Development (Cambridge)

Four genes encoding fork-head-domain-containing proteins (FD genes) have been isolated from a mouse 8.5 days post coitum (p.c.) embryo cDNA library. Two are mouse homologues of rat HNF-3β and HNF-3α. The other two are novel and have been named MF-1 and MF-2 (for mesoderm/mesenchyme fork head). Wholemount in situ hybridization of embryos between 6.5 and 9.5 days p. c. shows that each gene has a unique expression pattern. HNF-3β is expressed in the node, notochord, floor plate and gut, while HNF-3α is mainly in the definitive endoderm and gut, but also in the floor plate of the midbrain. These results suggest that HNF3β and HNF-3α, in addition to their known functions as transcriptional activators in adult liver, play a role in body axis formation, neural tube patterning and definitive endoderm formation during gastrulation. MF-1 RNA is present in non-notochordal mesoderm, and in neural-crest-derived head mesenchyme, while MF-2 transcripts are found in the sclerotomes of the somites and in head mesenchyme, including that from neural crest. Studies on gastrulation stage embryos suggest that the early temporal and spatial patterns of HNF-3β? MF1 and HNF-3α correlate with populations of cells undergoing commitment to different developmental fates. A model is proposed linking FD gene expression with gastrulation events in the mouse.

Previous genetic studies of intersegmental nerve b development have identified several cell-surface proteins required for correct axon guidance to appropriate target muscles. Here we provide evidence that the small GTPase Drac1 also plays a key role in this guidance process. Neuronal expression of the dominant negative mutation Drac1(N17) causes axons to bypass and extend beyond normal synaptic partners. This phenotype is consistently reproduced by pharmacological blockade of actin assembly. Genetic interactions between Drac1(N17) and the receptor-tyrosine phosphatase Dlar suggest that intersegmental nerve b guidance requires the integration of multiple, convergent signals.

Drosophila seven-up is an orphan receptor of the steroid receptor family that is required to specify photoreceptor neuron subtypes in the developing compound eye. Expression of seven-up is confined to four of the eight photoreceptor precursors, R3/R4/R1/R6. We show that misexpression of seven-up in any of the other cell types within the developing ommatidium interferes with their differentiation. Each cell type responds differently to seven-up misexpression. For example, ectopic expression in the non-neuronal cone cells using the sevenless promoter/enhancer (sev-svp) causes the cone cells to take on a neuronal identity. Ectopic expression of seven-up in R2/R5 using the rough enhancer (ro-svp) causes these neurons to lose aspects of their photoreceptor subtype identity while remaining neuronal. Each cell type appears to have a different developmental time window that is sensitive to misexpressed seven-up. The temporal order of responsiveness of each cell type to misexpressed seven-up is similar but not identical to the order of neuronal differentiation. This suggests that there are processes of specification that are distinct from the specification to become a photoreceptor neuron. We have identified members of the ras signaling pathway as suppressors of the cone cell to R7 neuron transformation caused by sev-svp. Suppression of the sev-svp phenotype can be achieved by decreasing the gene-dosage of any of the members of the ras-pathway. This suggests that the function of seven-up in the cone cells requires ras signaling. However, a decrease in ras signaling results in enhancement of the phenotype caused by the rosvp transgene. We discuss the relationship between decisions controlled by seven-up and those controlled by ras signaling.

klingon is a member of the Immunoglobulin superfamily and is expressed in a restricted pattern of neurons during embryonic neurogenesis and in the R7 photoreceptor precursor throughout its development. Starting from the H214 enhancer trap line, we identified a transcription unit, klingon, that encodes a putative protein of 528 amino acids and contains three C2-type Immunoglobulin-like domains followed by one fibronectin type III repeat. When Klingon is expressed in S2 tissue culture cells, it is associated with the cell membrane by a glycosyl-phosphatidylinositol linkage and can mediate homophilic adhesion. Genetic analysis has revealed that klingon is an essential gene that participates in the development of the R7 neuron. Ectopic expression of klingon in all neurons in a sevenless background can alter the position of the R8 rhabdomere.

We have investigated the mechanism that patterns dopamine expression among Caenorhabditis elegans male ray sensory neurons. Dopamine is expressed by the A-type sensory neurons in three out of the nine pairs of rays. We used expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFβ family signaling pathway, the DBL-1 pathway, dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability.

We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big,and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in theegl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-β and insulin pathways, respectively.

The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages,and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFκB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFκB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs)localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.

Các tế bào tiền thân sớm của các tiểu đảo tụy ở chuột đã được đặc trưng để xem xét lại mối quan hệ dòng họ khả thi của chúng với bốn loại tế bào tiểu đảo được tìm thấy trong các tiểu đảo trưởng thành. Insulin và glucagon lần đầu tiên được biểu hiện vào ngày phôi 9.5, và nhiều tế bào đồng thời biểu hiện hai dấu hiệu này, như đã được chỉ ra bởi phân tích vi mô ánh sáng và điện tử sử dụng hóa miễn dịch gắn nhãn kép. Việc ấp ủ tuyến tụy phôi với 1% glutaraldehyde, một thuốc cố định thường được sử dụng bởi các nhà vi thể điện tử, đã loại bỏ phản ứng này, qua đó giải thích những khó khăn được báo cáo trong việc phát hiện các tế bào tiền thân này. Sử dụng huyết thanh kháng thể đặc hiệu cho neuropeptide Y (NPY), một peptide có tính đồng hình đáng kể với polypeptide tụy (PP), chúng tôi cho thấy NPY lần đầu tiên xuất hiện cùng với phản ứng miễn dịch insulin và glucagon tại E9.5, và được đồng biểu hiện với glucagon ở phần lớn các tế bào trưởng thành. Như chúng tôi đã báo cáo trước đây, PP tự nó chỉ có thể phát hiện được bằng phương pháp hóa miễn dịch tế bào vào ngày sau sinh 1 với kháng thể cụ thể cho PP. Tuy nhiên, các kháng thể được tạo ra chống lại PP bò đã được chỉ ra qua dot blotting là nhận biết NPY với độ nhạy tương tự, cho thấy rằng một báo cáo gần đây về các tế bào tiền thân tiểu đảo chứa PP tại E9.5 (Herrera, P. L., Huarte, J., Sanvito, F., Meda, P., Orci, L. và Vassalli, J. D. (1991) Phát triển 113, 1257-1265), thực tế là phản ứng chéo với NPY. Dữ liệu hỗ trợ một mô hình trong đó các tế bào tiền thân sớm của tuyến tụy nội tiết đồng kích hoạt và đồng biểu hiện một tập hợp các hormone tế bào tiểu đảo và gen thần kinh, mà sự biểu hiện của chúng cả được tăng cường có chọn lọc và bị loại bỏ khi phát triển diễn ra, đi cùng với việc hạn chế các mẫu biểu hiện đặc trưng của các loại tế bào tiểu đảo trưởng thành.

The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. We demonstrate that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. We observed phenotypic similarities and genetic interactions among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. We propose that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.