Network explanations and explanatory directionality.

Network explanations raise foundational questions about the nature of scientific explanation. The challenge discussed in this article comes from the fact that network explanations are often thought to be non-causal, i.e. they do not describe the dynamical or mechanistic interactions responsible for some behaviour, instead they appeal to topological properties of network models describing the system. These non-causal features are often thought to be valuable precisely because they do not invoke mechanistic or dynamical interactions and provide insights that are not available through causal explanations. Here, I address a central difficulty facing attempts to move away from causal models of explanation; namely, how to recover the directionality of explanation. Within causal models, the directionality of explanation is identified with the direction of causation. This solution is no longer available once we move to non-causal accounts of explanation. I will suggest a solution to this problem that emphasizes the role of conditions of application. In doing so, I will challenge the idea that sui generis mathematical dependencies are the key to understand non-causal explanations. The upshot is a conceptual account of explanation that accommodates the possibility of non-causal network explanations. It also provides guidance for how to evaluate such explanations. This article is part of the theme issue 'Unifying the essential concepts of biological networks: biological insights and philosophical foundations'.

ß-Catenin is required for radial cell patterning and identity in the developing mouse cochlea.

Development of multicellular organs requires the coordination of cell differentiation and patterning. Critical for sound detection, the mammalian organ of Corti contains functional units arranged tonotopically along the cochlear turns. Each unit consists of sensory hair cells intercalated by nonsensory supporting cells, both specified and radially patterned with exquisite precision during embryonic development. However, how cell identity and radial patterning are jointly controlled is poorly understood. Here we show that ß-catenin is required for specification of hair cell and supporting cell subtypes and radial patterning of the cochlea in vivo. In 2 mouse models of conditional ß-catenin deletion, early specification of Myosin7-expressing hair cells and Prox1-positive supporting cells was preserved. While ß-catenin-deficient cochleae expressed FGF8 and FGFR3, both of which are essential for pillar cell specification, the radial patterning of organ of Corti was disrupted, revealed by aberrant expression of cadherins and the pillar cell markers P75 and Lgr6. Moreover, ß-catenin ablation caused duplication of FGF8-positive inner hair cells and reduction of outer hair cells without affecting the overall hair cell density. In contrast, in another transgenic model with suppressed transcriptional activity of ß-catenin but preserved cell adhesion function, both specification and radial patterning of the organ of Corti were intact. Our study reveals specific functions of ß-catenin in governing cell identity and patterning mediated through cell adhesion in the developing cochlea.

Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques.

Studies of specific tissue cell types are becoming increasingly important in advancing our understanding of cell biology and gene and protein expression. Prospective isolation of specific cell types is a powerful technique as it facilitates such investigations, allowing for analysis and characterization of individual cell populations. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and limited cell markers. In this chapter, we describe methods for selectively labeling and isolating different inner ear cell types from the neonatal mouse cochlea using fluorescence-activated cell sorting.

Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.

Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Making sense of Wnt signaling-linking hair cell regeneration to development.

Wnt signaling is a highly conserved pathway crucial for development and homeostasis of multicellular organisms. Secreted Wnt ligands bind Frizzled receptors to regulate diverse processes such as axis patterning, cell division, and cell fate specification. They also serve to govern self-renewal of somatic stem cells in several adult tissues. The complexity of the pathway can be attributed to the myriad of Wnt and Frizzled combinations as well as its diverse context-dependent functions. In the developing mouse inner ear, Wnt signaling plays diverse roles, including specification of the otic placode and patterning of the otic vesicle. At later stages, its activity governs sensory hair cell specification, cell cycle regulation, and hair cell orientation. In regenerating sensory organs from non-mammalian species, Wnt signaling can also regulate the extent of proliferative hair cell regeneration. This review describes the current knowledge of the roles of Wnt signaling and Wnt-responsive cells in hair cell development and regeneration. We also discuss possible future directions and the potential application and limitation of Wnt signaling in augmenting hair cell regeneration.

Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.

Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with µM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.

Normal hematopoietic stem cell function in mice with enforced expression of the Hippo signaling effector YAP1.

The Hippo pathway has recently been implicated in the regulation of organ size and stem cells in multiple tissues. The transcriptional cofactor yes-associated protein 1 (Yap1) is the most downstream effector of Hippo signaling and is functionally repressed by the upstream components of the pathway. Overexpression of YAP1 stimulates proliferation of stem and progenitor cells in many tissues, consistent with inhibition of Hippo signaling. To study the role of Hippo signaling in hematopoietic stem cells (HSCs), we created a transgenic model with inducible YAP1 expression exclusively within the hematopoietic system. Following 3 months induction, examination of blood and bone marrow in the induced mice revealed no changes in the distribution of the hematopoietic lineages compared to control mice. Moreover, the progenitor cell compartment was unaltered as determined by colony forming assays and immunophenotyping. To address whether YAP1 affects the quantity and function of HSCs we performed competitive transplantation experiments. We show that ectopic YAP1 expression does not influence HSC function neither during steady state nor in situations of hematopoietic stress. This is in sharp contrast to effects seen on stem- and progenitor cells in other organs and suggests highly tissue specific functions of the Hippo pathway in regulation of stem cells.

W41/W41 blastocyst complementation: a system for genetic modeling of hematopoiesis.

We report a rapid and highly efficient approach to generate mice in which the hematopoietic system is derived from embryonic stem (ES) cells. We show that ES cells injected into blastocysts from the c-kit-deficient W(41)/W(41) mouse strain have a clear advantage over the W(41)/W(41) blastocyst-derived inner cell mass cells in founding the hematopoietic system. Fetal liver hematopoietic stem cells from W(41)/W(41) blastocyst complementation embryos can be transplanted to establish large cohorts of bone marrow chimeras with hematopoiesis of practically pure ES-cell origin. Using ES cells with site-directed modifications, we show how this system can be used to drive inducible transgene expression in hematopoietic cells in a robust and reliable manner both in vitro and in vivo. The approach avoids the cost and time constraints associated with the creation of standard transgenic mouse strains while taking advantage of the sophisticated site-directed manipulations that are possible in ES cells.

Neonatal hematopoietic stem cell transplantation cures oc/oc mice from osteopetrosis.

OBJECTIVE: Infantile malignant osteopetrosis (IMO) is a rare autosomal recessive disorder affecting osteoclast function. Fifty percent of the patients have a mutation in the TCIRG1 gene coding for one subunit of an osteoclast proton pump. The only curative treatment is hematopoietic stem cell transplantation (SCT). The oc/oc mouse has a mutation in the gene homologous to TCIRG1 and its expected lifespan is only 3 to 4 weeks. Previous attempts to cure these mice with SCT have been unsuccessful. We wanted to determine if early hematopoietic SCT using enriched and MHC-matched stem cells can cure oc/oc mice from osteopetrosis. METHODS: One- and 8-day-old oc/oc and control mice were radiated with 200, 400, or 600 cGy and transplanted intraperitoneally with 1 or 5 x 10(6) normal lineage-depleted bone marrow cells. Blood, x-ray, and pathology analyses were performed on transplanted mice. RESULTS: All 1-day-old mice irradiated with 400 cGy and transplanted with 5 x 10(6) cells survived long term. An engraftment level of 20% is sufficient to correct most features of the disease. X-ray and histopathology examination of transplanted animals showed normalization of bone structure. However, although a correction of bone structure occurred, the transplanted oc/oc mice were smaller in size than their littermates. In contrast to untreated animals, oc/oc mice developed teeth after transplantation, but with abnormal structure and shape making them unusable. CONCLUSION: We have shown that this murine form of IMO is curable with neonatal SCT using enriched stem cells.