The brain of Drosophila is formed by approximately 100 lineages, each lineage being derived from a stem cell-like neuroblast that segregates from the procephalic neurectoderm of the early embryo. A neuroblast map has been established in great detail for the early embryo, and a suite of molecular markers has been defined for all neuroblasts included in this map (Urbach and Technau, 2003a). However, the expression of these markers was not followed into later embryonic or larval stages, mainly due to the fact that anatomical landmarks to which expression patterns could be related had not been defined. Such markers, in the form of stereotyped clusters of neurons whose axons project along cohesive bundles (“primary axon bundles” or “PABs”) are now available (Younossi-Hartenstein et al., 2006). In the present study we have mapped the expression of molecular markers in relationship to primary neuronal clusters and their PABs. The markers we analyzed include many of the genes involved in patterning of the brain along the anteroposterior axis (cephalic gap genes, segment polarity genes) and dorso-ventral axis (columnar patterning genes), as well as genes expressed in the dorsal protocerebrum and visual system (early eye genes). Our analysis represents an important step along the way to identify neuronal lineages of the mature brain with genes expressed in the early embryo in discrete neuroblasts. Furthermore, the analysis helped us to reconstruct the morphogenetic movements that transform the two-dimensional neuroblast layer of the early embryo into the three-dimensional larval brain and provides the basis for deeper understanding of how the embryonic brain develops.
Drosophila; embryonic brain; brain development; Hox genes; pair rule genes; segment polarity genes; head gap genes; retinal patterning genes; columnar patterning genes
Sphingosine-1-phosphate lyase (SPL) catalyzes the degradation of sphingosine-1-phosphate (S1P), a bioactive lipid that controls cell proliferation, migration and survival. Mice lacking SPL expression exhibit developmental abnormalities, runting and death during the perinatal period, suggesting that SPL plays a role in mammalian development and adaptation to extrauterine life. We investigated the pattern of SPL expression in the mouse embryo and placenta from day 8 to day 18. Our findings reveal that SPL is expressed in the developing brain and neural tube, Rathke’s pouch, first brachial arch, third brachial arch, optic stalk, midgut loops, and lung buds. Diffuse signal was high at E12, whereas a recognizable adult SPL pattern was evident by E15 and more intensely at E18, with strong expression in skin, nasal epithelium, intestinal epithelium, cartilage, thymus and pituitary gland. These findings suggest SPL may be involved in development of the mammalian central nervous system (CNS), anterior pituitary, trigeminal nerve, palate and facial bones, thymus and other organs. Our findings are consistent with the SPL expression pattern of the adult mouse and with congenital abnormalities observed in SPL mutant mice.
sphingosine-1-phosphate; sphingosine phosphate lyase; S1P lyase; Sgpl1; embryogenesis; sphingolipid
Foxi2 and Foxi3 are members of the Foxi class of Forkhead transcription factors. The Foxi transcription factor family has been shown to play roles in the development of the inner ear and pharyngeal arch derivatives in zebrafish. We describe the expression of Foxi2 and Foxi3 in chicken embryos during the first three days of embryonic development. Foxi3 is initially expressed broadly in the pre-placodal ectoderm surrounding the neural plate, which will give rise to all craniofacial sensory organs. It then becomes restricted to a region immediately anterior to the first pair of somites that will give rise to the otic and epibranchial placodes, before becoming down-regulated from this region and restricted to the ectoderm and endoderm of the pharyngeal arches. In contrast, Foxi2 is initially expressed broadly in cranial ectoderm with the striking exception of the otic placode, and ultimately becomes restricted to pharyngeal arch ectoderm. These expression patterns provide an insight into the roles of these transcriptional regulators during the development of the inner ear and pharyngeal arch derivatives.
Otic placode; Pharyngeal arch; Craniofacial development; Pre-placodal domain
Fmlns belong to the Formin family, catalysts of linear Actin polymerization with mostly unknown roles in vivo. In cell culture Fmnls are involved in cell migration and adhesion and the formation of different types of protrusions including filopodia and blebs, suggesting important roles during development. Moreover, Fmnls can act downstream of Rac and Cdc42, mediators of cytoskeletal changes as targets of important pathways required for shaping tissues. The zebrafish genome encodes five Fmnls. Here we report their tissue specific expression patterns during early development and pharyngula stages. The fmnls show overlapping and distinct expression patterns, which suggest that they could regulate similar processes during development, but may also have independent functions. In particular, we find a strong maternal contribution of all fmnls, but distinct expression patterns in the developing brain eye, ear, heart and vascular system.
Actin; vascular system; visual system; otic vesicles; brain
The Twirler mutation arose spontaneously and causes inner ear defects in heterozygous and cleft lip and/or cleft palate in homozygous mutant mice, providing a unique animal model for investigating the molecular mechanisms of inner ear and craniofacial development. Here we report the identification of a novel homeobox gene, Iroquois-related homeobox like-1 (Irxl1), from the Twirler locus. Irxl1 encodes a TALE-family homeodomain protein with its homeodomain exhibiting the highest amino acid sequence identity (54%) to those of invertebrate Iroquois and vertebrate Irx subfamily members. The putative Irxl1 protein lacks the Iro-box, a conserved motif in all known members of the Irx subfamily. Searching the databases showed that Irxl1 orthologs exist in Xenopus, chick, and mammals. In situ hybridization analyses of mouse embryos at various developmental stages showed that Irxl1 mRNA is highly expressed in the frontonasal process and palatal mesenchyme during primary and secondary palate development. In addition, Irxl1 mRNA is strongly expressed in mesenchyme surrounding the developing inner ear, in discrete regions of the developing mandible, in the dermamyotome during somite differentiation, and in a subset of muscular structures in late embryonic stages. The developmental expression pattern indicates that Irxl1 is a good candidate gene for the Twirler gene.
cleft lip; cleft palate; craniofacial development; dermamyotome; homeobox; homeodomain; Hox; intervertebral disc; Iroquois; Irx; Irxl1; mandible; palate development; secondary palate; skeletal muscle; somite; TALE; Twirler mutation
FGFs1 with similar sequences can play different roles depending on the model organisms examined. Determining these roles requires knowledge of spatio-temporal Fgf gene expression patterns. In this study, we report the cloning of chick Fgf5, 6 and 7, and examine their gene expression patterns by whole mount in situ hybridization. We show that Fgf5's spatio-temporally restricted expression pattern indicates a potentially novel role during inner ear development. Fgf6 and Fgf7, although belonging to different subfamilies with diverged sequences, are expressed in similar patterns within the mesoderm. Alignment of protein sequences and phylogenetic analysis demonstrate that FGF5 and FGF6 are highly conserved between chick, human, mouse and zebrafish. FGF7 is similarly conserved except for the zebrafish, which has considerably diverged.
Development; Fibroblast growth factor; otic placode; pharyngeal arch; pharyngeal endoderm
Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (Danio rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost RNA was noted during 12 hours post-fertilization (hpf). At 15 hpf, sost RNA was detected in the developing nervous system and in Kupffer’s vesicle. At 18, 20 and 22 hpf, expression in rhombic lip precursors was seen. By 24 hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28 hpf and then diminished in intensity through 44 hpf. At 28 hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48 hpf, sost RNA was clearly detected in the developing pharyngeal arch cartilage. Sost RNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120 hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.
Sclerostin; sost; skeleton; cartilage; brain
In the developing CNS, unique functional identities among neurons and glia are, in part, established as a result of successive transitions in gene expression programs within neural precursor cells. One of the temporal-identity windows within Drosophila CNS neural precursor cells or neuroblasts (NBs) is marked by the expression of a zinc-finger transcription factor (TF) gene, castor (cas). Our analysis of cis-regulatory DNA within a cas loss-of-function rescue fragment has identified seven enhancers that independently activate reporter transgene expression in specific sub-patterns of the wild-type embryonic cas gene expression domain. Most of these enhancers also regulate different aspects of cas expression within the larval and adult CNS. Phylogenetic footprinting reveals that each enhancer is made up of clusters of highly conserved DNA sequence blocks that are flanked by less-conserved inter-cluster spacer sequences. Comparative analysis of the conserved DNA also reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. Intra-species alignments show that two of the sub-pattern enhancers originated from an inverted duplication and that this repeat is unique to the cas locus in all sequenced Drosophila species. Finally we show that three of the enhancers differentially require cas function for their wild-type regulatory behavior. Cas limits the expression of one enhancer while two others require cas function for full expression. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it.
Drosophila CNS development; cis-regulatory DNA; sub-pattern enhancers; DNA sequence conservation; castor gene regulation
Rb1 is a tumor suppressor gene that regulates cell cycle progression through interactions with E2F transcription factors. In recent years, new roles for Rb1 in regulating cellular differentiation have also emerged. For example, it has been shown that Rb1 regulates osteoblast differentiation in a cell cycle independent manner, by binding to the transcription factor Runx2, and facilitating the up-regulation of late bone differentiation markers. Based on the facts that Runx2 also functions in tooth development, and that little is known about potential roles for Rb1 in mammalian tooth development, here we evaluated the expression of Rb1 mRNA in developmentally staged mouse teeth. Our data show that Rb1 mRNA is expressed in both dental epithelial and dental mesenchymal progenitor cells. In addition, Rb1 mRNA appears upregulated in differentiating ameloblasts and odontoblasts, suggesting roles for Rb1 in tooth differentiation.
Rb1; tooth development; dental epithelium; dental mesenchyme; cell-cell signaling
Sperm transport, maturation and storage are the essential functions of the epididymis. The epididymis in the mouse is structurally characterized by regional and segmental organization including caput, corpus and cauda epididymis that are comprised of ten segments. Although several growth factor signaling pathways have been discovered in the epididymis, how these converge onto the cell cycle components is unknown. To begin to elucidate the growth factor control of cell cycle events in the epididymis, we analyzed the expression of D-type cyclins at different postnatal ages. At 7d, cyclin D1 was mainly expressed in the cauda epithelium, by 14d its expression occurred in the epithelium of caput, corpus and cauda that persisted up to 21d. By 42d, cyclin D1 was mostly detectable in the principal cells of the caput and corpus (segments 1–7) but not in the cauda epididymis. Expression of cyclin D2, unlike that of cyclin D1, was evident only at 42d but not earlier, and was mostly confined to corpus and cauda epithelium. In contrast to both cyclin D1 and D2, cyclin D3 was expressed primarily in the interstitium at 7d and by 21d its expression was localized to the epithelium of the corpus and cauda epididymis. By 42d, expression of cyclin D3 peaked in segments 6–10 and confined to basal and principal cells of the corpus and apical cells of the cauda epithelium. Ki67 immunoreactivity confirmed absence of cell proliferation despite continued expression of D-type cyclins in the adult epididymis. Collectively, on the basis of our immunophenotyping and protein expression data, we conclude that the D-type cyclins are expressed in a development -, segment-, and cell- specific manner in the postnatal mouse epididymis.
Epididymis; Cyclin D1; Cyclin D2; Cyclin D3; Ki67
Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the 4 cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.
Xenopus; Mustn1; chondrogenesis; Sox9; morpholino; knockdown; craniofacial; somite
Zfhep/δEF1 is essential for embryonic development. We have investigated the expression pattern of Zfhep protein during mouse embryogenesis. We show expression of Zfhep in the mesenchyme of the palatal shelves, establishing concordance of expression with the reported cleft palate of the δEF1-null mice. Zfhep protein is strongly expressed in proliferating progenitors of the nervous system. In most regions of the brain, post-mitotic cells stop expressing Zfhep when they migrate out of the ventricular zone and differentiate. However, in the hindbrain, Zfhep protein is also highly expressed in post-mitotic migratory neuronal cells of the precerebellar extramural stream that arise from the neuroepithelium adjacent to the lower rhombic lip. Also, Zfhep is expressed as cells migrate from a narrow region of the pons ventricular zone towards the trigeminal nucleus. Co-expression with Islet1 shows that Zfhep is expressed in motor neurons of the trigeminal nucleus of the pons, but not in the inferior olive motor neurons at E12.5. Therefore, Zfhep is strongly expressed in a tightly regulated pattern in proliferating neural stem cells and a subset of neurons. Zfhep protein is also strongly expressed in trigeminal ganglia, and is moderately expressed in other cranial ganglia. In vitro studies have implicated Zfhep as a repressor of myogenesis, however, we find that Zfhep protein expression increases during muscle differentiation.
AREB6; brain nuclei; cleft; cleft palate; cranial ganglia; delta EF-1; ganglion; heart; hindbrain; medulla; muscle; myogenesis; migration; neurogenesis; neurogenin; pons; pontine nucleus; precerebellar; rhombic lip; trigeminal; ventricular zone; ZEB; Zfhx1a
Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is a rare inherited strabismus syndrome characterized by non-progressive ophthalmoplegia. We previously identified that CFEOM1 results from heterozygous missense mutations in KIF21A, which encodes a kinesin motor protein. Here we evaluate the expression pattern of KIF21A in human brain and muscles of control and CFEOM1 patients, and during human and mouse embryonic development. KIF21A is expressed in the cell bodies, axons, and dendrites of many neuronal populations including those in the hippocampus, cerebral cortex, cerebellum, striatum, and motor neurons of the oculomotor, trochlear, and abducens nuclei from early development into maturity, and its spatial distribution is not altered in the CFEOM1 tissues available for study. Multiple splice isoforms of KIF21A are identified in human fetal brain, but none of the reported CFEOM1 mutations are located in or near the alternatively spliced exons. KIF21A immunoreactivity is also observed in extraocular and skeletal muscle biopsies of control and CFEOM1 patients, where it co-localizes with triadin, a marker of the excitation-contractile coupling system. The diffuse and widespread expression of KIF21A in the developing human and mouse central and peripheral nervous system as well as in extraocular muscle does not account for the restricted ocular phenotype observed in CFEOM1, nor does it permit the formal exclusion of a myogenic etiology based on expression patterns alone.
We employ non-radioactive in situ hybridization techniques, which combine good tissue morphology preservation with high sensitivity of transcript detection, to map gene expression in the regenerating digestive tube of the sea cucumber Holothuria glaberrima. We investigated localization of transcripts of Wnt9, TCTP, Bmp1/Tll, the genes that have been previously known to be implicated in embryogenesis and cancer. The choice was determined by our long-term goal of trying to understand how the developmental regulatory pathways known to be involved in tumor development can be activated in post-traumatic regeneration without leading to malignant growth. The gene expression data combined with the available morphological information highlight the gut mesothelium (the outer layer of the digestive tube) as a highly dynamic tissue, whose cells undergo remarkable changes in their phenotype and gene expression in response to injury. This reversible transition of the gut mesothelium from a complex specialized tissue to a simple epithelium composed of rapidly proliferating multipotent cells seems to depend on the expression of genes from multiple developmental/cancer-related pathways.
The specification of temporal identity within single progenitor lineages is essential to generate functional neuronal diversity in Drosophila and mammals. In Drosophila, four transcription factors are sequentially expressed in neural progenitors (neuroblasts) and each regulates the temporal identity of the progeny produced during its expression window. The first temporal identity is established by the Ikaros-family zinc finger transcription factor Hunchback (Hb). Hb is detected in young (newly-formed) neuroblasts for about an hour and is maintained in the early-born neurons produced during this interval. Hb is necessary and sufficient to specify early-born neuronal or glial identity in multiple neuroblast lineages. The timing of hb expression in neuroblasts is regulated at the transcriptional level. Here we identify the cis-regulatory elements that confer proper hb expression in “young” neuroblasts and early-born neurons. We show that the neuroblast element contains clusters of predicted binding sites for the Seven-up transcription factor, which is known to limit hb neuroblast expression. We identify highly conserved sequences in the neuronal element that are good candidates for maintaining Hb transcription in neurons. Our results provide the necessary foundation for identifying trans-acting factors that establish the Hb early temporal expression domain.
Tbx2 and Tbx3 are closely related members of the T-box family of transcription factors that are important regulators during normal development as well as major contributors to human developmental syndromes when mutated. Although there is evidence for the involvement of Tbx2 and Tbx3 in pancreatic cancer, so far there are no reports characterizing the normal expression pattern of these genes in the pancreas. In this study, we examined spatial and temporal expression of Tbx2 and Tbx3 in mouse pancreas during development and in the adult using in situ hybridization and immunohistochemistry. Our results show that Tbx2 and Tbx3 are both expressed in the pancreatic mesenchyme throughout development beginning at embryonic day (E) 9.5. In addition, Tbx2 is expressed in pancreatic vasculature during development and in epithelial-derived endocrine and ductal cells during late fetal stages, postnatal development and in adult pancreas. In contrast, Tbx3 is expressed in exocrine tissue in the postnatal and adult pancreas. Further our results demonstrate that Tbx2 and Tbx3 are expressed in tumor-derived endocrine and exocrine cell lines, respectively. These dynamic changes in the expression pattern of these transcription factors lay the foundation for investigation of potential roles in pancreas development.
Tbx2; Tbx3; T-box; Pancreas; Islets
Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. In this paper, we demonstrate that sim is expressed in 3 clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in 3 clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which we term PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim+ neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5’-flanking, intronic, and 3’-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim+ brain cells, including all sim+ neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim+ brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim+ brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim+ brain neurons. Sequence analysis of existing sim mutant alleles identified 3 likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate Sim that have an early role in neuronal migration and a later role in axonogenesis.
Autoregulation; Axon guidance; Brain; Drosophila; Enhancer; Optic lobes; single-minded
The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.
Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.
Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are functionally important during vertebrate development. We have generated a zebrafish genetrap line that produces fluorescently tagged Crmp1 protein, which can be dynamically tracked in living fish at subcellular resolution. The results show that Crmp1 is expressed in numerous sites in the developing nervous system. Early expression is apparent in the forebrain, epiphysis, optic tectum and the developing spinal cord. In the larval brain, Crmp1 is expressed in several distinct brain regions, such as the telencephalon, habenula and cerebellum. In addition, it is expressed in the spinal cord in a manner that persists in the larva. The results suggest that this Crmp1 protein trap line offers a powerful tool to track selected neuronal populations at high resolution.
Crmp1; zebrafish; nervous system; development
Neuronal migration and growth cone motility are essential aspects of the development and maturation of the nervous system. These cellular events result from dynamic changes in the organization and function of the cytoskeleton, in part due to the activity of cytoskeletal motor proteins such as myosins. Although specific myosins such as Myo2 (conventional or muscle myosin), Myo1, and Myo5 have been well characterized for roles in cell motility, the roles of the majority of unconventional (other than Myo2) myosins in cell motility events have not been investigated. To address this issue, we have undertaken an analysis of unconventional myosins in zebrafish, a premier model for studying cellular and growth cone motility in the vertebrate nervous system. We describe the characterization and expression patterns of several members of the unconventional myosin gene family. Based on available genomic sequence data, we identified 18 unconventional myosin- and 4 Myo2-related genes in the zebrafish genome in addition to previously characterized myosin (-1, -2, -3, -5, -6, -7) genes. Phylogenetic analyses indicate that these genes can be grouped into existing classifications for unconventional myosins from mouse and man. In situ hybridization analyses using EST probes for 18 of the 22 identified genes indicate that 11/18 genes are expressed in a restricted fashion in the zebrafish embryo. Specific myosins are expressed in particular neuronal or neuroepithelial cell types in the developing zebrafish nervous system, spanning the periods of neuronal differentiation and migration, and of growth cone guidance and motility.
cytoskeleton; unconventional myosin; neuronal migration; axon guidance; growth cone motility; zebrafish; commissure; spinal cord; motor neuron; neural crest; somite; ear; eye; morphogenesis; in situ hybridization; phylogenetic tree; hindbrain; forebrain; midbrain; cranial muscles
The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms’ tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue–tissue interactions guiding multiple developmental processes.
Limk; Kidney; Heart; Epithelia-to-mesenchyme transition; Mesenchyme-to-epithelia transition; Eye; Testes
During embryogenesis, the rhombic lip of the fourth ventricle is the germinal origin of a diverse collection of neuronal populations that ultimately reside in the brainstem and cerebellum. Rhombic lip neurogenesis requires the bHLH transcription factor Atoh1 (Math1), and commences shortly after neural tube closure (E9.5). Within the rhombomere 1 – isthmus region, the rhombic lip first produces brainstem and deep cerebellar neurons (E9.5-E12), followed by granule cell precursors after E12. While Atoh1 function is essential for all of these populations to be specified, the downstream genetic programs that confer specific properties to early and late born Atoh1 lineages are not well characterized. We have performed a comparative microarray analysis of gene expression within early and later born cohorts of Atoh1 expressing neural precursors purified from E14.5 embryos using a transgenic labeling strategy. We identify novel transcription factors, cell surface molecules, and cell cycle regulators within each pool of Atoh1 lineages that likely contribute to their distinct developmental trajectories and cell fates. In particular, our analysis reveals new insights into the genetic programs that regulate the specification and proliferation of granule cell precursors, the putative cell of origin for the majority of medulloblastomas.
rhombic lip; Atoh1; Math1; cerebellum; neurogenesis; rhombomere 1
Dystrophin/dystrobrevin superfamily proteins play structural and signalling roles at the plasma membrane of many cell types. Defects in them or the associated multiprotein complex cause a range of neuromuscular disorders. Members of the dystrophin branch of the family form heterodimers with members of the dystrobrevin branch, mediated by their coiled-coil domains. To determine which combinations of these proteins might interact during embryonic development, we set out to characterise the gene expression pattern of dystrophin and dystrobrevin family members in zebrafish. γ-dystrobrevin (dtng), a novel dystrobrevin recently identified in fish, is the predominant form of dystrobrevin in embryonic development. Dtng and dmd (dystrophin) have similar spatial and temporal expression patterns in muscle, where transcripts are localized to the ends of differentiated fibres at the somite borders. Dtng is expressed in the notochord while dmd is expressed in the chordo-neural hinge and then in floor plate and hypochord. In addition, dtng is dynamically expressed in rhombomeres 2 and 4-6 of the hindbrain and in the ventral midbrain. α-dystrobrevin (dtna) is expressed widely in the brain with particularly strong expression in the hypothalamus and the telencephalon; drp2 is also expressed widely in the brain. Utrophin expression is found in early pronephros and lateral line development and utrophin and dystrophin are both expressed later in the gut. β-dystrobrevin (dtnb) is expressed in the pronephric duct and widely at low levels. In summary, we find clear instances of co-expression of dystrophin and dystrobrevin family members in muscle, brain and pronephric duct development and many examples of strong and specific expression of members of one family but not the other, an intriguing finding given the presumed heterodimeric state of these molecules.
muscle; zebrafish; notochord; midbrain; rhombomere; gene expression; utrophin; dystrophin; dystrobrevin; drp2; dystrotelin