Biology subjects

Stephen, D. N.

Publications and source records attributed to Stephen, D. N..

4 recordsLinked to original sources

bioRxiv2025

Transcription factors SP5 and SP8 drive primary cilia formation

While specific transcription factors are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multi-omics comparison of the transcriptomes of ciliated and non-ciliated embryonic cells to identify transcription factors upregulated in ciliated cells, and conditional genetics in mouse embryos and stem cells to demonstrate that SP5/8 regulate cilia formation and gene expression. In Sp5/8 mutant embryos primary and motile cilia are shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 is sufficient to induce primary cilia in unciliated cells. This work opens new avenues for studying cilia assembly using stem cell models and offers new insights into human ciliopathies.

developmental biology↗

bioRxiv2023

Engrailed transcription factors direct excitatory cerebellar neuron diversity and survival

The excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the cerebellar circuit. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the eCNm using scRNA-seq and spatial expression analysis and show they evolve during embryogenesis to resemble the adult. Furthermore, the eCNm is transcriptionally divergent from the rest of the eCN by E14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to death of a subset of embryonic eCNm. We demonstrate that mutation of En1/2 in embryonic eCNm results in cell death of specific posterior eCNm molecular subdomains and loss of TBR2 (EOMES) expression in an anterior subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the two other cerebellar excitatory neuron types. Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.

developmental biology↗

bioRxiv2020

ASCL1 drives induction of a transitory cell state required for repair of the injured neonatal brain

The underlying molecular foundation of neural progenitor diversity and plasticity is critical for understanding repair processes. The neonatal cerebellum with multiple progenitor populations has high regenerative potential. Upon ablation of cerebellar granule cell progenitors at birth, a normally gliogenic Nestin-expressing progenitor (NEP) in the Bergmann glia (Bg) layer (BgL-NEPs) undergoes adaptive reprograming to restore granule neuron production while a white matter NEP (WM-NEPs) reduces interneuron production. However, the cellular states and genes regulating the NEP fate switch are not known. Here using scRNA-seq and fate-mapping, we defined the molecular subtypes of NEPs and their lineages under homeostasis and repair. Five NEP populations comprising two molecular subtypes, Hopx-expressing gliogenic- and Ascl1-expressing neurogenic-NEPs were identified in both states. Furthermore, in the WM, distinct NEP populations generate interneurons or astrocytes, and amongst gliogenic-NEPs, astrocyte and Bg lineages are molecularly separable. Importantly, we uncovered that after injury a new transitory cellular state arises from Hopx-NEPs in the BgL that is defined by initiation of expression of the neurogenic gene Ascl1. Moreover, Ascl1 is required for adaptive reprogramming and the full regenerative capacity of the cerebellum. We thus define new populations of NEPs and identifed the transcription factor responsible for inducing a transitory cell critical for a glial to neural switch in vivo following injury.

developmental biology↗

bioRxiv2018

Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors

BackgroundThe cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known.\n\nMethodsWe used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atohl-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion.\n\nResultsMosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved.\n\nConclusionWe demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of conditional mutants affecting GCPs.

developmental biology↗