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issn:1567-133
1.  Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development 
Gene expression patterns : GEP  2009;9(6):444-453.
The development of the inner ear involves complex processes of morphological changes, patterning and cell fate specification that are under strict molecular control. SOX2 and SOX9 are SOX family transcription factors that are involved in the regulation of one or more of these processes. Previous findings have shown early expression of SOX9 in the otic placode and vesicle at E8.5–E9.5. Here we describe in detail, the expression pattern of SOX9 in the developing mouse inner ear beyond the otocyst stage and compare it with that of SOX2 from E9.5 to E18.5 using double fluorescence immunohistochemistry. We found that SOX9 was widely expressed in the otic epithelium, periotic mesenchyme and cartilaginous otic capsule. SOX2 persistently marked the prosensory and sensory epithelia. During the development of the sensory epithelia, SOX2 was initially expressed in all prosensory regions and later in both the supporting and hair cells up to E15.5, when its expression in hair cells gradually diminished. SOX9 expression overlapped with that of SOX2 in the prosensory and sensory region until E14.5 when its expression was restricted to supporting cells. This initial overlap but subsequent differential expression of SOX2 and SOX9 in the sensory epithelia, suggest that SOX2 and SOX9 may have distinct roles in molecular pathways that direct cells towards different cell fates.
doi:10.1016/j.gep.2009.04.003
PMCID: PMC3023882  PMID: 19427409
SOX2; SOX9; Inner ear; Otocyst; Hair cells; Sensory epithelia; Spiral ganglion
2.  Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology 
Autism susceptibility candidate 2 (Auts2) is a gene associated with autism and mental retardation, whose function is unknown. Expression of Auts2 mRNA and protein were studied in the developing mouse brain by in situ hybridization, immunohistochemistry, and western blotting. Auts2 mRNA was highly expressed in the developing cerebral cortex and cerebellum, regions often affected by neuropathological changes in autism, and a few other brain regions. On embryonic day (E) 12, Auts2 mRNA was expressed in the cortical preplate, where it colocalized with Tbr1, a transcription factor specific for postmitotic projection neurons. From E16 to postnatal day 21, Auts2 was expressed most abundantly in frontal cortex, hippocampus and cerebellum, including Purkinje cells and deep nuclei. High levels of Auts2 were also detected in developing dorsal thalamus, olfactory bulb, inferior colliculus and substantia nigra. Auts2 protein showed similar regional expression patterns as the mRNA. At the cellular level, Auts2 protein was localized in the nuclei of neurons and some neuronal progenitors.
doi:10.1016/j.gep.2009.11.005
PMCID: PMC2818569  PMID: 19948250
Auts2; Gene Expression; Patterning; Neurodevelopment; Autism
3.  Identification of differentially expressed genes in early inner ear development 
To understand the etiology of congenital hearing loss, a comprehensive understanding of the molecular genetic mechanisms underlying normal ear development is required. We are identifying genes involved in otogenesis, with the longer term goal of studying their mechanisms of action, leading to inner ear induction and patterning. Using Agilent microarrays, we compared the differential expression of a test domain (which consisted of the pre-otic placodal ectoderm with the adjacent hindbrain ectoderm and the underlying mesendodermal tissues) with a rostral control domain (which included tissue that is competent, but not specified, to express inner ear markers in explant assays). We identified 1,261 transcripts differentially expressed between the two domains at a 2-fold or greater change: 463 were upregulated and 798 were downregulated in the test domain. We validated the differential expression of several signaling molecules and transcription factors identified in this array using in situ hybridization. Furthermore, the expression patterns of the validated group of genes from the test domain were explored in detail to determine how the timing of their expression relates to specific events of otic induction and development. In conclusion, we identified a number of novel candidate genes for otic placode induction.
doi:10.1016/j.gep.2009.11.002
PMCID: PMC2818654  PMID: 19913109
Otic induction; inner ear; Wnt; Fgf; Notch; Hox; otic placode; microarray; gene expression screen
4.  Embryonic development of glial cells and myelin in the shark, Chiloscyllium punctatum 
Gene expression patterns : GEP  2009;9(8):572-585.
Glial cells are responsible for a wide range of functions in the nervous system of vertebrates. The myelinated nervous systems of extant elasmobranchs have the longest independent history of all gnathostomes. Much is known about the development of glia in other jawed vertebrates, but research in elasmobranchs is just beginning to reveal the mechanisms guiding neurodevelopment. This study examines the development of glial cells in the bamboo shark, Chiloscyllium punctatum, by identifying the expression pattern of several classic glial and myelin proteins. We show for the first time that glial development in the bamboo shark (Ch. punctamum) embryo follows closely the one observed in other vertebrates and that neural development seems to proceed at a faster rate in the PNS than in the CNS. In addition, we observed more myelinated tracts in the PNS than in the CNS, and as early as stage 32, suggesting that the ontogeny of myelin in sharks is closer to osteichthyans than agnathans.
doi:10.1016/j.gep.2009.09.001
PMCID: PMC2783534  PMID: 19733690
shark; glia; GFAP; myelin; glia development
5.  Development of Prostaglandin Endoperoxide Synthase Expression in the Ovine Fetal Central Nervous System and Pituitary 
Gene expression patterns : GEP  2009;9(8):603-611.
In this study, we tested the hypothesis that prostaglandin endoperoxide synthase -1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145 days of gestational age (DGA), 1 and 7 days postpartum lambs, and from adult ewes (n=4–5 per group). mRNA and protein were isolated from each region, and expression of Prostaglandin Synthase -1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA’s were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA’s were upregulated at 120 DGA. Tissue concentrations of PGE2 correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.
doi:10.1016/j.gep.2009.08.003
PMCID: PMC2784086  PMID: 19706338
parturition; prostanoid; neuroendocrinology; fetus; HPA axis; brain
6.  Trim43a, Trim43b, and Trim43c: novel mouse genes expressed specifically in mouse preimplantation embryos 
Gene expression patterns : GEP  2009;9(8):595-602.
We describe the identification and characterization of Trim43a, Trim43b, and Trim43c genes, whose expression are restricted to preimplantation stages and peak at the 8-cell to morula stage. We identified a 5 kb DNA fragment that covers upstream region of Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos.
doi:10.1016/j.gep.2009.08.002
PMCID: PMC2784184  PMID: 19703589
mouse preimplantation; gene expression; 8-cell; morula; fluorescent proteins; asymmetric PCR; promoter analysis
7.  Expression of five frizzleds during zebrafish craniofacial development 
Gene expression patterns : GEP  2009;9(7):520-527.
Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.
doi:10.1016/j.gep.2009.07.003
PMCID: PMC2748146  PMID: 19595791
zebrafish; frizzled 2; frizzled 6; frizzled 7a; frizzled 7b; frizzled 8a; cartilage; craniofacial; Wnt; planar cell polarity
8.  Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish 
Gene expression patterns : GEP  2009;9(7):490-496.
In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 hours post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.
doi:10.1016/j.gep.2009.07.006
PMCID: PMC2748168  PMID: 19616130
development; cell adhesion molecules; brain; retina; spinal cord
9.  Differential expression of Rfx1–4 during mouse spermatogenesis 
Gene expression patterns : GEP  2009;9(7):515-519.
The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1–4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1–3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.
doi:10.1016/j.gep.2009.07.004
PMCID: PMC2761754  PMID: 19596083
Alternate promoter; Gene regulation; Meiosis; Mouse; Real-time PCR; Spermatid; Spermatogenesis; Spermatogonia; Transcription factor; 5'-RACE
10.  An expression screen for RhoGEF genes involved in C. elegans gonadogenesis 
Gene expression patterns : GEP  2009;9(6):397-403.
The gonad in C. elegans is an important model system for understanding complex morphogenetic processes including cellular movement, cell fusion, cell invasion and cell polarity during development. One class of signaling proteins known to be critical for the cellular events underlying morphogenesis is the Rho family GTPases, particularly RhoA, Rac and Cdc42. In C. elegans orthologues of these genes have been shown to be important for gonad development. In our current study we have extended those findings by examining the patterns of 5’ cis-regulatory element (5’CRE) activity associated with nineteen putative guanine nucleotide exchange factors (GEFs) encoded by the C. elegans genome predicted to activate Rho family GTPases. Here we identify thirteen RhoGEF genes that are expressed during gonadogenesis and characterize the cells in which their 5’CREs are active. These data provide the basis for designing experiments to examine Rho GTPase activation during morphogenetic processes central to normal gonad development.
doi:10.1016/j.gep.2009.06.005
PMCID: PMC2726742  PMID: 19540360
C. elegans; gonadogenesis; RhoGEF; morphogenesis; organogenesis; anchor cell invasion; gonad development; Rho GTPase; rho; rac; cdc42; mig-2; ced-10; cdc-42; rho-1
11.  fezf2 expression delineates cells with proliferative potential and expressing markers of neural stem cells in the adult zebrafish brain 
Gene expression patterns : GEP  2009;9(6):411-422.
Fezf2 (also known as Fezl, ZNF312, or Zfp312) is an evolutionarily conserved forebrain-specific zinc finger transcription factor that is expressed during development and is implicated in patterning as well as neurogenesis in both zebrafish and mice. Despite these findings, the expression of fezf2 in the adult brain has not been well characterized, and fezf2 function in the adult brain remains unknown. The zebrafish has recently emerged as a new model system to study adult neurogenesis, given its similarity to mammalian systems and enhanced capability of undergoing adult neurogenesis. Through RNA in situ hybridization and using a fezf2 promoter-driven GFP transgenic line, we present data showing that fezf2 is expressed in radial glial progenitor cells of the telencephalic ventricular zone in the adult zebrafish brain, which co-express markers of neural stem cells and proliferation. Additionally, we identify the preoptic region and the hypothalamus as fezf2-expressing neurogenic regions in the adult zebrafish brain, where fezf2 labels progenitor cells as well as postmitotic neurons. Our findings establish Fezf2 as a novel marker for adult telencephalic ventricular progenitor cells that express markers of neural stem cells in zebrafish and lay a critical foundation for future investigation of Fezf2 function in the maintenance and differentiation of neural stem cells in the adult vertebrate brain.
doi:10.1016/j.gep.2009.06.002
PMCID: PMC2746713  PMID: 19524703
Fezf2; Radial glia; Ventricular progenitor cells; Telencephalon; Zebrafish; Transgenic; Adult neural stem cells; Adult neurogenesis; Olfactory bulb; Preoptic region; Hypothalamus
12.  Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension 
Gene expression patterns : GEP  2009;9(6):430-435.
Cell-cell signaling regulated by retinoic acid (RA), Wnt/β-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/β-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2−/− mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2−/− mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/β-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/β-catenin signaling as the body axis extends.
doi:10.1016/j.gep.2009.06.003
PMCID: PMC2791796  PMID: 19539783
Retinoic acid signaling; Wnt/β-catenin signaling; FGF signaling; Spry2; Pea3; Axin2; Fgf8; Wnt8a; Wnt3a; Tbx6; Cdx; axis extension
13.  FIAT is Co-expressed with its Dimerization Target ATF4 in early Osteoblasts, but not in Osteocytes 
Gene expression patterns : GEP  2009;9(5):335-340.
FIAT represses osteocalcin gene transcription by heterodimerizing with ATF4 and preventing it from binding to DNA. We report here the expression profiles of FIAT and ATF4 during osteoblastogenesis. Messenger RNA levels for the osteoblast transcriptional regulators Satb2, Runx2, Fiat and Atf4 were quantified using Real Time-reverse transcription PCR (RT-qPCR) and respective protein levels monitored by immunodetection in differentiating primary osteoblast cultures. Satb2, Fiat and Atf4 mRNA levels remained constant throughout the differentiation sequence, whereas Runx2 transcript levels were significantly increased by 12 days post-confluency. Using immunofluorescence, the SATB2, RUNX2, and ATF4 signals appeared to increase as a function of time in culture. FIAT protein expression was readily detected in early cultures, but signal intensity decreased thereafter. When immunoblotting was used to quantify the relative amounts of FIAT and ATF4 proteins, the expression levels of the two proteins were found to be inversely correlated. The decrease in FIAT protein levels coincided with increased binding of ATF4 to the osteocalcin gene promoter, and with increased osteocalcin expression measured by RT-qPCR or immunoblotting. Immunohistochemistry of long bones from mice at E16.5 and 2 days post-natal revealed that both proteins are initially expressed in osteoblasts. In adult bone, FIAT was detected in osteocytes, while ATF4 expression was observed in active osteoblasts and lining cells, but not in osteocytes. Taken together, these data support the idea that a stoichiometric excess of ATF4 over FIAT in mature osteoblasts releases ATF4 from sequestration by FIAT, thereby allowing ATF4 homodimerization and subsequent transactivation of the osteocalcin gene.
doi:10.1016/j.gep.2009.02.002
PMCID: PMC2692570  PMID: 19232401
ATF4; FIAT; leucine zipper transcription factors; osteoblasts; osteocytes; osteoblastogenesis; bone development
14.  BMP and BMP receptor expression during murine organogenesis 
Gene expression patterns : GEP  2009;9(5):255-265.
Cell-cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell-cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.
doi:10.1016/j.gep.2009.04.002
PMCID: PMC2709213  PMID: 19393343
Organogenesis; Bmp2; Bmp4; Bmp7; Bmpr1a; Bmpr1b; BmprII; Noggin; heart; lung; gut; stomach; limb bud; neural fold; somite
15.  Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues 
Gene expression patterns : GEP  2009;9(5):314-323.
The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E 11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchii and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.
doi:10.1016/j.gep.2009.02.006
PMCID: PMC2725439  PMID: 19250981
ADAMTS5; Adamts5; Aggrecanase; Development; Mouse; Transgenic; Arthritis; Osteoarthritis; Aggrecan; Versican; LacZ; Beta-galactosidase; In situ hybridization; Peripheral nerve; Schwann cell; Smooth muscle; Skeletal muscle; Sympathetic ganglia; Inter-digital mesenchyme; Dorsal Root Ganglia; Choroid Plexus; Cartilage; Tendon; Fibroblast
16.  Expression Of Nucleosomal Protein HMGN1 In The Cycling Mouse Hair Follicle 
Gene expression patterns : GEP  2009;9(5):289-295.
Here we examine the expression pattern of HMGN1, a nucleosome binding protein that affects chromatin structure and activity, in the hair follicle and test whether loss of HMGN1 affects the development or cycling of the follicle. We find that at the onset of hair follicle development, HMGN1 protein is expressed in the epidermal placode and in aggregated dermal fibroblasts. In the adult hair follicle, HMGN1 is specifically expressed in the basal layer of epidermis, in the outer root sheath, in the hair bulb, but not in the inner root sheath and hair shaft. The expression pattern of HMGN1 is very similar to p63, suggesting a role for HMGN1 in the transiently amplifying cells. We also find HMGN1 expression in some, but not all hair follicle stem cells as detected by its colocalization with Nestin and with BrdU label-retaining cells. The appearance of the skin and hair follicle of Hmgn1-/- mice was indistinguishable from that of their Hmgn1+/+ littermates. We found that in the hair follicle the expression of HMGN2 is very similar to HMGN1 suggesting functional redundancy between these closely related HMGN variants.
doi:10.1016/j.gep.2009.03.002
PMCID: PMC2738608  PMID: 19303948
chromatin; HMGN; p63; hair follicle
17.  Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system 
Gene expression patterns : GEP  2009;9(7):475-489.
A long-standing problem in development is understanding how progenitor cells transiently expressing genes contribute to complex anatomical and functional structures. In the developing nervous system an additional level of complexity arises when considering how cells of distinct lineages relate to newly established neural circuits. To address these problems, we used both cumulative marking with Cre/loxP and Genetic Inducible Fate Mapping (GIFM), which permanently and heritably marks small populations of progenitors and their descendants with fine temporal control using CreER/loxP. A key component used in both approaches is a conditional phenotyping allele that has the potential to be expressed in all cell types, but is quiescent because of a loxP flanked Stop sequence, which precedes a reporter allele. Upon recombination, the resulting phenotyping allele is ‘turned on’ and then constitutively expressed. Thus, the reporter functions as a high fidelity genetic lineage tracer in vivo. Currently there is an array of reporter alleles that can be used in marking strategies, but their recombination efficiency and applicability to a wide array of tissues has not been thoroughly described. To assess the recombination/marking potential of the reporters, we utilized CreERT under the control of a Wnt1 transgene (Wnt1-CreERT) as well as a cumulative, non-inducible En1Cre knock-in line in combination with three different reporters: R26R (LacZ reporter), Z/EG (EGFP reporter), and Tau-Lox-STOP-Lox-mGFP-IRES-NLS-LacZ (membrane-targeted GFP/nuclear LacZ reporter). We marked the Wnt1 lineage using each of the three reporters at embryonic day (E) 8.5 followed by analysis at E10.0, E12.5, and in the adult. We also compared cumulative marking of cells with a history of En1 expression at the same stages. We evaluated the reporters by whole-mount and section analysis and ascertained the strengths and weaknesses of each of the reporters. Comparative analysis with the reporters elucidated complexities of how the Wnt1 and En1 lineages contributed to developing embryos and axonal projection patterns of neurons derived from these lineages.
doi:10.1016/j.gep.2009.07.007
PMCID: PMC2855890  PMID: 19616131
En1Cre; Wnt1-CreERT; cell lineage; neural circuits; midbrain; spinal cord; trigeminal ganglia; conditional phenotyping alleles; Genetic Inducible Fate Mapping
18.  Identification of differentially expressed genes in the zebrafish hypothalamus - pituitary axis 
Gene expression patterns : GEP  2009;9(4):200-208.
The vertebrate hypothalamic-pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in adenohypophysis ontogenesis, little is known about hypothalamic and neurohypophysial development or when the HP matures and becomes functional. To identify markers of the HP, we constructed subtractive cDNA libraries between adult zebrafish hypothalamus and pituitary. We identified previously published genes and ESTs and novel zebrafish genes, some of which were predicted by genomic database analysis. We also analyzed expression patterns of these genes and found that several are expressed in the embryonic and larval hypothalamus, neurohypophysis, and/or adenohypophysis. Expression at these stages makes these genes useful markers to study HP maturation and function.
doi:10.1016/j.gep.2008.12.007
PMCID: PMC2804439  PMID: 19166982
Adenohypophysis; cell differentiation; hormone; hypothalamic-pituitary axis; hypothalamus; neurohypophysis; organ maturation; pituitary; subtractive cDNA library
19.  Characterization of spatial and temporal expression pattern of SCG10 during zebrafish development 
Gene expression patterns : GEP  2009;9(4):231-237.
SCG10 (Superior Cervical Ganglia 10, STMN2) is a member of the stathmin family of proteins. Stathmins regulate microtubule dynamics by inhibiting polymerization and promoting their depolymerization. SCG10 is believed to be a neuronal-specific stathmin that is enriched in the growth cones of developing neurons and plays a role in regulating neurite outgrowth. In all species examined so far, SCG10 is expressed in both the CNS and PNS.
We have cloned two zebrafish SCG10 homologues and have determined the temporal and spatial expression pattern of both of these genes by RT-PCR and in situ hybridization. RT-PCR shows that both transcripts are expressed maternally and zygotically through at least 5 days. In situ hybridization analysis reveals that both SCG10 orthologues have dynamic, spatial expression patterns that are nearly identical to each other. Initially, these orthologues are expressed in discrete areas of the forebrain, midbrain, and hindbrain, as well as in the anterior and posterior lateral line ganglia and transiently in the spinal cord Rohon-Beard neurons. From 48hpf onwards, the level of expression of both genes increases and becomes mainly restricted to the anterior CNS (the forebrain region, retina, optic tectum and hindbrain), and to the cranial ganglia. From 72 to 96 hpf, SCG10 genes are also expressed in the developing neurons in the gut and in the surrounding intestinal mesenchyme. Our results provide a starting point for future studies that will investigate the in vivo function of SCG10 orthologues in zebrafish neural development.
doi:10.1016/j.gep.2008.12.010
PMCID: PMC2670952  PMID: 19272335
SCG10; STMN2; zgc:92905; zgc:110132; stathmins; zebrafish; danio rerio; neuronal development; central nervous system; anterior lateral line ganglia; posterior lateral line ganglia; cranial ganglia; Rohon-Beard neurons; peripheral nervous system; enteric neurons; sympathetic chains

Results 1-19 (19)