide insights into how conserved transcriptional networks govern tissue development.

Methods: ptx-1 mutants were generated using CRISPR/Cas9 with two gRNAs targeting sites 350 base pairs apart within the ORF, cloned into pCFD4 via Gibson assembly and verified by Sanger sequencing. The pCFD4-gRNA1-2 construct was microinjected into nos-Cas9 embryos (Genentivision), and G0s were crossed to balancer flies. F1 progeny were screened for deletions using PCR and Sanger sequencing. Embryos were fixed in heptane/formaldehyde, devitellinized with methanol, and stained in PBSTB with primary antibodies: anti-Crb, 22C10, BP102, and rabbit anti-β-gal (to mark mutant homozygotes). For chromogenic detection, goat anti-mouse-biotin and DAB/NiCl/H₂O₂ were used; for fluorescence, Alexa 488 and Alexa 568 conjugates were used. Samples were dehydrated through ethanol and methyl salicylate and mounted in AquaPoly/Mount. Images were acquired using a Leica DM2500 microscope (20X/40X, NA 0.8).

Results: We found that loss of ptx-1 does not affect central or peripheral nervous system development, nor gut morphology. Immunostaining with CNS (BP102), PNS (22C10), and apical (Crb) markers revealed no significant differences between ptx-1 null mutants and wild-type embryos. Co-staining with β-gal allowed for clear identification of homozygous mutants. This suggests a potential functional redundancy with other genes.

Discussion: Our findings suggest that while ptx-1 is highly expressed in regions undergoing midgut constriction, it is not essential for CNS, PNS, or gut morphology. This may indicate redundancy with other transcription factors. The generation of CRISPR/Cas9 ptx-1 mutants provides a valuable tool for dissecting its functional domains. Future studies will examine midgut cell cycle status using Fly-FUCCI and identify downstream targets and regulatory mechanisms with ChIP-seq.