Project Summary/Abstractor
In the retina, various neuronal types are confined to three somatic layers and connected in two plexiform layers.
Defects in neuronal positioning or connections result in vision impairment or blindness. While migration of
neurons to their destined layers is well studied, the influence of segregated nuclear layers on the formation of
neuronal connections remains unclear. This project aims to delineate the transcriptional control of neuronal
position and connection. We use starburst amacrine cells (SACs)—the cholinergic interneurons in the retina—
as a model to address this question. SACs are crucial components of the direction-selective (DS) circuit and
consist of two closely related subtypes. OFF SACs in the inner nuclear layer (INL) establish dendritic
stratifications and connections in the OFF layer (S2) of the inner plexiform layer (IPL), whereas ON SACs in the
ganglion cell layer (GCL) ramify their dendrites in the ON layer (S4) of the IPL. My previous research identified
the transcription factor Fezf1 that is specifically expressed by ON SACs and determines their somatic positions
in the GCL. In the Fezf1 constitutive knockout (Fezf1-/-), ON SACs are mislocalized to the INL alongside OFF
SACs. To overcome the neonatal lethality associated with Fezf1-/-, we generated a new Fezf1 conditional allele
to facilitate the study of postnatal development of SACs. We made three observations: (1) Similar to Fezf1-/-,
early embryonic deletion of Fezf1 results in mislocalization of ON SACs to the GCL, but late deletion allows them
to remain in the GCL (2) In both early and late Fezf1 deletions, SAC dendrites no longer separate into two distinct
layers but remain intermingled. (3) Following altered SAC dendritic stratification, ON and OFF direction-selective
ganglion cells (ooDSGCs) and ON DSGCs redirect their dendritic arbors from making contacts with SACs in S2
and S4 (ooDSGCs) or S4 (ON DSGCs) to contacting S2 alone. These findings collectively support the central
hypothesis that Fezf1 sequentially controls the somatic position, dendritic stratification, and synaptic connections
of SACs. We therefore propose two specific aims. Aim 1 aims to determine how Fezf1 mediates dendritic
stratification of SACs. We will elucidate the morphological changes of developing SACs via image-tracing and
assess the sufficiency of Fezf1 in rescuing the dendritic defects via in utero electroporation. We will determine
the regulatory network of Fezf1 using combined ATAC-seq and RNA-seq assays. Lastly, we will test the role of
repulsive molecules Slit2/Nell2/Robo2 in separating the dendritic arbors between ON and OFF SACs. Aim 2
aims to determine how the altered dendritic stratification of SACs redirects dendritic innervation of ooDSGCs
and ON DSGCs. We will use genetic labeling and image-tracing to determine the specificity of targeting defects
to DSGC types but not other RGC types. We will then use RNA-seq and AAV-mediated genetic manipulation to
determine the molecular mechanism that mediates the dendritic mistargeting of DSGCs. This project will
comprehensively dissect the transcriptional control of neuronal positioning and connection formation of SACs
and generate a new molecular basis for the assembly of the direction-selective circuit.