A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. The functional units within the kidneys are epithelial tubules called nephrons. These tubules take in filtrate from the blood or coelom and selectively reabsorb nutrients through evolutionarily conserved nephron segments, leaving waste product to be eliminated in the urine. Genes coding for functional transporters are segmentally expressed, enabling nephrons to function as selective filters. The developmental patterning program that generates these segments is of great interest. The Xenopus embryonic kidney, the pronephros, has served as a valuable model to identify genes involved in nephron formation and patterning. Prior work has defined the gene expression profiles of Xenopus epithelial nephron segments via in situ hybridization strategies, but our understanding of the cellular makeup of the Xenopus pronephric kidney remains incomplete. Here, we scrutinize the cellular composition of the Xenopus pronephric nephron through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney, this study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and expression profiles. The data highlight significant conservation in podocytes, proximal and distal tubule cells and divergence in cellular composition underlying the evolution of the corticomedullary axis, while emphasizing the Xenopus pronephros as a model for physiology and disease.

ARF small GTPases in the developmental function mediated by ARF regulators GNOM and VAN3

ARF small GTPases are molecular switches acting in intracellular trafficking. Their cycles of activity are controlled by regulators, ARF Guanine nucleotide Exchange Factors (ARF-GEFs) and ARF GTPase Activating Proteins (ARF-GAPs). The ARF-GEF GNOM (GN) and the ARF-GAP VAN3 share a prominent function in auxin-mediated developmental patterning, but the ARFs which they might control were not identified. We conducted a loss-of-function and localization-based screening of the ARF/ARF-LIKE gene family in Arabidopsis thaliana with the primary aim of identifying functional partners of GN and VAN3, while extending the limited understanding of this gene group as a whole. We identified a function of ARLA1 in branching angle control. Mutants lacking the variably localized ARLB1, ARFB1, ARFC1, ARFD1, and ARF3, even in high order combinations, do not exhibit any evident phenotypes. Loss of function arfa1 phenotypes support a major role of ARFA1 in growth and development overall, but patterning defects typical to gn loss of function are not found. ARFA1 are not localized at the plasma membrane, where GN and VAN3 carry out developmental patterning function according to current models. Taken together, putative ARF partners of GN and VAN3 in developmental patterning cannot be conclusively identified.

Developmental patterning function of GNOM ARF-GEF mediated from the plasma membrane

The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.

Developmental Patterning and Neurogenetic Gradients of Nurr1 Positive Neurons in the Rat Claustrum and Lateral Cortex

The claustrum is an enigmatic brain structure thought to be important for conscious sensations. Recent studies have focused on gene expression patterns, connectivity, and function of the claustrum, but relatively little is known about its development. Interestingly, claustrum-enriched genes, including the previously identified marker Nurr1, are not only expressed in the classical claustrum complex, but also embedded within lateral neocortical regions in rodents. Recent studies suggest that Nurr1 positive neurons in the lateral cortex share a highly conserved genetic expression pattern with claustrum neurons. Thus, we focus on the developmental progression and birth dating pattern of the claustrum and Nurr1 positive neurons in the lateral cortex. We comprehensively investigate the expression of Nurr1 at various stages of development in the rat and find that Nurr1 expression first appears as an elongated line along the anterior-posterior axis on embryonic day 13.5 (E13.5) and then gradually differentiates into multiple sub-regions during prenatal development. Previous birth dating studies of the claustrum have led to conflicting results, therefore, we combine 5-ethynyl-2′-deoxyuridine (EdU) labeling with in situ hybridization for Nurr1 to study birth dating patterns. We find that most dorsal endopiriform (DEn) neurons are born on E13.5 to E14.5. Ventral claustrum (vCL) and dorsal claustrum (dCL) are mainly born on E14.5 to E15.5. Nurr1 positive cortical deep layer neurons (dLn) and superficial layer neurons (sLn) are mainly born on E14.5 to E15.5 and E15.5 to E17.5, respectively. Finally, we identify ventral to dorsal and posterior to anterior neurogenetic gradients within vCL and DEn. Thus, our findings suggest that claustrum and Nurr1 positive neurons in the lateral cortex are born sequentially over several days of embryonic development and contribute toward charting the complex developmental pattern of the claustrum in rodents.

Clathrin-mediated endocytosis-independent function of VAN3 ARF GTPase Activating Protein at the plasma membrane

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. The Arabidopsis thaliana ARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally in van3-1 knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal quantitative regulatory dynamics from a complex genetic network in vivo. We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent expression of giant and hunchback and delayed repression of Kruppel. In contrast, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a previously unreported role for Bicoid in suppressing knirps expression. Acute modulation of transcription factor concentration while simultaneously recording output gene activity represents a powerful approach for studying how gene circuit elements are coupled to cell identification and complex body pattern formation in vivo.

Root Patterning: Tuning SHORT ROOT Function Creates Diversity in Form

Roots have a fundamental role in plant growth and adaptation to different environments. Diversity in root morphology and architecture enables plants to acquire water and nutrients in contrasting substrate conditions, resist biotic and abiotic stress, and develop symbiotic associations. At its most fundamental level, morphology is determined by discrete changes in tissue patterning. Differences in the number and arrangement of the cell layers in the root can change tissue structure, as well as root length and girth, affecting important productivity traits. Therefore, understanding the molecular mechanisms controlling variation in developmental patterning is an important goal in biology. The ground tissue (GT) system is an ideal model to study the genetic basis of morphological diversity because it displays great interspecific variability in cell layer number. In addition, the genetic circuit controlling GT patterning in Arabidopsis thaliana has been well described, although little is known about species with more complex root anatomies. In this review, we will describe the Arabidopsis model for root radial patterning and present recent progress in elucidating the genetic circuitry controlling GT patterning in monocots and the legume Medicago truncatula (Mt), species that develop roots with more complex anatomies and multilayered cortex.

Visualizing the organization and differentiation of the male-specific nervous system of C. elegans

Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode C. elegans, where male animals contain a little studied set of 93 male-specific neurons. To make these neurons amenable for future study, we describe here how a multicolor reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male specific neurons. We used NeuroPAL to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes and for cellular fate analysis by analyzing the effect of removal of several developmental patterning genes on neuronal identity acquisition. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage. This coordinated wave of differentiation, which we call “just-in-time" differentiation, couples neuronal maturation programs with the appearance of sexual organs.

Basal cells in the epidermis and epidermal differentiation

A definite identification of epidermal stem cells is not known and the mechanism of epidermal differentiation is not fully understood. Toward both of these quests, considerable information is available from the research on lineage tracing and clonal growth analysis in the basal layer of the epidermis, on the hair follicle and interfollicular epidermal stem cells, and on Wnt signaling along with its role in developmental patterning and cell differentiation. In this paper, literature on the aforementioned research has been collated and analyzed. In addition, models of basal layer cellular composition and epidermal differentiation have been presented.

Vascular and Lymphatic Malformations: Perspectives From Human and Vertebrate Studies

Vascular malformations, affecting ≈1% to 1.5% of the population, comprise a spectrum of developmental patterning defects of capillaries, arteries, veins, and/or lymphatics. The majority of vascular malformations occur sporadically; however, inherited malformations exist as a part of complex congenital diseases. The malformations, ranging from birthmarks to life-threatening conditions, are present at birth, but may reveal signs and symptoms—including pain, bleeding, disfigurement, and functional defects of vital organs—in infancy, childhood, or adulthood. Vascular malformations often exhibit recurrent patterns at affected sites due to the lack of curative treatments. This review series provides a state-of-the-art assessment of vascular malformation research at basic, clinical, genetic, and translational levels.