Diverse axonal morphologies of individual callosal projection neurons reveal new insights into brain connectivity.

In the mature brain, functionally distinct areas connect to specific targets, mediating network activity required for function. New insights are still occurring regarding how specific connectivity occurs in the developing brain. Decades of work have revealed important insights into the molecular and genetic mechanisms regulating cell type specification in the brain. This work classified long-range projection neurons of the cerebral cortex into three major classes based on their primary target (e.g. subcortical, intracortical, and interhemispheric projections). However, painstaking single-cell mapping reveals that long-range projection neurons of the corpus callosum connect to multiple and overlapping ipsilateral and contralateral targets with often highly branched axons. In addition, their scRNA transcriptomes are highly variable, making it difficult to identify meaningful subclasses. This work has prompted us to reexamine how cortical projection neurons that comprise the corpus callosum are currently classified and how this stunning array of variability might be achieved during development.

An Early Cortical Progenitor-Specific Mechanism Regulates Thalamocortical Innervation.

The cortical subplate is critical in regulating the entry of thalamocortical sensory afferents into the cortex. These afferents reach the subplate at embryonic day (E)15.5 in the mouse, but "wait" for several days, entering the cortical plate postnatally. We report that when transcription factor LHX2 is lost in E11.5 cortical progenitors, which give rise to subplate neurons, thalamocortical afferents display premature, exuberant ingrowth into the E15.5 cortex. Embryonic mutant subplate neurons are correctly positioned below the cortical plate, but they display an altered transcriptome and immature electrophysiological properties during the waiting period. The sensory thalamus in these cortex-specific Lhx2 mutants displays atrophy and by postnatal day (P) 7, sensory innervation to the cortex is nearly eliminated leading to a loss of the somatosensory barrels. Strikingly, these phenotypes do not manifest if LHX2 is lost in postmitotic subplate neurons, and the transcriptomic dysregulation in the subplate resulting from postmitotic loss of LHX2 is vastly distinct from that seen when LHX2 is lost in progenitors. These results demonstrate a mechanism operating in subplate progenitors that has profound consequences on the growth of thalamocortical axons into the cortex.SIGNIFICANCE STATEMENT Thalamocortical nerves carry sensory information from the periphery to the cortex. When they first grow into the embryonic cortex, they "wait" at the subplate, a structure critical for the guidance and eventual connectivity of thalamic axons with their cortical targets. How the properties of subplate neurons are regulated is unclear. We report that transcription factor LHX2 is required in the progenitor "mother" cells of the cortical primordium when they are producing their "daughter" subplate neurons, in order for the thalamocortical pathway to wait at the subplate. Without LHX2 function in subplate progenitors, thalamocortical axons grow past the subplate, entering the cortical plate prematurely. This is followed by their eventual attrition and, consequently, a profound loss of sensory innervation of the mature cortex.

Spatio-Temporal Autophagy Tracking with a Cell-Permeable, Water-Soluble, Peptide-Based, Autophagic Vesicle-Targeted Sensor.

Autophagy is an essential cellular degradation process. Impaired autophagy has been linked to multiple disorders, including cancer and neurodegeneration. Tracking the autophagic flux in living cells will provide mechanistic insights into autophagy and will allow rapid screening of autophagy modulators as potential therapeutics. Imaging autophagy to track the autophagic flux demands a cell-permeable probe that can specifically target autophagic vesicles and report on the extent of autophagy. Existing fluorescent protein-based probes for imaging autophagy target autophagic vesicles but are cell-impermeable and degrade with the progress of autophagy resulting in ambiguous information on the later stages of autophagy. Although small-molecule-based autophagy probes can be cell-permeable, they are mostly water-insoluble and often target lysosomes instead of autophagic vesicles leading to incomplete evidence of the early stages of the process. Hence, there is a major gap in the ability to link the imaging data obtained by applying fluorescent sensors to the real extent of autophagy in living cells. To address these challenges, we have combined the desirable features of targetability and cell permeability to develop a novel water-soluble, cell-permeable, visible-light excitable, peptide-based, fluorescent sensor, HCFP, for imaging autophagy and tracking the autophagic flux. The probe readily enters living cells within 30 min of incubation, distinctly targets autophagic vesicles, and spatio-temporally tracks the entire autophagy pathway in living cells via a ratiometric pH-sensitive detection scheme. The salient features of the probe combining targetability with cell permeability should provide an edge in high-throughput screening of autophagy modulators by tracking autophagy live.

LDB1 Is Required for the Early Development of the Dorsal Telencephalon and the Thalamus.

LIM domain binding protein 1 (LDB1) is a protein cofactor that participates in several multiprotein complexes with transcription factors that regulate mouse forebrain development. Since Ldb1 null mutants display early embryonic lethality, we used a conditional knockout strategy to examine the role of LDB1 in early forebrain development using multiple Cre lines. Loss of Ldb1 from E8.75 using Foxg1Cre caused a disruption of midline boundary structures in the dorsal telencephalon. While this Cre line gave the expected pattern of recombination of the floxed Ldb1 locus, unexpectedly, standard Cre lines that act from embryonic day (E)10.5 (Emx1Cre) and E11.5 (NesCre) did not show efficient or complete recombination in the dorsal telencephalon by E12.5. Intriguingly, this effect was specific to the Ldb1 floxed allele, since three other lines including floxed Ai9 and mTmG reporters, and a floxed Lhx2 line, each displayed the expected spatial patterns of recombination. Furthermore, the incomplete recombination of the floxed Ldb1 locus using NesCre was limited to the dorsal telencephalon, while the ventral telencephalon and the diencephalon displayed the expected loss of Ldb1. This permitted us to examine the requirement for LDB1 in the development of the thalamus in a context wherein the cortex continued to express Ldb1. We report that the somatosensory VB nucleus is profoundly shrunken upon loss of LDB1. Our findings highlight the unusual nature of the Ldb1 locus in terms of recombination efficiency, and also report a novel role for LDB1 during the development of the thalamus.

Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.