osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

IGDB-2, an Ig/FNIII protein, binds the ion channel LGC-34 and controls sensory compartment morphogenesis in C. elegans.

Sensory organ glia surround neuronal receptive endings (NREs), forming a specialized compartment important for neuronal activity, and reminiscent of glia-ensheathed synapses in the central nervous system. We previously showed that DAF-6, a Patched-related protein, is required in glia of the C. elegans amphid sensory organ to restrict sensory compartment size. LIT-1, a Nemo-like kinase, and SNX-1, a retromer component, antagonize DAF-6 and promote compartment expansion. To further explore the machinery underlying compartment size control, we sought genes whose inactivation restores normal compartment size to daf-6 mutants. We found that mutations in igdb-2, encoding a single-pass transmembrane protein containing Ig-like and fibronectin type III domains, suppress daf-6 mutant defects. IGDB-2 acts in glia, where it localizes to glial membranes surrounding NREs, and, together with LIT-1 and SNX-1, regulates compartment morphogenesis. Immunoprecipitation followed by mass spectrometry demonstrates that IGDB-2 binds to LGC-34, a predicted ligand-gated ion channel, and lgc-34 mutations inhibit igdb-2 suppression of daf-6. Our findings reveal a novel membrane protein complex and suggest possible mechanisms for how sensory compartment size is controlled.

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans.

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Some, but not all, retromer components promote morphogenesis of C. elegans sensory compartments.

The endings of sensory receptor cells often lie within specialized compartments formed by glial cells. The main sensory organ of Caenorhabditis elegans, the amphid, provides a powerful setting for studying glial compartment morphogenesis. Our previous studies showed that amphid compartment size is controlled by opposing activities of the Nemo-like kinase LIT-1, which promotes compartment expansion, and the Patched-related protein DAF-6, which restricts compartment growth. From a genetic screen for mutations able to suppress the bloated sensory compartments of daf-6 mutants, we identified an allele of the sorting nexin gene snx-1. SNX-1 protein is a component of the retromer, a protein complex that facilitates recycling of transmembrane proteins from the endosome to the Golgi network. We find that snx-1 functions cell autonomously within glia to promote sensory compartment growth, and that SNX-1 protein is enriched near the surface of the sensory compartment. snx-1 interacts genetically with lit-1 and another regulator of compartment size, the Dispatched-related gene che-14. Mutations in snx-3 and vps-29, also retromer genes, can suppress daf-6 defects. Surprisingly, however, remaining retromer components seem not to be involved. Our results suggest that a novel assembly of retromer components is important for determining sensory compartment dimensions.

Genomic sequencing has a high diagnostic yield in children with congenital anomalies of the heart and urinary system.

Background: Congenital heart defects (CHD) and congenital anomalies of the kidney and urinary tract (CAKUT) account for significant morbidity and mortality in childhood. Dozens of monogenic causes of anomalies in each organ system have been identified. However, even though 30% of CHD patients also have a CAKUT and both organs arise from the lateral mesoderm, there is sparse overlap of the genes implicated in the congenital anomalies for these organ systems. We sought to determine whether patients with both CAKUT and CHD have a monogenic etiology, with the long-term goal of guiding future diagnostic work up and improving outcomes. Methods: Retrospective review of electronic medical records (EMR), identifying patients admitted to Rady Children's Hospital between January 2015 and July 2020 with both CAKUT and CHD who underwent either whole exome sequencing (WES) or whole genome sequencing (WGS). Data collected included demographics, presenting phenotype, genetic results, and mother's pregnancy history. WGS data was reanalyzed with a specific focus on the CAKUT and CHD phenotype. Genetic results were reviewed to identify causative, candidate, and novel genes for the CAKUT and CHD phenotype. Associated additional structural malformations were identified and categorized. Results: Thirty-two patients were identified. Eight patients had causative variants for the CAKUT/CHD phenotype, three patients had candidate variants, and three patients had potential novel variants. Five patients had variants in genes not associated with the CAKUT/CHD phenotype, and 13 patients had no variant identified. Of these, eight patients were identified as having possible alternative causes for their CHD/CAKUT phenotype. Eighty-eight percent of all CAKUT/CHD patients had at least one additional organ system with a structural malformation. Conclusions: Overall, our study demonstrated a high rate of monogenic etiologies in hospitalized patients with both CHD and CAKUT, with a diagnostic rate of 44%. Thus, physicians should have a high suspicion for genetic disease in this population. Together, these data provide valuable information on how to approach acutely ill patients with CAKUT and CHD, including guiding diagnostic work up for associated phenotypes, as well as novel insights into the genetics of CAKUT and CHD overlap syndromes in hospitalized children.

A Leptin Receptor Antagonist Attenuates Adipose Tissue Browning and Muscle Wasting in Infantile Nephropathic Cystinosis-Associated Cachexia.

Mice lacking the functional cystinosin gene (Ctns-/-), a model of infantile nephropathic cystinosis (INC), exhibit the cachexia phenotype with adipose tissue browning and muscle wasting. Elevated leptin signaling is an important cause of chronic kidney disease-associated cachexia. The pegylated leptin receptor antagonist (PLA) binds to but does not activate the leptin receptor. We tested the efficacy of this PLA in Ctns-/- mice. We treated 12-month-old Ctns-/- mice and control mice with PLA (7 mg/kg/day, IP) or saline as a vehicle for 28 days. PLA normalized food intake and weight gain, increased fat and lean mass, decreased metabolic rate and improved muscle function. It also attenuated perturbations of energy homeostasis in adipose tissue and muscle in Ctns-/- mice. PLA attenuated adipose tissue browning in Ctns-/- mice. PLA increased gastrocnemius weight and fiber size as well as attenuated muscle fat infiltration in Ctns-/- mice. This was accompanied by correcting the increased expression of muscle wasting signaling while promoting the decreased expression of myogenesis in gastrocnemius of Ctns-/- mice. PLA attenuated aberrant expressed muscle genes that have been associated with muscle atrophy, increased energy expenditure and lipolysis in Ctns-/- mice. Leptin antagonism may represent a viable therapeutic strategy for adipose tissue browning and muscle wasting in INC.

C. elegans daf-6 encodes a patched-related protein required for lumen formation.

Sensory organs are often composed of neuronal sensory endings accommodated in a lumen formed by ensheathing epithelia or glia. Here we show that lumen formation in the C. elegans amphid sensory organ requires the gene daf-6. daf-6 encodes a Patched-related protein that localizes to the luminal surfaces of the amphid channel and other C. elegans tubes. While daf-6 mutants display only amphid lumen defects, animals defective for both daf-6 and the Dispatched gene che-14 exhibit defects in all tubular structures that express daf-6. Furthermore, DAF-6 protein is mislocalized, and lumen morphogenesis is abnormal, in mutants with defective sensory neuron endings. We propose that amphid lumen morphogenesis is coordinated by neuron-derived cues and a DAF-6/CHE-14 system that regulates vesicle dynamics during tubulogenesis.

Functional genomics of the cilium, a sensory organelle.

Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Biedl syndrome (BBS). To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the ciliogenic transcription factor, DAF-19. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm.

Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM.