Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

BACKGROUND: Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. RESULTS: We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. CONCLUSIONS: The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Therian origin of INSL3/RXFP2-driven testicular descent in mammals.

Introduction: During early development in most male mammals the testes move from a position near the kidneys through the abdomen to eventually reside in the scrotum. The transabdominal phase of this migration is driven by insulin-like peptide 3 (INSL3) which stimulates growth of the gubernaculum, a key ligament connecting the testes with the abdominal wall. While all marsupials, except the marsupial mole (Notoryctes typhlops), have a scrotum and fully descended testes, it is unclear if INSL3 drives this process in marsupials especially given that marsupials have a different mechanism of scrotum determination and position relative to the phallus compared to eutherian mammals. Methods: To understand if INSL3 plays a role in marsupial testicular descent we have sequenced and curated the INSL3 gene and its receptor (RXFP2) in a range of marsupials representing every order. Furthermore, we looked at single cell RNA-seq and qPCR analysis of INSL3 in the fat-tailed dunnart testis (Sminthopsis crassicaudata) to understand the location and timing of expression during development. Results: These data show a strong phylogenetic similarity between marsupial and eutherian orthologues, but not with monotreme INSL3s which were more similar to the ancestral RLN3 gene. We have also shown the genomic location of INSL3, and surrounding genes is conserved in a range of marsupials and eutherians. Single cell RNA-seq and qPCR data show that INSL3 mRNA is expressed specifically in Leydig cells and expressed at higher levels during the testicular descent phase in developing marsupials. Discussion: Together, these data argue strongly for a therian origin of INSL3 mediated testicular descent in mammals and suggests that a coordinated movement of the testes to the abdominal wall may have preceded externalization in marsupials and therian mammals.