A new dawn for the study of cell type evolution.

Animal evolution is influenced by the emergence of new cell types, yet our understanding of this process remains elusive. This prompts the need for a broader exploration across diverse research organisms, facilitated by recent breakthroughs, such as gene editing tools and single-cell genomics. Essential to our understanding of cell type evolution is the accurate identification of homologous cells. We delve into the significance of considering developmental ontogeny and potential pitfalls when drawing conclusions about cell type homology. Additionally, we highlight recent discoveries in the study of cell type evolution through the application of single-cell transcriptomics and pinpoint areas ripe for further exploration.

Follicle-innervating Aδ-low threshold mechanoreceptive neurons form receptive fields through homotypic competition.

The mammalian somatosensory system is comprised of multiple neuronal populations that form specialized, highly organized sensory endings in the skin. The organization of somatosensory endings is essential to their functions, yet the mechanisms which regulate this organization remain unclear. Using a combination of genetic and molecular labeling approaches, we examined the development of mouse hair follicle-innervating low-threshold mechanoreceptors (LTMRs) and explored competition for innervation targets as a mechanism involved in the patterning of their receptive fields. We show that follicle innervating neurons are present in the skin at birth and that LTMR receptive fields gradually add follicle-innervating endings during the first two postnatal weeks. Using a constitutive Bax knockout to increase the number of neurons in adult animals, we show that two LTMR subtypes have differential responses to an increase in neuronal population size: Aδ-LTMR neurons shrink their receptive fields to accommodate the increased number of neurons innervating the skin, while C-LTMR neurons do not. Our findings suggest that competition for hair follicles to innervate plays a role in the patterning and organization of follicle-innervating LTMR neurons.

c-Kit Receptor Maintains Sensory Axon Innervation of the Skin through Src Family Kinases.

Peripheral somatosensory neurons innervate the skin and sense the environment. Whereas many studies focus on initial axon outgrowth and pathfinding, how signaling pathways contribute to maintenance of the established axon arbors and terminals within the skin is largely unknown. This question is particularly relevant to the many types of neuropathies that affect mature neuronal arbors. We show that a receptor tyrosine kinase (RTK), c-Kit, contributes to maintenance, but not initial development, of cutaneous axons in the larval zebrafish before sex determination. Downregulation of Kit signaling rapidly induced retraction of established axon terminals in the skin and a reduction in axonal density. Conversely, misexpression of c-Kit ligand in the skin in larval zebrafish induced increases in local sensory axon density, suggesting an important role for Kit signaling in cutaneous axon maintenance. We found Src family kinases (SFKs) act directly downstream to mediate Kit's role in regulating cutaneous axon density. Our data demonstrate a requirement for skin-to-axon signaling to maintain axonal networks and elucidate novel roles for Kit and SFK signaling in this context. This Kit-SFK signaling axis offers a potential pathway to therapeutically target in sensory neuropathies and to further explore in other neurobiological processes.SIGNIFICANCE STATEMENTThe skin is full of small nerve endings that sense different environmental stimuli. How these nerve endings grow and reach a specific area of the skin during development has been the focus of many studies. In contrast, the cellular and molecular mechanisms required to maintain the function and health of these structures is relatively unknown. We discovered that a specific receptor in sensory neurons, c-Kit, is required to maintain the density of nerve endings in the skin. Furthermore, we found that a molecular target of c-Kit, Src family kinases (SFKs), is necessary for this role. Thus, c-Kit/SFK signaling regulates density and maintenance of sensory nerve endings in the skin and may have important roles in neural disease and regeneration.

Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay.

PARP6, a member of a family of enzymes (17 in humans) known as poly-ADP-ribose polymerases (PARPs), is a neuronally enriched PARP. While previous studies from our group show that Parp6 is a regulator of dendrite morphogenesis in rat hippocampal neurons, its function in the nervous system in vivo is poorly understood. Here, we describe the generation of a Parp6 loss-of-function mouse model for examining the function of Parp6 during neurodevelopment in vivo. Using CRISPR-Cas9 mutagenesis, we generated a mouse line that expressed a Parp6 truncated variant (Parp6TR) in place of Parp6WT. Unlike Parp6WT, Parp6TR is devoid of catalytic activity. Homozygous Parp6TR do not exhibit obvious neuromorphological defects during development, but nevertheless die perinatally. This suggests that Parp6 catalytic activity is important for postnatal survival. We also report PARP6 mutations in six patients with several neurodevelopmental disorders, including microencephaly, intellectual disabilities, and epilepsy. The most severe mutation in PARP6 (C563R) results in the loss of catalytic activity. Expression of Parp6C563R in hippocampal neurons decreases dendrite morphogenesis. To gain further insight into PARP6 function in neurons we also performed a BioID proximity labeling experiment in hippocampal neurons and identified several microtubule-binding proteins (e.g., MAP-2) using proteomics. Taken together, our results suggest that PARP6 is an essential microtubule-regulatory gene in mice, and that the loss of PARP6 catalytic activity has detrimental effects on neuronal function in humans.

Immunohistochemical and Genetic Labeling of Hairy and Glabrous Skin Innervation.

Cutaneous innervation is an essential component of the mammalian sensory nervous system. During development, genetically and morphologically diverse subtypes of sensory neurons use distinct molecular pathways to innervate end organs or form free nerve endings in glabrous and hairy skin. Peripheral neurons can be damaged by acute injury or degenerate due to chronic conditions including diabetes and chemotherapy, leading to peripheral neuropathy. The analysis of skin and cutaneous innervation can be applied to many research endeavors, from developmental neuroscience to pharmaceutical testing. Due to the natural hydrophobicity and heterogenous makeup of the skin (dense, keratinized cells as well as sparse, extracellular-matrix-bound cells), its histological analysis presents unique challenges compared to that of many other tissues. This series of protocols describes histological methods for generalized immunohistochemistry and subtype-specific genetic labeling of sensory neurons in mouse skin in both whole-mount and section formats. We provide detailed methodology of tissue preparation for hairy and glabrous skin, several types of labeling, and counting of hair follicles in flat-mounted mouse skin. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cryosectioning and immunostaining of mouse hairy skin Alternate Protocol 1: Alternate method for preparation and fixation of mouse hairy skin Basic Protocol 2: Sectioning of mouse paw glabrous skin Basic Protocol 3: Whole-mount immunolabeling of mouse skin Basic Protocol 4: Sparse labeling of skin-innervating neurons with a Cre-dependent membrane-bound alkaline phosphatase reporter Alternate Protocol 2: Sparse labeling of skin-innervating neurons with a Cre-dependent fluorescent reporter Basic Protocol 5: Oil Red O staining of skin.

Multiple Representations of Space by the Cockroach, Periplaneta americana.

When cockroaches are trained to a visual-olfactory cue pairing using the antennal projection response (APR), they can form different memories for the location of a visual cue. A series of experiments, each examining memory for the spatial location of a visual cue, were performed using restrained cockroaches. The first group of experiments involved training cockroaches to associate a visual cue (CS-green LED) with an odor cue (US) in the presence or absence of a second visual reference cue (white LED). These experiments revealed that cockroaches have at least two forms of spatial memory. First, it was found that during learning, the movements of the antennae in response to the odor influenced the cockroaches' memory. If they use only one antenna, cockroaches form a memory that results in an APR being elicited to the CS irrespective of its location in space. When using both antennae, the cockroaches resulting memory leads to an APR to the CS that is spatially confined to within 15° of the trained position. This memory represents an egocentric spatial representation. Second, the cockroaches simultaneously formed a memory for the angular spatial relationships between two visual cues when trained in the presence of a second visual reference cue. This training provided the cockroaches an allocentric representation or visual snapshot of the environment. If both egocentric and the visual snapshot were available to the cockroach to localize the learned cue, the visual snapshot determined the behavioral response in this assay. Finally, the split-brain assay was used to characterize the cockroach's ability to establish a memory for the angular relationship between two visual cues with half a brain. Split-brain cockroaches were trained to unilaterally associate a pair of visual cues (CS-green LED and reference-white LED) with an odor cue (US). Split-brain cockroaches learned the general arrangement of the visual cues (i.e., the green LED is right of the white LED), but not the precise angular relationship. These experiments provide new insight into spatial memory processes in the cockroach.

Mutations and Modeling of the Chromatin Remodeler CHD8 Define an Emerging Autism Etiology.

Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder with a strong but complex genetic component. Recent family based exome-sequencing strategies have identified recurrent de novo mutations at specific genes, providing strong evidence for ASD risk, but also highlighting the extreme genetic heterogeneity of the disorder. However, disruptions in these genes converge on key molecular pathways early in development. In particular, functional enrichment analyses have found that there is a bias toward genes involved in transcriptional regulation, such as chromatin modifiers. Here we review recent genetic, animal model, co-expression network, and functional genomics studies relating to the high confidence ASD risk gene, CHD8. CHD8, a chromatin remodeling factor, may serve as a "master regulator" of a common ASD etiology. Individuals with a CHD8 mutation show an ASD subtype that includes similar physical characteristics, such as macrocephaly and prolonged GI problems including recurrent constipation. Similarly, animal models of CHD8 disruption exhibit enlarged head circumference and reduced gut motility phenotypes. Systems biology approaches suggest CHD8 and other candidate ASD risk genes are enriched during mid-fetal development, which may represent a critical time window in ASD etiology. Transcription and CHD8 binding site profiles from cell and primary tissue models of early development indicate that CHD8 may also positively regulate other candidate ASD risk genes through both direct and indirect means. However, continued study is needed to elucidate the mechanism of regulation as well as identify which CHD8 targets are most relevant to ASD risk. Overall, these initial studies suggest the potential for common ASD etiologies and the development of personalized treatments in the future.