The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons.

Asymmetric activation of Dll4-Notch signaling by Foxn4 and proneural factors activates BMP/TGFß signaling to specify V2b interneurons in the spinal cord.

During development of the ventral spinal cord, the V2 interneurons emerge from p2 progenitors and diversify into two major subtypes, V2a and V2b, that play key roles in locomotor coordination. Dll4-mediated Notch activation in a subset of p2 precursors constitutes the crucial first step towards generating neuronal diversity in this domain. The mechanism behind the asymmetric Notch activation and downstream signaling events are, however, unknown at present. We show here that the Ascl1 and Neurog basic helix-loop-helix (bHLH) proneural factors are expressed in a mosaic pattern in p2 progenitors and that Foxn4 is required for setting and maintaining this expression mosaic. By binding directly to a conserved Dll4 enhancer, Foxn4 and Ascl1 activate Dll4 expression, whereas Neurog proteins prevent this effect, thereby resulting in asymmetric activation of Dll4 expression in V2 precursors expressing different combinations of proneural and Foxn4 transcription factors. Lineage tracing using the Cre-LoxP system reveals selective expression of Dll4 in V2a precursors, whereas Dll4 expression is initially excluded from V2b precursors. We provide evidence that BMP/TGFß signaling is activated in V2b precursors and that Dll4-mediated Notch signaling is responsible for this activation. Using a gain-of-function approach and by inhibiting BMP/TGFß signal transduction with pathway antagonists and RNAi knockdown, we further demonstrate that BMP/TGFß signaling is both necessary and sufficient for V2b fate specification. Our data together thus suggest that the mosaic expression of Foxn4 and proneural factors may serve as the trigger to initiate asymmetric Dll4-Notch and subsequent BMP/TGFß signaling events required for neuronal diversity in the V2 domain.

A critical role for sFRP proteins in maintaining caudal neural tube closure in mice via inhibition of BMP signaling.

Both the BMP and Wnt pathways have been implicated in directing aspects of dorsal neural tube closure and cell fate specification. However, the mechanisms that control the diverse responses to these signals are poorly understood. In this study, we provide genetic and functional evidence that the secreted sFRP1 and sFRP2 proteins, which have been primarily implicated as negative regulators of Wnt signaling, can also antagonize BMP signaling in the caudal neural tube and that this function is critical to maintain proper neural tube closure and dorsal cell fate segregation. Our studies thus reveal a novel role for specific sFRP proteins in balancing the response of cells to two critical extracellular signaling pathways.

Wnt signaling inhibitors regulate the transcriptional response to morphogenetic Shh-Gli signaling in the neural tube.

Shh-Gli signaling controls cell fates in the developing ventral neural tube by regulating the patterned expression of transcription factors in neural progenitors. However, the molecular mechanisms that limit target gene responses to specific domains are unclear. Here, we show that Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated by positive Shh-Gli signaling and negative Tcf repressor activity. Our data indicate that the dorsal limit of Nkx2.2 is controlled by Tcf4-mediated transcriptional repression, and not by a direct requirement for high-level Shh-Gli signaling, arguing against a simple model based on differential Gli factor affinities in target genes. These results identify a transcriptional mechanism that integrates graded Shh and Wnt signaling to define progenitor gene expression domains and cell fates in the neural tube.

A Cre transgenic line for studying V2 neuronal lineages and functions in the spinal cord.

During spinal neurogenesis, the p2 progenitor domain generates at least two subclasses of interneurons named V2a and V2b, which are components of the locomotor central pattern generator. The winged-helix/forkhead transcription factor Foxn4 is expressed in a subset of p2 progenitors and required for specifying V2b interneurons. Here, we report the generation of a Foxn4-Cre BAC transgenic mouse line that drives Cre recombinase expression mimicking endogenous Foxn4 expression pattern in the developing spinal cord. We used this transgenic line to map neuronal lineages derived from Foxn4-expressing progenitors and found that they gave rise to all neurons of the V2a, V2b, and the newly identified V2c lineages. These data suggest that Foxn4 may be transiently expressed by all p2 progenitors and that the Foxn4-Cre line may serve as a useful genetic tool not only for lineage analysis but also for functional studies of genes and neurons involved in locomotion.

QTc Prolongation Risk Evaluation in Female COVID-19 Patients Undergoing Chloroquine and Hydroxychloroquine With/Without Azithromycin Treatment.

Women have higher risk for developing TdP in response to ventricular repolarization prolonging drugs. Hundreds of trials are administering chloroquine and hydroxychloroquine with/without azithromycin to COVID-19 patients. While an overall prolonged QTc has been reported in COVID-19 patients undergoing these treatments, the question on even higher QTc elevation risk in thousands of female COVID-19 patients undergoing these treatments remains unanswered. We therefore explore data reported and shared with us to evaluate safety and efficacy of antimalaria pharmacotherapies in female COVID-19 patients. Although we observed longer mean QTc intervals in female patients in 2 of the 3 cohorts reviewed, the sex disproportionality in COVID-19 hospitalizations precludes a clear sex mediated QTc interval elevation risk association in the female COVID-19 patients undergoing acute treatment regimens. Adoption of study designs that include observation of sex mediated differential triggering of cardiac electrical activity by these drugs is warranted.

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord.

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development.

The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function.

The X protein of hepatitis B virus or HBx is a multifunctional regulatory protein that carries the fame of a promiscuous transactivator. Although, the N-terminal 'A' region of HBx (amino acids 1-20) is the most conserved region among mammalian hepadnavirus genomes, it has been found to be dispensable for transactivation function [Proc. Natl. Acad. Sci. U.S.A. 93, 1996, 5647]. To elucidate its biological role, DNA sequence corresponding to the A region of X gene was amplified by polymerase chain reaction and cloned as a 72 base pair HBx mutant X17. In order to augment the intracellular biochemical stability of the expressed protein, the monomeric X17 was multimerized and 2-10 units long tandem repeats of the A region (X17-n) were cloned in a mammalian expression vector. Expression of the X17 constructs was confirmed by in vitro transcription and translation, as well as by RT-PCR after transfection in hepatoma cells. The function of X17 was investigated using the chloramphenicol acetyl transferase reporter constructs of viral (RSV-LTR, HIV1-LTR and HBx) and cellular gene promoters (c-Jun and epidermal growth receptor). Not only did the X17 multimers inhibit the HBx-mediated transactivation of all the reporter genes, but also their basal activities. The inhibition was dependent on the amount of X17 plasmid transfected in cells as well as on the number of repeat units present in the X17 expression vectors. Further, the X17-related inhibition of transactivation was not a cytotoxic effect. Thus, our data suggests that the N-terminal 'A' domain of HBx has a negative regulatory function.

Prox1 regulates a transitory state for interneuron neurogenesis in the spinal cord.

Proper central nervous system (CNS) function depends critically on the generation of functionally distinct neuronal types in specific and reproducible positions. The generation of neuronal diversity during CNS development involves a fine balance between dividing neural progenitors and the differentiated neuronal progeny that they produce. However, the molecular mechanisms that regulate these processes are still poorly understood. Here, we show that the Prox1 transcription factor, which is expressed transiently and specifically in spinal interneurons, plays an important role in neurogenesis. Using both gain- and loss-of-function approaches, we find that Prox1 is capable of driving neuronal precursors out of the cell cycle and can initiate limited expression of neuronal proteins. Using RNAi approaches, we show that Prox1 function is required to execute a neurogenic differentiation program downstream of Mash1 and Ngn2. Our studies demonstrate an important, spinal interneuron-specific role for Prox1 in controlling steps required for both cell-cycle withdrawal and differentiation.

Foxn4 acts synergistically with Mash1 to specify subtype identity of V2 interneurons in the spinal cord.

Neuronal subtype diversification is essential for the establishment of functional neural circuits, and yet the molecular events underlying neuronal diversity remain largely to be defined. During spinal neurogenesis, the p2 progenitor domain, unlike others in the ventral spinal cord, gives rise to two intermingled but molecularly distinct subtypes of interneurons, termed V2a and V2b. We show here that the Foxn4 winged helix/forkhead transcription factor is coexpressed with the bHLH factor Mash1 in a subset of p2 progenitors. Loss of Foxn4 function eliminates Mash1 expression and V2b neurons and causes a fate-switch to V2a neurons, whereas the absence of Mash1 displays a similar but less severe phenotype. Overexpression of Foxn4 alone in spinal neural progenitors promotes the V2a fate at the expense of the V2b fate, whereas Mash1 suppresses both the V2a and V2b fates. However, coexpression of both Foxn4 and Mash1 promotes the V2b fate while inhibiting the V2a fate, indicating that Foxn4 cooperates with Mash1 to specify the identity of V2b neurons from bipotential p2 progenitors.