SHH signaling gradients tune hypothalamic nuclei identity in human organoids Open Access

Abstract

The hypothalamus is a critical region that integrates hormonal, metabolic, and neuronal signals to regulate the autonomic nervous system. Key nuclei within the hypothalamus, including ARC, PVN, and VMH, form interconnected circuits that coordinate physiological and behavioral responses to nutritional state. Despite their central role in metabolic regulation, the developmental mechanisms that generate distinct hypothalamic nuclei identities remain poorly understood. Here, we establish a human hypothalamic organoid platform capable of generating distinct hypothalamic nuclei identities and assembling them into multi-domain structures. Quantitative transcriptional analysis revealed that modulation of SHH pathway activity shifts hypothalamic marker expression, generating gene expression profiles associated with dorsal and ventral hypothalamic domains. These findings indicate that SHH signaling gradients function as tunable morphogen cues that direct hypothalamic nuclei specification in vitro. To further investigate interactions between hypothalamic domains, organoids patterned under distinct SHH signaling conditions were assembled into hypothalamic assembloids, revealing that the assembled organoids maintained clear spatial boundaries while remaining integrated within a shared structure. The persistence of these nuclei-like domains suggests that early patterning signals establish stable hypothalamic identities prior to assembly. Together, this study demonstrates that morphogen-dependent patterning can generate distinct hypothalamic nuclei identities in human organoids and provides a tractable in vitro platform for investigating human hypothalamic development, feeding-related neural circuitry, and the molecular mechanisms that govern nuclei-specific identity and connectivity.

Table of Contents

1 Abstract 1

2 Introduction 2

3 Results 4

3.1 Generation of ventral and dorsal hypothalamic organoids 4

3.2 SHH signaling gradients tune hypothalamic nuclei identity in human organoids 6

3.3 Assembly of differently patterned hypothalamic organoids generates nuclei-like boundaries 9

4 Discussion 11

5 Methods 13

5.1 Human iPSC maintenance 13

5.2 Generation of human cortical organoids (HCO) 14

5.3 Generation of ventral and dorsal hypothalamic spheroids (vhThS and dhThS) 14

5.4 qPCR analysis 15

5.5 Bulk RNA sequencing and transcriptomic analysis 16

5.6 Immunohistochemistry 16

5.7 96-well plate organoids and SHH gradient modulation experiment 17

5.8 Viral labeling and assembloid generation 17

6 Reference 19

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular Biology, Neuroscience
Keyword	Organoid Sonic-hedgehog signaling pathway Assembloid Hypothalamus
Committee Chair / Thesis Advisor	Fikri Birey, Emory University
Committee Members	Arri Eisen, Emory University Matthew Weinschenk, Emory University Jimena Andersen, Emory University

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