Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells

Lizards regenerate amputated tails but fail to recapitulate the dorsoventral patterning achieved during embryonic development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. However, adult ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tubes. Both NTs and ETs contain neural stem cells (NSCs), but only embryonic NSC populations differentiate into roof plate identities when protected from endogenous Hedgehog signaling. NSCs were isolated from parthenogenetic lizard embryos, rendered unresponsive to Hedgehog signaling via CRISPR/Cas9 gene knockout of smoothened (Smo), and implanted back into clonally-identical adults to regulate tail regeneration. Here we report that Smo knockout embryonic NSCs oppose cartilage formation when engrafted to adult ETs, representing an important milestone in the creation of regenerated lizard tails with dorsoventrally patterned skeletal tissues.

Characterization of a novel Lbx1 mouse loss of function strain

Adolescent Idiopathic Scoliosis (AIS) is the most common type of spine deformity affecting 2-3% of the population worldwide. The etiology of this disease is still poorly understood. Several GWAS studies have identified single nucleotide polymorphisms (SNPs) located near the gene LBX1 that is significantly correlated with AIS risk. LBX1 is a transcription factor with roles in myocyte precursor migration, cardiac neural crest specification, and neuronal fate determination in the neural tube. Here, we further investigated the role of LBX1 in the developing spinal cord of mouse embryos using a CRISPR-generated mouse model expressing a truncated version of LBX1 (Lbx1Δ). Homozygous mice died at birth, likely due to cardiac abnormalities. To further study the neural tube phenotype, we used RNA-sequencing to identify 410 genes differentially expressed between the neural tubes of E12.5 wildtype and Lbx1Δ/Δ embryos. Genes with increased expression in the deletion line were involved in neurogenesis and those with broad roles in embryonic development. Many of these genes have also been associated with scoliotic phenotypes. In comparison, genes with decreased expression were primarily involved in skeletal development. Subsequent skeletal and immunohistochemistry analysis further confirmed these results. This study aids in understanding the significance of links between Lbx1 function and AIS susceptibility.

Perfecting the Imperfect: Introducing dorsoventral patterning into adult regenerating lizard tails with gene-edited embryonic neural stem cells.

Lizards are able to regrow amputated tails, but the lizard tail regenerative process fails to recapitulate the dorsoventral patterning achieved during embryonic tail development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. Embryonic NTs are, themselves, dorsoventrally patterned, with Pax7+ Shh- dorsal roof plate domains that restrict cartilage skeletal formation induced by Pax7- Shh+ floor domains to ventral tail regions. However, adult regenerated tail ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tube skeletons. Both NTs and ETs contain populations of neural stem cells (NSCs), but only embryonic NSC populations are able to differentiate into roof plate identities and neurons. Embryonic NSCs transplanted into regenerated tail ETs retain the capacity to form roof domains but are ultimately ventralized by the unchecked hedgehog signaling of regenerated lizard tail environments. We hypothesized that only the simultaneous repression of hedgehog signaling and enhancement of NCS roof plate differentiation capacity would induce patterning in lizard ETs and, hence, regenerated cartilage. This was tested through the use of a novel genetic engineering process in which NSCs are isolated from embryos of the parthenogenetic lizard Lepidodactylus lugubris, gene-edited in vivo, and implanted back into clonally-identical adults to regulate tail regeneration. Embryonic lizard NSC lines unresponsive to hedgehog stimulation were generated through the use of CRISPR/Cas9 technologies to knockout (KO) the signaling regulator smoothened (Smo). Exogenous Smo KO NSCs were injected into adult tail spinal cords, where they engrafted to endogenous ependymal cell populations and contributed to dorsal domains in regenerated tail ETs. Embryonic Smo KO NSCs maintained roof plate identities in vivo, and lizards treated with edited NSCs regrew tails that lacked cartilage in dorsal regions. These studies represent an important milestone in the creation of the first regenerated lizard tails with dorsoventrally patterned ETs and skeletal tissues.

Cytoneme delivery of sonic hedgehog from ligand-producing cells requires Myosin 10 and a dispatched-BOC/CDON co-receptor complex

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known. Herein, we reveal that the actin motor Myosin 10 promotes vesicular transport of Sonic Hedgehog (SHH) morphogen in mouse cell cytonemes, and that SHH morphogen gradient organization is altered in neural tubes of Myo10-/- mice. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid, receptor-dependent signal response that occurs within seconds of ligand delivery. This activity is dependent upon a novel Dispatched (DISP)-BOC/CDON co-receptor complex that functions in ligand-producing cells to promote cytoneme occurrence and facilitate ligand delivery for signal activation.

Diethylhexyl phthalate induces teratogenic effects through oxidative stress response in a chick embryo model

Diethylhexyl phthalate (DEHP) is known as a persistent environmental pollutant. However, the possible effects of DEHP on human neural tube defects (NTDs) remain elusive. We set out to investigate the exposure of DEHP in human and explore the association of DEHP and NTDs. The level of DEHP in maternal urine was measured and analyzed by GC-MS. To further validate the results in human NTDs, chick embryos were used as animal models. Viability, reactive oxygen species (ROS) level, oxidative stress indicators and apoptosis were detected in DEHP-treated chick embryos. Our research revealed that the detection ratio of positive DEHP and its metabolites in maternal urine were observed dramatically higher in NTDs population than that in normal controls (P < 0.01, P < 0.05, respectively). Moreover, DEHP treatment (10−6 M) led to developmental toxicity in chick embryos via accelerating oxidative stress response and cell apoptosis, and changing the level of oxidative stress-related indicators. Moreover, high dose choline (100 μg/μl) could partially restrain the toxicity effects induced by DEHP. Our data collectively imply that the incidence of NTDs may closely associate with DEHP exposure, which disturbs the development of neural tubes by enhancing oxidative stress.

Role of FGF signalling in neural crest cell migration during early chick embryo development

SummaryFibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

Introduction to Neuroanatomy

This chapter focuses on learning the origination and organization of the nervous system and how to draw all the various elements that comprise it. Instructions are given for drawing the cerebrum, basal ganglia, thalamus, limbic systems, brainstem, cranial nerves, vertebral column, spinal cord, peripheral nervous system (PNS), autonomic nervous system (ANS), formation of neural plates, and neural tubes. Additionally, the chapter addresses how the elements of the nervous system are related to each other, notes their function, and outlines their respective sensorimotor and cognitive activities.