FGF signaling through FGFR1 is required for olfactory bulb morphogenesis

During development, the embryonic telencephalon is patterned into different areas that give rise to distinct adult brain structures. Several secreted signaling molecules are expressed at putative signaling centers in the early telencephalon. In particular, Fgf8 is expressed at the anterior end of the telencephalon and is hypothesized to pattern it along the anteroposterior (AP) axis. Using a CRE/loxP genetic approach to disrupt genes in the telencephalon, we address the role of FGF signaling directly in vivo by abolishing expression of the FGF receptor Fgfr1. In the Fgfr1-deficient telencephalon, AP patterning is largely normal. However, morphological defects are observed at the anterior end of the telencephalon. Most notably, the olfactory bulbs do not form normally. Examination of the proliferation state of anterior telencephalic cells supports a model for olfactory bulb formation in which an FGF-dependent decrease in proliferation is required for initial bulb evagination. Together the results demonstrate an essential role for Fgfr1 in patterning and morphogenesis of the telencephalon.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling

ObjectivesTo explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model.MethodsMorpholino oligonucleotide was used to induce Dnmt1 deficiency. RNA sequencing, in situ hybridization (ISH), whole genomic bisulfide sequencing (WGBS) and immunostaining were used to investigate the morphologic alterations and mechanisms.ResultsWe found that downregulation of Dnmt1 induced decreased number of neuromasts and repressed cell proliferation of primordium in the developing posterior lateral line system of zebrafish. The ISH data uncovered that Fgf signalling pathway was inhibited and the expression of chemokine members cxcr4b, cxcr7b and cxcl12a were interfered, while lef1 expression was increased after inhibiting Dnmt1. Additionally, Dnmt1 downregulation led to malformed otoliths and deformed semicircular canals, and hair cell differentiation in utricle and saccule was inhibited severely. The in situ staining of otic placode markers pax2/5 and fgf 3/8/10 was decreased when Dnmt1 downregulated. The WGBS analysis demonstrated that the global methylation status was markedly downregulated, and cell cycle genes were among those most differently expressed between Dnmt1 morphants and the controls. Further ISH analysis confirmed the findings by RNA‐seq and WGBS assay that cdkn1a and tp53 were both upregulated after knockdown of Dnmt1.ConclusionOur results revealed that Dnmt1 is essential for the development of zebrafish auditory organ through regulating cell cycle genes together with Wnt and Fgf signalling pathways.

     

Dp1 is required for extra-embryonic development

Release of E2F1/DP1 heterodimers from repression mediated by the retinoblastoma tumor suppressor (pRB) triggers cell cycle entry into S phase, suggesting that E2F1 and DP1 proteins must act in unison, either to facilitate or to suppress cell-cycle progression. In stark contrast to the milder phenotypes that result from inactivation of E2Fs, we report that loss of Dp1 leads to death in utero because of the failure of extra-embryonic development. Loss of Dp1 compromises the trophectoderm-derived tissues - specifically, the expansion of the ectoplacental cone and chorion, and endoreduplication in trophoblast giant cells. Inactivation of p53 is unable to rescue the Dp1-deficient embryonic lethality. Thus, DP1 is absolutely required for extra-embryonic development and consequently embryonic survival, consistent with E2F/DP1 normally acting to promote growth in vivo.     

The auditory sensory epithelium: the instrument of sound perception

The auditory sensory epithelium is the specialized region of the cochlear epithelium that transduces sound. It is composed of a highly ordered, repeated array of mechanosensory hair cells and nonsensory supporting cells that run along the length of the cochlea. On the apical surface of the hair cells is a specialized structure called the hair bundle that deflects in response to sound vibration, resulting in depolarization of the hair cell and neurotransmitter release. Formation of the auditory sensory epithelium during embryogenesis involves strict control of both cell proliferation and cell patterning. Misregulation of these events can lead to congenital hearing loss, and damage to the auditory sensory epithelium during adult life can lead to adult-onset deafness. This paper reviews recent data on the formation of the auditory sensory epithelium during embryogenesis, the identification of components of the sound transduction apparatus, and advances in the treatment of hearing impairment.     

Hairless is required for the development of adult sensory organ precursor cells in Drosophila

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled us to identify genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted us to investigate the cellular development of sensilla in these same positions in larvae and pupae and thereby establish the developmental basis for the mutant phenotypes. We have found that H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, we were unable at the appropriate stages of development to detect sensillum-specific cell types, the precursor cell divisions that generate them, or the primary precursor cells themselves. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development.     

Hmga1 is required for normal sperm development

The Hmgi protein family of chromosomal architectural factors is extensively studied for its roles in embryogenesis and its association with benign mesenchymal tumors. Although the biochemical function of Hmga1 has been studied in vitro, to provide in vivo insight into its biological function, a targeted disruption of Hmga1 was initiated. Chimeric founder mice were derived from embryonic stem (ES) cells harboring a targeted mutation in a single Hmga1 allele. These 14 different chimeric founders produced 494 black progeny. Since none of these 494 progeny were agouti, none of them were derived from ES cells. Control injections of the wild-type ES cell lines resulted in ES cell derived agouti mice, indicating that the ES cells were totipotent. Therefore, our results indicate that one intact Hmga1 allele was not sufficient for germ-line transmission of the ES cells. Seven chimeric founder mice that were examined histologically demonstrated aberrant regions in their reproductive organs. Aberrant regions of seminiferous tubules were reduced in diameter, demonstrated vacuolated Sertoli cells, and had an absolute deficiency of sperm. While the Hmga1(+/-) ES cells were shown to contribute to the formation of the epididymides, they did not significantly contribute to the testes of chimeric founder mice. No sperm isolated from any of the Hmga1(+/-) chimeric mice were shown to arise from the ES cells, as none of them contained the targeted disruption of the Hmga1 gene. Our results suggest that both alleles of Hmga1 are required for normal sperm production in the mouse.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.     

Pkd1 is required for male reproductive tract development

Reproductive tract abnormalities and male infertility have higher incidence in ADPKD patients than in general populations. In this work, we reveal that Pkd1, whose mutations account for 85% of ADPKD cases, is essential for male reproductive tract development. Disruption of Pkd1 caused multiple organ defects in the murine male reproductive tract. The earliest visible defect in the Pkd1^?/? reproductive tract was cystic dilation of the efferent ducts, which are derivatives of the mesonephric tubules. Epididymis development was delayed or arrested in the Pkd1^?/? mice. No sign of epithelial coiling was seen in the null mutants. Disruption of Pkd1 in epithelium alone using the Pax2-cre mice was sufficient to cause efferent duct dilation and coiling defect in the epididymis, suggesting that Pkd1 is critical for epithelium development and maintenance in male reproductive tract. In-depth analysis showed that Pkd1 is required to maintain tubulin cytoskeleton and important for Tgf-β/Bmp signal transduction in epithelium of male reproductive tract. Altogether, our results for the first time provide direct evidence for developmental roles of Pkd1 in the male reproductive tract and provide new insights in reproductive tract abnormalities and infertility in ADPKD patients.     

PTBP1 is required for embryonic development before gastrulation

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.     

The FGFR pathway is required for the trunk-inducing functions of Spemann's organizer.

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.     

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.     

Prefoldin 5 is required for normal sensory and neuronal development in a murine model

Molecular chaperones and co-chaperones are crucial for cellular development and maintenance as they assist in protein folding and stabilization of unfolded or misfolded proteins. Prefoldin (PFDN), a ubiquitously expressed heterohexameric co-chaperone, is necessary for proper folding of nascent proteins, in particular, tubulin and actin. Here we show that a genetic disruption in the murine Pfdn5 gene, a subunit of prefoldin, causes a syndrome characterized by photoreceptor degeneration, central nervous system abnormalities, and male infertility. Our data indicate that a missense mutation in Pfdn5, may cause these phenotypes through a reduction in formation of microtubules and microfilaments, which are necessary for the development of cilia and cytoskeletal structures, respectively. The diversity of phenotypes demonstrated by models carrying mutations in different PFDN subunits suggests that each PFDN subunit must confer a distinct substrate specificity to the prefoldin holocomplex.     

ZEN-4/MKLP1 is required to polarize the foregut epithelium

BACKGROUND: Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. Here, we describe a novel function during tubulogenesis for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. RESULTS: Our analyses demonstrate that ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. CONCLUSION: We suggest that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ.