Jari Berkhout¹, Peter Burbach², Roger Adan², Ahmed Mahfouz³, Onno Meijer⁴, 

(1) Leiden University Medical Center, Leiden, Netherlands. (2) University Medical Center Utrecht, Utrecht, Netherlands.

Introduction:

The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are closely related neurohormonal peptides. AVP regulates arterial blood pressure and water homeostasis, while the best-known function of OXT is the induction of parturition and lactation. AVP and OXT are produced in magnocellular neurons (MCNs) in the hypothalamus. Development of both neuron types necessitates expression of transcription factors (TFs) OTP, SIM1, ARNT2 and POU3F2. However, the presumed TF(s) involved in the divergence of the AVP⁺ and OXT⁺ lineages have not been described as of yet.

Methods:

We used the developmental single-cell dataset from Romanov et al. (2020), comprising of mouse hypothalamus tissue at several developmental timepoints. To understand the gene regulatory processes leading to final cell type differentiation, we used pySCENIC on these developmental clusters. We then applied the CellRank 2 pipeline, utilizing RNA velocity, pseudotime, and optimal-transport analysis to infer cell fate probabilities, and subsequently visualize expression dynamics.

Results:

We identified RORA, EBF3, FOXP1, FOXP2, and BCL11B as TFs with possible relevance for AVP⁺ and OXT⁺ MCN divergence. Expression dynamics show enrichment of EBF3 and BCL11B in the Avp⁺ lineage, while FOXP1 and FOXP2 are enriched in the Oxt⁺lineage. The expression dynamics of RORA are unclear, and incongruent with RORA regulon activity.

Discussion:

Several candidate TFs – both novel and previously proposed – were found for divergence of AVP⁺ and OXT⁺ lineages. Due to the in-silico nature of our analysis, further experimental work is warranted to validate our candidate TFs.