Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression

Highlights

• •Glucocorticoids increase PAX6⁺EOMES⁺ basal progenitors and upper-layer neurons
• •ZBTB16 is necessary and sufficient for the glucocorticoid effects on neurogenesis
• •ZBTB16 activates gyrified species-enriched processes in a lissencephalic cortex
• •Glucocorticoid excess during neurogenesis relates to beneficial postnatal outcomes

Summary

Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to study the cell-type-specific effects of glucocorticoids on neurogenesis. We show that glucocorticoids increase a specific type of basal progenitors (co-expressing PAX6 and EOMES) that has been shown to contribute to cortical expansion in gyrified species. This effect is mediated via the transcription factor ZBTB16 and leads to increased production of neurons. A phenome-wide Mendelian randomization analysis of an enhancer variant that moderates glucocorticoid-induced ZBTB16 levels reveals causal relationships with higher educational attainment and altered brain structure. The relationship with postnatal cognition is also supported by data from a prospective pregnancy cohort study. This work provides a cellular and molecular pathway for the effects of glucocorticoids on human neurogenesis that relates to lasting postnatal phenotypes.

Introduction

Prenatal development affects postnatal health. The “developmental origin of health and disease (DOHaD) hypothesis”¹ proposes that environmental exposures during critical prenatal periods have lasting effects on cells and tissues, including on the central nervous system (CNS),² impacting lifelong human health.

Glucocorticoids (GCs) are steroid hormones that play a vital role in CNS development during pregnancy.³ GCs regulate fetal organ development, particularly for the lungs and brain.⁴ Their levels are very tightly regulated during gestation in a species-specific pattern. Corticosterone, the main GC in mice, increases sharply late in gestation, whereas the levels of cortisol, the main GC in humans, rise progressively starting at the beginning of the second trimester.⁵

GC levels outside the physiological range and/or time window, resulting from the therapeutic use of synthetic GCs (sGCs) or from maternal endocrine and stress-related disorders, impact fetal development.³ The placenta acts as a protective barrier for endogenous maternal GCs, but stress and depression can reduce placental cortisol metabolism.⁶ Furthermore, sGCs readily cross the placenta,⁷ leading to higher exposure of the fetus. sGCs, either betamethasone or dexamethasone (dex), are most commonly prescribed from 22 to 33 gestational weeks (GWs) in pregnancies at high risk for preterm delivery to facilitate fetal lung maturation, increasing survival rates.^8,9 More than 1 in 10 babies are born prematurely every year, a number that amounts to ∼15 million preterm births (<GW37),¹⁰ of which ∼615,000 are born extremely preterm (<GW28),¹¹ highlighting the clinical and societal importance of prenatal sGC use.

Deviation from the physiological range of prenatal GCs can have lasting postnatal effects on brain structure and behavior, as seen in large epidemiological studies,¹² and data from animal models support direct GC effects.³ In fact, although the molecular and cellular effects of GCs on the term and adult brain are well characterized in rodents,¹³ their impact on early brain development, especially during the human neurogenic period (extending until GW28¹⁴ in humans and thus in the time frame of sGC administration for extremely preterm births), remains largely unexplored.

To address this, we combined experiments in human cerebral organoids (hCOs) and mouse embryos with human genetic analyses and mechanistically linked enhanced prenatal sGC exposure to human cortical neurogenesis and lasting effects on cognitive abilities and brain structure.

Results

GCs increase the number of basal progenitors

To study GC regulation of neurogenic trajectories in the neocortex, we treated hCOs¹⁵ with 100 nM dex for 7 days, a dose and time consistent with therapeutic guidelines followed in clinical settings⁸ (see STAR Methods). This treatment was initiated at day 43 (Figure 1A) in hCOs derived from two independent induced pluripotent stem cell (iPSC) lines, HPS0076 and No.1. Days 40–50 were chosen as a time range for when hCOs are actively performing neurogenic processes with all the progenitor cell types present while the birth of deep-layer neurons is peaking and the birth of upper-layer neurons has started.¹⁶ First, we analyzed the effects of dex on different progenitor cell types defined by the expression of paired box 6 (PAX6) and eomesodermin (EOMES, also known as T-box brain protein 2 or TBR2). PAX6 is highly expressed in radial glia (RG) cells; EOMES, but not PAX6, is expressed in intermediate basal progenitors (IPs); whereas both can be expressed in certain basal progenitors (BPs). Dex consistently led to a significant increase of PAX6⁺EOMES⁺ BPs (Figures 1B and 1C) in hCOs derived from both iPSC lines. These PAX6⁺EOMES⁺ cells were localized at the basal side of the germinal zones in the subventricular-like zone (SVZ; Figures 1D, S1A, and S1B). Moreover, we confirmed these effects of dex by analyzing the number of progenitor subtypes in No.1 hCOs with flow cytometry analysis (FCa). We observed a significant increase (+11%) in PAX6⁺EOMES⁺ BPs when hCOs were treated with dex compared with vehicle (veh; Figures S2A–S2C). Co-administration of the GC receptor (GR) antagonist RU486 supported that dex effects are mainly mediated by the GR and not the mineralocorticoid receptor (Figures S2D–S2F). Furthermore, the increased numbers of these BPs seem to contribute to germinal layer expansion as seen by increased PAX6⁺ zone thickness in dex-treated ventricles (Figure S1C).